Beat Ernst, Gerald W. Hart, Pierre Sinay
Carbohydrates in Chemistry and Biology
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Beat Ernst, Gerald W. Hart, Pierre Sinay
Carbohydrates in Chemistry and Biology Part I
Chemistry of Saccharides
VOl. 1
Chemical Synthesis of Glycosides and Glycomimetics
@WILEY-VCH Weinheim - New York * Chichester Brisbane - Singapore * Toronto
Beat Emst, Gerald W. Hart, Pierre Sinay
Carbohydrates in Chemistry and Biology Part I
Chemistry of Saccharides
VOl. 2
Enzymatic Synthesis of Glycosides and Carbohydrate-Receptor Interaction
Weinheim - New York - Chichester Brisbane - Singapore - Toronto
Beat Ernst, Gerald W. Hart, Pierre Sinay
Carbohydrates in Chemistry and Biology Part I1
Biology of Saccharides
VOl. 3
Biosynthesis and Degradation of Glycoconjugates
Weinheim - New York * Chichester Brisbane - Singapore * Toronto
Beat Ernst, Gerald W. Hart, Pierre Sinay
Carbohydrates in Chemistry and Biology Part I1
Biology of Saccharides
VOl. 4
Lectins and Saccharide Biology
@WILEY-VCH Weinheim - New York * Chichester Brisbane * Singapore * Toronto
Prof. Dr. B. Ernst Institut fur Molekulare Pharmazie Universitat Basel Klingenbergstrasse SO 4051 Basel Switzerland
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Prof. Dr. G. W. Hart Dept. of Biological Chemistry Johns Hopkins University School of Medicine 725 N. Wolf St. Rm. 401 Hunterian Baltimore, MD 21205-2185 USA ___p
Prof. Dr. P. Sinay Dept. de Chimie, URA 1686 Ecole Normale SupCrieure 24 rue Lhomond 75231ParisCedex05 France
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This book was carefully produced. Nevertheless, editors, authors and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.
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Cover picture: 3,6-dideoxyhexose binding to antibody se155.4 Courtesy of David Bundle, University of Alberta
Library of Congress Card No.: applied for A catalogue record for this book is available from the British Library. Die Deutsche Bibliothek - CIP Cataloguing-in-Publication-Data A catalogue record for this publication is available from Die Deutsche Bibliothek ISBN 3-527-295 11-9
0 WILEY-VCH Verlag GmbH, D-69469 Weinheim (Federal Republic of Germany). 2000 Printed on acid-free and chlorine-free paper. All rights reserved (including those of translation in other languages). No part of this book may be reproduced in any form - by photoprinting, microfilm, or any other means - nor transmitted or translated into machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Composition: Asco Typesetters, Hongkong. Printing: betz-druck gmbH, D-6429 1Darmstadt. Bookbinding: Wilhelm Osswald & Co., D-67433 Neustadt. Printed in the Federal Republic of Germany.
About the Editors
Beat Ernst studied chemistry at the ETH in Zurich, Switzerland, where he completed his PhD-thesis about novel tricyclic hydrocarbons under the guidance of Professors Oskar Jeger and Camille Ganter. After his post-doctoral research on tirandamycinic acid with Professor R. E. Ireland at caltech in Pasadena, he joined the Central Research Laboratories of Ciba-Geigy (now Novartis) in Basel where he headed the Carbohydrate Research Section. In 1998 he became Professor of Molecular Pharmacy at the University of Basel, Switzerland. His major research interests are Carbohydrate Chemistry and Glycobiologymainly the chemical and enzymatic syntheses of oligosaccharides, investigation of carbohydrate/lectin interactions and carbohydrate mimics. In 1991 he was awarded the Werner-Price of the Swiss Chemical Society, and in 1993 he became CIBA-fellow for his research contributions.
Gerald Warren Hart is Director and DeLamar Professor of Biological Chemistry at Johns Hopkins University in Baltimore, MD. He studied biology, and chemistry at Washburn University and completed his PhD-thesis in developmental biology at Kansas State University before he was appointed assistant professor of Biological Chemistry at Johns Hopkins University School of Medicine. From 1993 to 1997 he held a chair at the department of Biochemistry and Molecular Genetics at the University of Alabama before he returned to Johns Hopkins University. In 1989 he founded the journal Glycobiology which became the leading journal in the field. His research results and contributions to the field of glycobiology are honored with many prices and awards-President of the Society of Glycobiology, Member of the Council of the ASBMB, First International Glycoconjugate Organization Award, just to name a few.
VI
About the Editors Pierre Sinay studied chemistry at Ecole Nationale Superieure des Industries Chimiques, Nancy, France, and received his PhD from the University of Nancy. After his post-doctoral research at Harvard University he became Professor at the University of Orleans before he was appointed professor of organic chemistry at Pierre et Marie Curie University and Ecole Normale Superieure in Paris. His main research focuses on the chemical synthesis of oligosaccharides of biological relevance and on the development of new synthetic methods in the carbohydrate field. He is Editor-in-Chief of the journal Carbohydrate Letters and he has published numerous scientific papers and patents. In 1987 he was elected president of the organic chemistry division of the French Chemical Society. In 1978 he was awarded the Le Be1 Price of the French Chemical Society, in 1996 he was triple-honoured with the Desnuelles Price, the Bethellot Medal and with the election as corresponding member of the French Academy of Sciences.
Contents
Part I
.
Vol 1
Chemistry of Saccharides Chemical Synthesis of Glycosides and Glycomimetics List of Contributors ...................................................
LV
Abbreviations Used in Volumes 1 and 2 ...........................
LXIII
I
Chemical Synthesis of Glycosides ..................................
1
1
Introduction to Volumes 1 and 2 .......................................
3
2
Trichloroacetimidates .................................................. Richard R. Schmidt and Karl-Heinz Jung Introduction ............................................................ Methods ................................................................ 0-Glycosides ............... ......................................... Synthesis of Oligosaccharides ......................................... p-Glucosides, p.Galactosides, a.Rhanmosides. etc.................... Aminosugar Trichloroacetimidates.................................... p-Mannosides ......................................................... 2-Deoxyglycosides .................................................... Miscellaneous Compounds ........................................... Complex Oligosaccharides............................................ Inositol Glycosides.................................................... Glycosylation of Sphingosine Derivatives and Mimics .............. Glycosylation of Amino Acids ....................................... Polycyclic and Macrocyclic Glycosides ..............................
5
2.1 2.2 2.3 2.3.1
2.3.2 2.3.3 2.3.4 2.3.5
5 6 7 7 7 8 13 13 14 14 36 38 40 42
VIII
Contents
2.3.6 2.3.7 2.4 2.5 2.6 2.7
Glycosides of Phosphoric and Carboxylic Acids .................... Solid-Phase Synthesis................................................. S-Glycosides .......................................................... N- and P-Glycosides.................................................. C-Glycosides.......................................................... Conclusion and Outlook ............................................. References ............................................................
3
Iterative Assembly of Glycals and Glycal Derivatives: The Synthesis of Glycosylated Natural Products and Complex Oligosaccharides... Lawrence J . Williams. Robert M . Garbaccio. and Samuel J . Danishefsky Introduction .......................................................... Ciclamycin 0 .......................................................... Allosamidin ........................................................... KS-502 and Rebeccamycin ........................................... Extension to Thioethyl Donors ...................................... LewisY ................................................................. Globo H .............................................................. KH-1 .................................................................. Concluding Remarks ................................................. Acknowledgments .................................................... References ............................................................
3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9
4
4.1 4.2 4.2.1 4.2.2 4.2.3 4.3 4.3.1 4.3.2
4.4 4.4.1 4.4.2 4.4.3
44 45 49 51 51 53 53
61
61 64 66 69 74 76 82 86 90 90 90
Thioglycosides ........................................................ 93 Stefan Oscarson Introduction .......................................................... 93 Synthesis of Thioglycosides .......................................... 94 From Anomeric Acetates............................................. 94 From Glycosyl Halides ............................................... 95 Protecting Group Manipulations in Thioglycosides ................. 96 Glycosylations with Thioglycoside Donors .......................... 97 A Two-step Procedure: Transformation of Thioglycosides into 97 Other Types of Glycosyl Donors ..................................... 99 Direct Activation of Thioglycoside Donors ......................... 99 Heavy Metal Salt Promoters ......................................... Halonium, sulfonium and carbonium type promoters ............... 100 Single-ElectronActivation ........................................... 106 Other Types of Donors With an Anomeric Sulfur .................. 108 Applications of Thioglycosides....................................... 110 Block Syntheses, Orthogonal Glycosylations ........................ 110 Thioglycosides as Acceptors.......................................... 110 Thioglycosides as Both Donors and Acceptors ...................... 111 Intramolecular Glycosidations ....................................... 112 Solid Phase Synthesis ................................................. 113 Reference6 ............................................................ 113
Contents 5
5.1 5.2 5.3 5.3.1 5.3.2 5.3.3 5.3.4 5.3.5
5.3.6 5.4 5.4.1 5.4.2 5.4.3 5.4.4
6
6.1 6.2 6.3 6.4 6.5 6.6 6.7 7
7.1 7.2 7.3 7.4 7.4.1
IX
Glycosylation Methods: Use of Phosphites ........................... 117 Zhiyuan Zhang and Chi-Huey Wong Introduction ........................................................... 117 118 Preparation of Glycosyl Phosphites .................................. Glycosylation using Glycosyl Phosphites ............................ 119 119 Mechanism ............................................................ Low Temperature-Dependent Stereoselectivity ...................... 121 Glycosylation of Sialyl Phosphites ................................... 122 Glycosylation of C-2-Acylated Glycosyl Phosphites ................. 123 Glycosylation with C-2-O-Benzylated Glycosyl Phosphites ......... 124 Glycosylation using Glucosyl Phosphites with a Benzyl Group at C-2 .................................................................... 124 Glycosylation using Galactosyl and Fucosyl Phosphites with a 125 Benzyl Group at C-2.................................................. Glycosylation using other Glycosyl Phosphites with a Benzyl Group at C-2 .......................................................... 126 Glycosylation with 2-Deoxy Glycosyl Phosphites ................... 127 128 Other Applications of Glycosyl Phosphites .......................... Synthesis of CMP-NeuAc ..... .................................... 129 Synthesis of GDP-Fucose ............................................ 129 Formation of Glycosyl Phosphonate. ................................ 131 Transformation to other Types of Glycosyl Donor ..................131 131 Phosphate ............................................................. Phosphorimidate ...................................................... 131 132 References............................................................. Glycosylation Methods: Use of n-Pentenyl Glycosides ............... 135 Bert Fraser.Reid. G. Anilkumar. Mark R. Gilbert. Subodh Joshi. and Ralf Kraehmer Introduction ........................................................... 135 Fundamental Reactions .............................................. 135 Determination of Relative Reactivities ............................... 138 n-Pentenyl Orthoesters as Glycosyl Donors .......................... 141 144 n-Pentenyl Orthoesters as Latent C2 Esters .......................... 146 Protecting Groups .................................................... Solid-Phase Iterative Couple-Deprotect-Couple Strategy .......... 146 153 References .............................................................. Glycosylidene Diazirines .............................................. Andrea Vasella. Bruno Bernet. Martin Weber. and Wolfgang Wenger Introduction ........................................................... Synthesis of Glycosylidene Diazirines ................................ Stability of the Glycosylidene Diazirines ............................. Glycosidation by Glycosylidene Diazirines .......................... General Aspects .......................................................
155 155 155 158 158 158
X 7.4.2 7.4.3 7.5 7.6 7.7
8
8.1 8.2 8.2.1 8.2.2 8.2.3 8.2.4 8.2.5 8.2.6 8.2.7 8.2.8 8.2.9 8.3 8.3.1 8.3.2 8.3.3 8.3.4 8.3.5
Contents
Glycosidation of Strongly Acidic Hydroxy Compounds ............ 162 Glycosidation of Phenols ............................................. 162 163 Glycosidation of Fluorinated Alcohols .............................. Glycosylation of Weakly Acidic Hydroxy Compounds ............. 163 163 Glycosidation of Monovalent Alcohols .............................. Glycosidation of Diols and Triols ................................... 164 Synthesis of Spirocyclopropanes ..................................... 168 Addition to Aldehydes and Ketones ................................. 170 Exploratory Use of Diazirines: Formation of Glycosyl Phosphines, Stannanes. N.Sulfonylamines. Esters. Boranes. and Alanes. and of 1.l-Difluorides ........................................................ 171 Acknowledgments .................................................... 174 174 References ............................................................ Glycosylation Methods: Alkylations of Reducing Sugars ............ 177 Jun-ichi Tamura Introduction .......................................................... 177 Anomeric O-Alkylation .............................................. 177 Anomeric O-Alkylation of Ribofuranose with Primary Triflates: Effect of the Protecting Group at 0 - 5 of Ribofuranose ............. 178 Anomeric O-Alkylation of Mannofuranose with Primary Triflates: 179 The Crown Ether Effect .............................................. Anomeric O-Alkylation of Gluco- and Galactopyranoses with Primary Triflates: High P-Selectivity as a Result of the Reactive Anomeric p-Anion .................................................... 180 Anomeric O-Alkylation of Acyl-Protected Nucleophiles with Primary Triflates ...................................................... 181 Anomeric O-Alkylation of Mannopyranose with Primary Triflates: Possibility of Intramolecular Complexation of the Nucleophile .... 184 Anomeric O-Alkylation of KDO with Primary Triflates ............ 185 Anomeric O-Alkylation of Some Protected Aldoses with Primary 186 Triflate ................................................................ Anomeric O-Alkylation of Unprotected Aldoses with Primary 187 Triflate, Bromides, and Cyclic Sulfates .............................. Anomeric O-Alkylation with Secondary Triflates and Nonaflate ... 188 Glycosylation via the Locked Anomeric Configuration ............. 189 Synthesis of Methyl. Allyl. and Benzyl Glycosides via Stannylene Acetals ................................................................ 189 Epimerization at C-2 by the Locked Anomeric Configuration 189 Method ............................................................... The Locked Anomeric Configuration Method for Rhamnosyl Stannylene Acetal ..................................................... 190 The Locked Anomeric Configuration Method for Mannosyl Stannylene Acetal: Isomerization of Acetal [25. 261 ................. 190 The Locked Anomeric Configuration Method for Stannylene Acetal with the Glucose Configuration [25. 261 ..................... 191
Contents
XI
8.4
Conclusion ............................................................ References .............................................................
9
Other Methods of Glycosylation ...................................... 195 Luigi Panza and Luigi Lay 195 Introduction and Summary ........................................... Highlights ............................................................. 195 Enol Ethers ........................................................... 197 Endo-En01 Ethers ..................................................... 198 Exo-Enol Ethers ...................................................... 201 Endo-Glycals ......................................................... 202 Exo-Glycals .......................................................... 204 Vinyl Glycosides ...................................................... 206 1-Hydroxy Sugars ..................................................... 209 Acidic Activation ..................................................... 210 Acidic Activation With Additional Reagents ........................ 211 Dehydrative Glycosylation ........................................... 212 In the Presence of the Acceptor From the Beginning ................213 214 Mitsunobu Glycosylation ............................................. I-0-Silyl Glycosides .................................................. 215 Esters and Related Derivatives ....................................... 216 216 Esters .................................................................. Sugar Carbonates and Derivatives ................................... 221 Orthoesters and Oxazolines........................................... 223 Phosphorus and Sulfur Derivatives .................................. 229 References ............................................................. 233
9.1 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 9.2 9.2.1 9.2.2 9.2.3 9.2.4 9.3 9.3.1 9.3.2 9.3.3 9.3.4
10 10.1 10.2 10.3 10.4 10.4.1 10.4.2 10.5
10.5.1
192 193
Polymer-Supported Synthesis of Oligosaccharides.................... 239 Jiri J. Krepinsky and Stephen P . Douglas Introduction ........................................................... 239 General Reflections ................................................... 240 Polymer Supports ..................................................... 246 One-Phase Systems (Syntheses in Solution) .......................... 247 248 Polyethyleneglycol,. monomethylether (MPEG) ..................... 249 Linear Polystyrene .................................................... Linkers ................................................................ 250 Succinoyl Diester ..................................................... 250 Dioxyxylyl Diether (DOX) ........................................... 252 254 Chemistry Investigations ............................................. Two-Phase Systems (Syntheses on Solid Supports) ..................255 Controlled Pore Glass ................................................ 256 256 Cross-Linked Polystyrene ............................................. Polyethylene Grafts on Cross-Linked Polystyrene. ..................256 Linkers ................................................................ 259 259 Dialkyl- or Diaryl-Silyl ............................................... Thioglycoside Linkers ................................................ 259
XI1
10.6 10.7 10.8 10.9 11 11.1 11.1.1 11.1.2
11.1.3 11.2 11.2.1
11.2.2 11.3 11.3.1
11.3.2
Contents
Linkers Cleavable by Photolysis ..................................... Examples of Syntheses ............................................... Combinatorial Libraries .............................................. Capping ............................................................... Concluding Remarks ................................................. References ............................................................
260 260 261 262 262 262
Glycopeptide Synthesis in Solution and on the Solid Phase .......... 267 Horst Kunz and Michael Schultz Introduction .......................................................... 267 Which Protecting Groups are Suitable for Carbohydrates (Table 2)? ............................................................. 269 Which Glycosylation Methods are Useful for the Formation of Glycopeptides?........................................................ 271 Formation of Asparagine N-Glycosides ............................. 271 a-Fucosylation ........................................................ 271 272 Formation of the P-Lactosamine Linkage ........................... a-Sialylation .......................................................... 272 Glycopeptides Containing Particularly Sensitive Linkages .......... 272 Acid Sensitivity ....................................................... 273 273 Base Sensitivity ....................................................... Synthesis of Glycopeptides in Solution .............................. 274 0-Glycopeptides ...................................................... 274 Glycopeptides Carrying N-Acetylgalactosamine (Tn-Antigen)...... 274 Glycopeptides Carrying the T-Antigen (Gal-GalNAc) ............. 276 Glycopeptides Carrying the Sialyl T Antigen (NeuAca2,6[Galpl,31GalNAc) ....................................... 278 279 Glycopeptides Carrying 0-GlcNAc .................................. N-Glycopeptides ...................................................... 280 N-Glycopeptides Carrying Natural Saccharide Side-Chains ........ 280 N-Glycopeptides with Lewis-Type Saccharide Side-Chains ......... 285 Glycopeptide Synthesis on the Solid Phase .......................... 286 0-Glycopeptides ...................................................... 287 Glycopeptides Carrying N-Acetylgalactosamine (Tn-Antigen)...... 287 0-Glycopeptides Carrying the T Antigen (Gal-GalNAc) .......... 290 0-Glycopeptides Carrying the Sialyl Tn Antigen (NeuNAc~a2, 6. GalNAc) .............................................................. 291 0-Glycopeptides Carrying the 2, 3.Sialyl T Antigen ................. 293 0-Glycopeptides Carrying 0-GlcNAc Side-Chains .................294 295 0-Glycopeptides Carrying 0-Linked Fucose ........................ 0-Glycopeptides Carrying a Sialyl Lewis Antigen Structure ........ 296 N-Glycopeptides ...................................................... 297 The Construction of N-Glycopeptide Libraries on the Solid 298 Phase .................................................................. Sequential N-Glycopeptide Synthesis on the Solid Phase with Oligosaccharides from Natural Sources.............................. 299
Contents
XI11
11.4
Conclusion ............................................................ References .............................................................
12
Glycolipid Synthesis ................................................... 305 Hidehuru Ishida Introduction ........................................................... 305 Synthesis of Ganglio-Series Gangliosides ............................ 305 305 Retrosynthetic Analysis of Ganglioside G D l a ...................... Preparation of Sialylgalactose Donor as Building Block ............ 306 Construction of Oligosaccharide ..................................... 308 Transformation of Oligosaccharide into Glycolipid .................310 Synthesis of Polysialo Ganglio-Series Gangliosides.................. 311 Retrosynthetic Analysis of GQl b .................................... 313 313 Preparation of Building Block ........................................ Construction of Oligosaccharide ..................................... 314 315 Conclusion ............................................................ References ............................................................. 316
12.1 12.2 12.2.1 12.2.2 12.2.3 12.2.4 12.3 12.3.1 12.3.2 12.3.3 12.4 13
13.1 13.2 13.2.1
13.2.2 13.2.3 13.2.4 13.2.5
13.2.6 13.2.7 13.2.8 13.2.9 13.3 13.4
Stereoselective Synthesis of P-Mannosides............................ Vince Pozsguy Introduction ........................................................... Chemical Methods .................................................... Glycosylation with Mannosyl Donors ............................... Mannosylation using Insoluble Promoters ........................... The Sulfonate Approach .............................................. Intramolecular Mannosylation ....................................... Other Mannosyl Donor-Based Methods ............................. Epimerization of p-Glucopyranosides at C-2 ........................ The Oxidation-Reduction Approach ................................ Direct Inversion ....................................................... The 2-Ulosyl Donor Method ......................................... Anomeric O-alkylation ............................................... Alkylation of l-O-Metal Complexes ................................. The Stannylene Acetal Method ...................................... Miscellaneous Methods ............................................... Radical Inversion of the Anomeric Chirality of a-DMannopyranosides .................................................... Reductive Cleavage of Cyclic Orthoesters ........................... De nouo Syntheses .................................................... 2-Acetamido-2-deoxy-~-~-mannopyranosides ....................... Aryl p-D-mannopyranosides.......................................... l-Thio-P-D-mannopyranosides ....................................... P-D-Mannopyranosylamines .......................................... Enzymatic Synthesis .................................................. Conclusions ........................................................... References.............................................................
300 300
319 319 320 320 320 322 324 327 329 329 329 331 332 332 332 333 333 334 334 335 336 336 337 337 338 338
XIV 14
14.1 14.2 14.3 14.4 14.4.1 14.4.2 14.4.3 14.5 14.5.1 14.5.2 14.5.3
15
15.1 15.2 15.2.1 15.2.2 15.2.3 15.2.4 15.2.5 15.2.6 15.2.7 15.3 15.4 15.5 15.5.1 15.5.2 15.5.3 15.5.4 15.6 15.6.1 15.6.2 15.6.3 15.6.4 15.6.5
Contents
Special Problems in Glycosylation Reactions: Sialidations ........... 345 Mukoto Kiso. Hidehuru Ishida and Hirorni It0 Introduction .......................................................... 345 Sialidation by the Koenigs-Knorr Method .......................... 345 347 Sialidation Using an Auxiliary Group at C-3 ....................... Sialidation Using 2.Thioglycosides. Xanthates. or Phosphites of 349 Sialic Acids in Acetonitrile ........................................... Thioglycosides ........................................................ 349 Xanthates and Phosphites ............................................ 356 359 Reaction Mechanism ................................................. Further Solutions to the Problem .................................... 359 Combination of C-3 Auxiliary and Sterically less Hindered Sugar Acceptors ............................................................. 359 Combination of C-3 Auxiliary and Specific Activation of the 360 Anomeric Center C-2 ................................................. Thioglycoside of N ,N.Diacetylneuraminic Acid and Combination 363 with C-3 Auxiliary .................................................... References ............................................................ 364 Special Problems in Glycosylation Reactions: 2-Deoxy Sugars ...... 367 Aluin Veyri2res Introduction .......................................................... 367 Electrophilic Additions to Glycals: Mechanistic Aspects and Applications to the Synthesis of 2-Deoxyglycosides . ................ 368 Protonation of Glycals ............................................... 369 370 Enzyme-Catalyzed Additions to Glycals............................. Halogenation of Glycals ............................................. 370 372 Bromo- and Iodoalkoxylation of Glycals ............................ 377 Epoxidation of Glycals ............................................... Addition of Sulfur Based Electrophiles to Glycals .................. 379 Addition of Selenium Based Electrophiles to Glycals ............... 382 The Cycloaddition Way to Glycosyl Transfer ....................... 384 385 Fluoroglycosylation of Glycals ....................................... 386 Glycosyl Donors with a C-2 Heteroatom ............................ 2-Bromo-2-deoxyglycosyl bromides .................................. 386 2-Deoxy-2-(thiophenyl)-glycosylfluorides ........................... 387 388 2,6.Anhydro-2-Thi o-Glycosyl Donors ............................... 1,2-Di-O-Acetyl-P-Hexopyranoses and N-Formylglucosamine Derivatives ............................................................ 392 2-Deoxyglycosyl Donors ............................................. 393 394 2-Deoxy-Hexopyranoses ............................................. Tert-Butyldimethylsilyl 2-Deoxyglycosides .......................... 394 1-0-Acyl- and Acetimidyl-2-Deoxy.Hexopyranoses ................. 394 395 2-Deoxyglycosyl Bromides and Fluorides ........................... S-(2-Deoxyglycosyl)phosphorodithioates............................ 396
Contents 15.6.6 15.6.7 15.6.8 5.7 5.7.1 5.7.2
6
16.1 16.2 16.2.1 16.2.2 16.3 16.3.1 16.3.2 16.3.3 16.3.4 16.4 16.4.1 16.4.2 16.4.3 16.5
17
17.1 17.2 17.3 17.4 17.5 17.5.1 17.5.2 17.6 17.7 17.8
XV
2-Deoxyglycosyl Phosphates. Phosphoramidites and Phosphites .... 397 2-Deoxy Thioglycosides .............................................. 398 399 2-Deoxyglycosyl Sulfoxides........................................... Other Approaches to 2-Deoxyglycosides............................. 400 Cyclization of Acyclic Sugars ........................................ 401 Use of Alkoxy-Substituted Anomeric Radicals ...................... 402 References............................................................. 403 Orthogonal Strategy in Oligosaccharide Synthesis ...................407 Osurnu Kun ie Introduction ........................................................... 407 Analysis of the Strategic Aspects of Oligosaccharide Synthesis ..... 408 General Aspects ....................................................... 408 The Pursuit of Efficiency in Oligosaccharide Synthesis ..............408 The Introduction of the Orthogonal Glycosylation Strategy ........ 410 Limitation of Current Concepts ...................................... 410 The Orthogonal Coupling Concept .................................. 412 413 What is Orthogonality Anyway? ..................................... Orthogonal Glycosylation and Solid-Phase Oligosaccharide 414 Synthesis .............................................................. The Orthogonal Glycosylation Strategy ............................. 414 Orthogonal Chain Elongation of Homo-Oligosaccharides: 414 Synthesis of Chito-Oligosaccharides [ 191 ............................. Orthogonal Coupling for Hetero-Oligomer Synthesis [22]...........418 Application to Polymer-Supported Synthesis [26] ...................420 421 Conclusions and Prospects ........................................... Acknowledgments .................................................... 424 References ............................................................. 424 Protecting Groups: Effects on Reactivity. Glycosylation Stereoselectivity. and Coupling Efficiency ............................ 427 Luke G. Green and Steven V. Ley Introduction ........................................................... 427 Glycosidic Mechanism ................................................ 428 Electronic and Torsional Effects ..................................... 430 Influence of Protecting Group on Donor Reactivity ................. 431 Stereoselectivity ....................................................... 436 Neighboring-Group Participation .................................... 436 437 Reactivity Control .................................................... influence of the Protecting Group on the Acceptor .................441 Steric Effects on Glycosylation ....................................... 443 444 Conclusions ........................................................... Acknowledgments .................................................... 445 References ............................................................. 446
XVI 18
18.1 18.2 18.2.1 18.2.2 18.3 18.4 19 19.1 19.2 19.2.1 19.2.2 19.2.3 19.2.4 19.2.5 19.2.6 19.3 19.3.1 19.3.2 19.3.3 19.3.4 19.3.5 19.3.6 19.4 19.4.1 19.4.2 19.4.3 19.5
Contents
Intramolecular Glycosidation Reactions .............................. 449 Jacob Madsen and Mikael Bols Introduction .......................................................... 449 Reactions in which the Tether Participates in the Reaction ......... 450 Tethering to the Glycosyl Donor .................................... 450 Carbon Tethers ....................................................... 450 Silicon Tethers ........................................................ 454 Tethering to the Leaving Group ..................................... 459 Reactions in which the Tether does not Participate in the Reaction 459 Conclusion ............................................................ 464 References ............................................................ 465 Classics In Total Synthesis of Oligosaccharides and Glycoconjugates....................................................... 467 Jean-Maurice Mallet and Pierre Sinay Introduction .......................................................... 467 Syntheses of Nod factors ............................................. 467 Introduction .......................................................... 467 The K . C . Nicolaou Synthesis (1992) [3]............................. 468 The J.-M. Beau Synthesis (1994) [ 121 ................................ 471 The T . Ogawa Synthesis (1994) [ 161 ................................. 475 The Y . Z . Hui Synthesis (1992) [ 181 ................................. 477 Conclusion ............................................................ 480 Synthesis of the Antithrombin-Binding Pentasaccharide Sequence in Heparin (1984) [ 19, 201 ............................................... 480 Introduction .......................................................... 480 An Overview of the Synthesis of the Protected Pentasaccharide 73 ..................................................................... 481 Synthesis of the Disaccharidic Bromide Donor 68 .................. 483 Synthesis of the Disaccharidic Acceptor 69 .......................... 484 Synthesis of the Protected Pentasaccharide 73 ....................... 484 Synthesis of the Active Site of Heparin .............................. 485 Total Synthesis of VIM-2 Ganglioside [31] .......................... 485 Introduction .......................................................... 485 The Total Synthesis of VIM-2-a General Strategy ................ 486 Preparation of the Key Protected Octasaccharide 87 ................ 487 Epilogue .............................................................. 490 References ............................................................ 491
I1
Synthesis of Oligosaccharide Mimics .................................
493
20
Synthesis of C-Oligosaccharides...................................... Troels Skrydstrup. Boris Vauzeilles. and Jean-Marie Beau Introduction .......................................................... The Anionic Approach ............................................... C5-Alkynyl Anions ...................................................
495
20.1 20.2 20.2.1
495 496 496
Contents
XVII
20.2.2 20.2.3 20.2.4 20.2.5 20.3 20.3.1 20.3.3 20.4 20.5
C1-Glycal Carbanions ................................................ 500 Anomeric Samarium Species ......................................... 502 C-Branched Carbanions .............................................. 506 C6-Phosphoranes ..................................................... 508 The Radical Approach ............................................... 511 Intermolecular Anomeric Radical Addition ......................... 511 513 Intramolecular Anomeric Radical Addition ......................... The Partial de Nuuo Approach ....................................... 518 The Cycloaddition and Rearrangement Approach ..................527 528 References .............................................................
21
531 Synthesis of Oligosaccharide Mimics: S-Analogs..................... Jon K. Fairweather and Hugues Driguez Introduction ........................................................... 531 General Synthesis ..................................................... 532 Preparation of Thioglycoses .......................................... 532 l-Thioglycoses ........................................................ 532 2-, 3-, 4-, 5.. or 6-Thioglycoses ....................................... 532 Selective S-Deprotection of Thioglycoses ............................ 533 Glycosylation Methods ............................................... 534 Establishment of 1,6-Thio Linkages .................................. 534 6-Thiodisaccharides ................................................... 534 6-Thiooligosaccharides ............................................... 538 538 Branched Thiocyclodextrins .......................................... Establishment of I , 4-Thio Linkages .................................. 541 1,4-Thiodisaccharides ................................................. 541 General Approaches .................................................. 541 SN~-Displacement on Triflates ....................................... 541 546 1,4-Thiooligosaccharides ............................................. Conventional Approaches ............................................ 546 548 Chemoenzymatic Approaches ........................................ 549 Michael Addition to Unsaturated Acceptors ........................ Solid-Support Synthesis........ ................................... 550 551 Establishment of 1, 3.Thio Linkages .................................. 1,3-Thiodisaccharides ................................................. 551 Conventional Methods ............................................... 551 Cyclic Sulfamidate and Aziridine .................................... 551 1,3-Thiooligosaccharides ............................................. 552 Establishment of 1,2.Thio Linkages .................................. 553 1,2-Thiodisaccharides................................................. 553 Conventional Methods ............................................... 554 Other Approaches .................................................... 555 Establishment of I , l-Thio Linkages .................................. 557 558 Establishment of Mixed Thio linkages ............................... Thiooligosaccharides and Proteins ................................... 558 The Conformation of Thiooligosaccharides in Solution ............. 558
21.1 21.2 21.2.1
21.3 21.3.1 21.3.2 21.3.3 21.4 21.4.1 21.4.2
21.5 21.5.1 21.5.2 21.6 21.6.1 21.7 21.8 21.9 21.9.1
XVIII
Contents
21.9.2
Enzyme-Substrate Interactions ...................................... a-Glucan- Active Enzymes ............................................
21.9.3 21.10
P-Glucan-Active Enzymes ............................................ Lectin-Ligand Interactions .......................................... Conclusion ............................................................ Acknowledgments .................................................... References ............................................................
22 22.1 22.2 22.2.1 22.2.2 22.3 22.4 22.4.1 22.4.2 22.4.3 22.4.4
Saccharide-Peptide Hybrids .......................................... Huns Peter Wessel Introduction .......................................................... Carbohydrate Amino Acids .......................................... Natural Carbohydrate Amino Acids ................................. Synthetic Carbohydrate Amino Acids ............................... Amide-Linked Carbohydrate Polymers .............................. Amide-Linked Carbohydrate Oligomers ............................. Solution Synthesis .................................................... Solid-Phase Synthesis................................................. Biological Activity .................................................... Conformational Properties ........................................... References ............................................................
565
Index ...................................................................
11
Part I
Chemistry of Saccharides
Vol. 2
Enzymatic Synthesis of Glycosides and CarbohydrateReceptor Interaction
111
Enzymatic Synthesis of Glycosides ..................................
23
On the Origin of Oligosaccharide Species-Glycosyltransferases in Action ................................................................ Dirk H . van den Eijnden Introduction ......................................................... Protein N-Glycosylation: Pre-assembly of Oligosaccharide-PPDolichol and en bloc Transfer ...................................... Trimming of the Polypeptide-Bound Oligosaccharide .............. Folding and Quality Control ........................................ Committed Steps in the Formation of Complex-Type Oligosaccharide Chains and Branching ............................. Topology of the Reaction Catalyzed by a Typical GlcNAcT ...... Elongation and Termination Reactions in the truns-Golgi ......... Activity with Branched Substrates ..................................
23.1 23.2 23.3 23.4 23.5 23.6 23.7 23.8
560 560 561 562 562 562 562
565 566 566 567 572 574 574 578 579 582 583
587 589 589 591 592 593 594 596 596 598
Contents
23.9 23.10 23.11 23.12 23.13 23.14 23.15 23.16 23.17 23.18 23.19 23.20 23.21 23.22 23.23 23.24 23.25 23.26 23.27 23.28 23.29 24 24.1 24.2 24.2.1
24.2.2
24.3 24.4
XIX
600 Branch Specificity .................................................... Essential Requirements for Activity with LacNAc ................. 601 Further Terminal Reactions in Complex-Type Oligosaccharide 602 Synthesis ............................................................. Specific Modifications of Polylactosaminoglycans .................. 603 The Invariable Core of N-linked Oligosaccharide Chains, and 606 Site- and Protein-Specific Processing ................................ Comparison of the Synthesis of Type 1 (Gal(P1-3)GlcNAcP-R) 607 and Type 2 (Gal(P1-4)GlcNAcP-R) Chains ........................ The LacdiN Ac Pathway of Complex-Type Oligosaccharide Synthesis ................... ....................................... 607 608 Protein O-Glycosylation ............................................. Glycosyltransferase Families ........................................ 608 610 Sialyltransferase Family ............................................. 611 a2-Fucosyltransferase Family ....................................... 612 a3/4-Fucosyltransferase Family ..................................... a3-Galactosyl/N-Acetylgalactosaminyltransferase(Histo-Blood Group ABO) Family ................................................ 613 P6-N- Acetylglucosaminyltransferase Family ........................ 613 Polypeptide N-Acetylgalactosaminyltransferase Family ............ 614 P4-N-AcetylgalactosaminyltransferaseFamily ...................... 615 P4-Galactosyltransferase Family .................................... 615 P3-Galactosyltransferase Family .................................... 617 P3-Glucuronyltransferase Family ................................... 617 Glycosyltransferases Standing Alone ................................ 617 618 Concluding Remarks ................................................ References............................................................ 618 Synthesis of Sugar Nucleotides ...................................... Reinhold Ohrlein Introduction .......................................................... Synthesis of Sugar Nucleotides ...................................... Chemical Synthesis .................................................. UDP-Activated Donors ............................................. CMP-Activated Sugars.............................................. GDP-Activated Donors ............................................. Comments ............................................................ Chemo-Enzymatic Synthesis ........................................ Uridine Diphosphate-Activated Donor Sugars ..................... CMP-Activated Sugars.............................................. GDP-activated sugars ............................................... Comments ............................................................ In situ Generation of Sugar Nucleotides ............................ Comments ............................................................ Outlook .............................................................. References............................................................
62.5 62.5 626 626 626 629 632 634 635 635 637 639 640 641 641 644 644
XX 25
25.1 25.2 25.2.1 25.2.2 25.2.3 25.2.4 25.2.5 25.2.6 25.2.7 25.3 25.3.1 25.3.2 25.3.3 25.3.4 25.4 25.5
26 26.1 26.2 26.3 26.4 26.4.1 26.4.2 26.4.3 26.4.4 26.4.5 26.5
27
27.1 27.2 27.2.1
Contents
Enzymatic Glycosylations with Glycosyltransferases ................ Ossi Renkonen Introduction ......................................................... In vitro Synthesis of the Core Region of 0.Glycans ................ Initialization of O-Glycan Biosynthesis ............................. Synthesis of Core 1 .................................................. Synthesis of Core 2 .................................................. Synthesis of Core 3 and Core 4 ..................................... In vitro Extension of Core 1 Glycans ............................... In vitro Extension of Core 2 Glycans ............................... Extension of Core 3 and Core 4 Glycans ........................... Enzymatic in vitro Synthesis of Polylactosamine Backbones ....... Enzymatic Synthesis of the Primary Chains of Blood Group i-Type ................................................................ Distal Branching of i-Type Polylactosamine Backbones ........... Central Branching of i-Type Polylactosamine Backbones.......... P4-Galactosylation in Polylactosamine Backbones ................. a3-Sialylation of N-Acetyllactosaminoglycansat the Terminal Gal a3-Fucosylation of Lactosamine Saccharides....................... References ........................................................... Recycling of Sugar Nucleotides in Enzymatic Glycosylation........ Kathryn M . Koeller and Chi-Huey Wong Introduction ......................................................... Glycosyltransferases of the Leloir Pathway and their Sugar Nucleotide Substrates ............................................... Design of Regeneration Systems .................................... Practical Regeneration Systems ..................................... UDP-Galactose ..................................................... Other UDP-Sugars .................................................. CMP-NeuAc ........................................................ GDP-Sugars ......................................................... Other Carbohydrate-Based Regeneration Systems ................. Conclusion ........................................................... References ........................................................... Enzymatic Glycosylations with Non-Natural Donors and Acceptors............................................................. Xiangping Qian. Keiko Sujino. and Monica M . Palcic Introduction ......................................................... Enzymatic Glycosylations ........................................... Galactosylations ..................................................... P 1.4-Galactosyltransferase .......................................... al.3-Galactosyltransferase ..........................................
647 647 648 648 648 649 650 650 651 651 651 652 653 654 656 656 657 659 663 663 663 665 666 666 669 671 676 680 682 683
685 685 686 686 686 688
Contents
27.2.2
27.2.3 27.2.4 27.3
28
28.1 28.2 28.3 28.3.1
28.3.2 28.4 28.4.1
28.4.2
29
29.1 29.2 29.3 29.4 29.5 29.6
XXI
Fucosylations ........................................................ Human Milk a173/4.Fucosyltransferase ............................. FucT I11 and VI ..................................................... FucT V ............................................................... Sialylations ........................................................... a2,3-Sialyltransferase and a2,6.Sialyltransferase .................... N.Acetylglucosaminylation .......................................... N-Acetylglucosaminyltransferase I, 11. and I11...................... N-Acetylglucosaminyltransferase V ................................. Summary ............................................................. Acknowledgments ................................................... References ............................................................
690 690 692 692 692 692 696 696 698 700 700 700
Solid-Phase Synthesis with Glycosyltransferases .................... Claudine AugC. Christine Le Narvor. and Andri Lubineau Introduction .......................................................... General Aspects ...................................................... Enzymatic Synthesis on Insoluble Supports ......................... Enzymatic Synthesis of Oligosaccharides ........................... Use of an Amino-Functionalized Water-Compatible Polyacrylamide Gel .................................................. Use of a Sepharose Matrix .......................................... Use of Controlled-Pore Glass ....................................... Enzymatic Synthesis of Glycopeptides .............................. Use of Controlled-Pore Glass ....................................... Use of Polyethylene Glycol Polyacrylamide (PEGA)............... Enzymatic Synthesis of Oligosaccharides and Glycoconjugates on Soluble Supports ..................................................... Enzymatic Synthesis of Oligosaccharides ........................... Use of Water-Soluble Amino-Substituted Poly(viny1 alcohol) ..... Use of Water-Soluble Glycopolymer Synthesized by Polymerization ....................................................... Enzymatic Synthesis of Glycolipids on Water-Soluble Polyacrylamide-Poly (N-acryloxysuccinimide) (PAN) .............. References ............................................................
705
Glycosidase-Catalysed Oligosaccharide Synthesis ................... David J. Vocadlo and Stephen G. Withers Introduction .......................................................... Background on Glycosidases ........................................ Basic Mechanisms ................................................... Synthesis by the ‘Thermodynamic’ Approach ...................... The Kinetic Approach ............................................... Recent Developments and New Directions ......................... References ............................................................
705 705 707 707 707 708 711 712 712 715 715 715 715 717 718 722 723 723 723 724 724 728 732 838
XXII 30
30.1 30.2 30.2.1 30.2.2 30.2.3 30.2.4 30.3 30.3.1 30.3.2 30.3.3 30.4 30.4.1 30.4.2 30.4.3
Contents
Production of Heterologous Oligosaccharides by Recombinant Bacteria (Recombinant Oligosaccharides) ........................... Roberto A . Geremia and Eric Samain Introduction ......................................................... Concept and Methodology of Heterologous (‘Recombinant’) Oligosaccharide Production in E. coli............................... Biosynthesis of Nod Factors ........................................ Expression Systems and Cloning Strategy .......................... High Cell-Density Cultivation ...................................... Purification of Recombinant Oligosaccharides ..................... Examples of Recombinant Oligosaccharides ....................... Production of Chitin Oligosaccharides in E . coli Expressing NodC ................................................................ Production of Nod Factor Precursors .............................. Production of Derivatives of N-Acetyllactosamine ................. Conclusions and Future Perspectives ............................... Production of Labeled Chitin Oligosaccharides to Study Their Interactions with Proteins ........................................... Improvement of Oligosaccharide Production, and Metabolic Engineering .......................................................... Production of More Complex Oligosaccharides .................... Acknowledgments ................................................... References ...........................................................
845 845 847 847 849 851 852 852 852 853 855 856 856 858 858 859 859
IV
Carbohydrate-Protein Interactions..................................
861
31
Protein-Carbohydrate Interaction: Fundamental Considerations ... Nikki F. Burkhalter. Sarah M . Dimick. and Eric J . Toone Introduction ......................................................... Association in Aqueous Solution ................................... Gas Phase Non Covalent Interactions .............................. Dipole-Dipole Interactions ......................................... Dipole-Induced Dipole ............................................. Dispersive Interactions .............................................. Specific Forces: Hydrogen Bonding and n - ( ~Bonding .............. The Effect of Water on Intermolecular Interactions ................ Coulombic Stabilization ............................................. Hydrogen Bonding .................................................. Dispersive Interactions .............................................. ‘Hydrophobic’ Interactions .......................................... The Evaluation of Protein-Carbohydrate Binding ................. Precipitin Assays ..................................................... Enzyme-Linked Lectin Assay (ELLA).............................. Isothermal Titration Microcalorimetry ............................. The Interpretation of Calorimetric Data ............................
863
31.1 31.2 31.2.1
31.2.2
3 1.2.3 31.3 31.3.1 31.3.2 31.3.3 31.4
863 864 864 864 866 867 868 869 870 871 872 872 876 877 878 878 882
Contents XXIII 31.4.1 3 1.4.2 31.4.3 31.5 31.6 31.6.1 31.6.2 31.6.3
32
32.1 32.2 32.3 32.4 32.5 32.6 32.7 32.8 32.9 32.9.1 32.9.2 32.9.3 32.9.4 32.10 32.10.1 32.10.2
Solvation/Desolvation ............................................... Solvation Entropy ................................................... Translational/Rotational Entropy ................................... Other Contributions to Thermodynamics of Association .......... Proton Transfer ...................................................... Salt Effects/Binding Site Reorganization ........................... van’t Hoff versus Calorimetric Enthalpies .......................... The Thermodynamics of Protein-Carbohydrate lnteraction ....... The Role of Multivalency in Protein-Carbohydrate Interaction ... Phenomenology ...................................................... The Energetic Consequence of Ligand Linkage .................... Enthalpic Contributions to AGi ..................................... Entropic Contributions to AGi ...................................... A Molecular Basis for the Cluster Glycoside Effect ................ Acknowledgments ................................................... References............................................................ Structural Analysis of Oligosaccharides: FAB.MS. ES-MS and MALDI-MS ......................................................... Anne Dell. Howard R. Morris. Richard Easton. Stuart Haslam. Maria Panico. Mark Sutton.Smitlz. Andrew J . Reason. and Kay-Hooi Khoo Introduction .......................................................... Fast Atom Bombardment-Mass Spectrometry (FAB-MS) ......... Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry (MALDI-TOF-MS) .................................. Electrospray-Mass Spectrometry (ES-MS).......................... Appearance of Mass Spectra Obtained in FAB.MS. MALDI-MS and ES-MS Experiments ............................................ Assignment of Mass Values ......................................... Derivatisation ........................................................ Fragmentation Pathways ............................................ Protocols for MS Analysis........................................... Protocol l-Sample Loading for FAB-MS Analysis ............... Protocol 2-Sample Loading for NanoES-MS and MS-MS Analysis on the Q.TOF .............................................. Protocol 3-Sample Loading for LC-ES-MS and LC-ES-MS-MS on the Q.TOF ........................................................ Protocol 4-Sample Loading for MALDI-MS Analysis ........... Applications of FAB.MS. MALDI-MS and ES-MS in Glycobiology ......................................................... Case Study 1-Molecular Weight Profiling of Polysaccharides by MALDI-MS ......................................................... Case Study 2-Analysis of Glycoproteins by LC-ES-MS and FAB-MS .....................................................
882 883 884 885 885 885 886 887 901 901 905 906 907 910 911 911
915
915 915 917 918 919 921 921 922 924 924 925 925 925 926 926
XXIV 32.10.3 32.10.4 32.10.5 32.10.6 32.11 33 33.1 33.2 33.2.1 33.2.2 33.2.3 33.2.4 33.2.5
33.3 33.3.1 33.3.2 33.4 33.4.1 33.4.2 33.5 33.5.1 33.5.2 33.5.3 33.5.4 33.5.5 33.6 34
34.1 34.2 34.3 34.3.1
Contents Case Study 3-Characterization of a Novel N-Glycan by FAB-MS and FAB.MS.MS ......................................... Case Study 4-High Sensitivity Sequencing of a Novel Glycopeptide by Q-TOF ES-MS-MS and MALDI.MS ............ Case Study 5-FAB-MS Screening of Biological Samples for Glycan Content ...................................................... Case Study 6-MS Analysis of Mycobacterial Glycoconjugates . . Concluding Remarks ................................................ References ........................................................... Conformational Analysis in Solution by NMR ...................... S. W. Homans Introduction ......................................................... Solution Conformations of Oligosaccharides ....................... The NMR Technique ................................................ Conformational Parameters in Oligosaccharides ................... Conformational Restraints .......................................... I3C Isotopic Enrichment ............................................ Additional Conformational Restraints .............................. Exchangeable Protons ............................................... Heteronuclear Overhauser Effects................................... 13C- c Coupling-Constants ....................................... Dipolar Couplings ................................................... Experimental Restraints in Conformational Analysis .............. Restraining Protocol ................................................. Biharmonic Restraints ............................................... Time-Dependent Restraints ......................................... Dynamical Simulated Annealing .................................... Analysis of Oligosaccharide Dynamics ............................. Monte-Carlo Simulations ........................................... Molecular Dynamics Simulations ................................... A Case Study on Neu5Aca2-3GalP1-4Glc ......................... Resonance Assignments in Neu5Aca2-3Galfll-4Glc ............... ROE Connectivities ................................................. ‘Global Minimum’ Conformation of Neu5Aca2-3GalP1-4Glc .... Conformational Dynamics of Neu5Aca2-3Gal~1-4Glc............ Short-range vs Long-range Restraints .............................. Conclusions .......................................................... References ........................................................... Oligosaccharide Conformations by Diffraction Methods ............ Serge Pdrez. Catherine Gautier. and Anne Imberty Introduction ......................................................... General Analysis .................................................... Crystalline Conformations of Disaccharide Moieties .............. The Disaccharides ...................................................
930 933 935 942 944 945 947 947 947 947 948 949 949 950 950 952 953 954 955 955 955 957 957 958 959 959 959 960 960 961 962 963 966 966 969 969 970 973 973
Contents 34.3.2 34.4 34.5 34.6 34.7 34.8
35 35.1 35.2 35.3 35.4 35.5 35.6 35.7 35.8 36
36.1 36.2 36.3 36.4 36.5 36.6 36.7 36.8 36.9
37 37.1 37.1.1 37.1.2 37.2
XXV
The Analogs (S. C. N. ....). ......................................... Hydrogen Bonding in Crystalline Oligosaccharides ................ Packing Features ..................................................... Selected Examples ................................................... Crystalline Conformations of Oligosaccharides Complexed with Lectins................................................................ Concluding Remarks ................................................ References ............................................................
985 987 988 990 992 996 998
Transfer NOE Experiments for the Study of Carbohydrate-Protein Interactions ........................................................... 1003 Thomas Peters Introduction .......................................................... 1003 1004 The Transfer NOE Experiment ..................................... 1006 Measurement of trNOEs .......................................... Bioactive Conformations of Carbohydrate Ligands From trNOE Experiments .......................................................... 1008 Spin Diffusion may Generate Misleading Distance Constraints ... 1009 The Conformation of Sialyl Lewis' Bound to E-selectin ...........1011 Interaction of Bacterial Lipopolysaccharide Fragments with Monoclonal Antibodies .............................................. 1016 Conclusions and Future Directions ................................. 1019 References ............................................................ 1021 Carbohydrate-Protein Interactions: Use of the Laser Photo Chemically Induced Dynamic Nuclear Polarization(C1DNP)-NMR Technique ............................................................ 1025 Hans-Christian Siebert and Johannes F. G. Vliegenthart Introduction .......................................................... 1025 The CIDNP Method ................................................. 1026 CIDNP-related Molecular Modelling ............................... 1027 Applications .......................................................... 1027 Hevein-like Lectins .................................................. 1029 Galactoside-binding Lectins from Plant and Animal Origin ....... 1032 Sialidase from Clostridium Perfringens (Wild Type and Mutants) . 1037 CIDNP Analysis of Glycoproteins .................................. 1039 Conclusions .......................................................... 1040 Acknowledgments ................................................... 1041 1042 References ............................................................ Biacore ............................................................... 1045 Wolfgang Jager Introduction .......................................................... 1045 Real-time Analysis by Surface Plasmon Resonance ................1045 Information in a Sensorgram ........................................ 1047 Experimental Procedures ............................................ 1048
XXVI
Contents
37.2.1 31.2.2 37.2.3 37.2.4 37.2.5 37.3 37.3.1 37.3.2 37.3.3
Immobilization of Biomolecules at the Sensor Surface ............. 1048 Surface Regeneration ................................................ 1050 Interaction Analysis and Controls .................................. 1051 1052 Determination of Kinetic Rate Constants .......................... Affinity Determination .............................................. 1053 1054 Application Areas ................................................... Selectin Binding to a Glycoprotein Ligand ......................... 1054 Oligosaccharide Characterization ................................... 1055 In situ Modification of Immobilized Carbohydrates ................1056 References ........................................................... 1056
V
Carbohydrate-Carbohydrate Interactions ...........................
38
Carbohydrate-Carbohydrate Interactions ........................... 061 Dorothe Spillmann and Max M . Burger Introduction ......................................................... 1061 From Structural Components to Cell Recognition ................. 1063 Carbohydrate-Carbohydrate Interactions as Part of Structural Components ......................................................... 1063 Extracellular Matrix of Seaweeds-Agarose, Carrageenan and 1063 Alginate .............................................................. Cell Walls ............................................................ 1064 Mammalian Extracellular Matrix Components .................... 1066 Carbohydrate-Carbohydrate Interactions as Part of Recognition 1068 Keys? ................................................................. Carbohydrate Interactions in Invertebrates-The Marine Sponge Microciona prolifera as a Model System ............................ 1069 Carbohydrate Interactions in Vertebrates-Embryonal and Tumor 1071 Cells .................................................................. Repulsive Carbohydrate-Carbohydrate Interactions ............... 1072 Molecular Aspects of Carbohydrate Interactions ...................1074 1074 Polyvalence to Inforce Weak Interactions .......................... Arrangement of Motifs and the Possibility to Control Specificity . 1075 Molecular Basis of Carbohydrate-Carbohydrate Interactions ..... 1076 Experimental Approaches ........................................... 1078 General Considerations ............................................. 1078 Affinity Interactions ................................................. 1079 1079 Cell Binding Studies ................................................. Aggregation of de n o w Complexes ................................. 1081 Affinity Chromatography ........................................... 1082 Distribution between Compartments ............................... 1082 Microscopy .......................................................... 1083 Electron Microscopy ................................................ 1083 Atomic Force Microscopy .......................................... 1083 1084 Crystallography ......................................................
38.1 38.2 38.2.1
38.2.2
38.3 38.3.1 38.3.2 38.3.3 38.4 38.4.1 38.4.2
38.4.3 38.4.4
059
Contents XXVII
38.4.5 38.4.6 38.4.7 38.4.8
Mass Spectrometry .................................................. Nuclear Magnetic Resonance ....................................... Molecular Modelling ................................................ Tools ................................................................. Synthetic Oligosaccharides .......................................... Antibodies against Carbohydrate Motifs ........................... Cells .................................................................. References ............................................................
1085 1085 1086 1086 1086 1087 1088 1088
V1
Carbohydrate-Nucleic Acid Interactions ............................
1093
39
Carbohydrate-Nucleic Acid Interactions ............................ Heinz E . Moser Introduction .......................................................... Carbohydrates Binding to DNA .................................... Ene-Diyne Antibiotics and Antitumor Agents ...................... Esperamicins ......................................................... Calicheamicins ....................................................... Anthracyclins ........................................................ Pluramycins and Aureolic Acids .................................... Carbohydrates Binding to RNA .................................... Aminoglycosides ..................................................... References ............................................................
1095
39.1 39.2 39.2.1 39.2.2 39.2.3 39.3 39.3.1
Index ...................................................................
Part I1
Biology of Saccharides
Vol . 3
Biosynthesis and Degradation of Glycoconjugates Introduction to Volumes 3 and 4 ....................................... Abbreviations Used in Volumes 3 and 4 .............................
1095 1096 1096 1096 1100 1106 1111 1112 1113 1120 11
V LV
I
Biosynthesis of Glycoconjugates .......................................
1
1
Metabolism of Sugars and Sugar Nucleotides ......................... Hudson H . Freeze Introduction ............................................................ Basic Principles ........................................................ Transporters Deliver Monosaccharides to Cells ...................... Intracellular Sources of Sugars ........................................ Salvage .................................................................
3
1.1 1.2 1.3 1.4 1.4.1
3 3 4 5 5
XXVIII Contents 1.4.2
1.5 1.6 1.7 2
2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10
3
3.1 3.2 3.3 3.4 3.5
Activation and Interconversion of Monosaccharides................. Glycogen ............................................................... Glucose ................................................................ Glucuronic acid ........................................................ Iduronic acid ........................................................... Xylose .................................................................. Mannose ............................................................... Fucose .................i ............................................... Galactose ............................................................. N-Acetylglucosamine................................................. N-Acetylgalactosamine............................................... Sialic acids ............................................................ Sugar Nucleotide Transporters ....................................... Control of Sugar Nucleotide Levels ................................. Possible Future Directions ........................................... References ............................................................
6 6 7 8 8 8 8 9 10 10 10 11 11 13 13 14
Nucleotide Sugar Transporters ....................................... Rita Gerardy-Schahn and Matthias Eckhardt Introduction .......................................................... General Considerations .............................................. The Requirement for Nucleotide Sugar Transporters and Their Mechanism of Function: A Comprehensive Overview of the Last 20 Years .............................................................. Molecular Cloning of Nucleotide Sugar Transporters .............. The Structure of Nucleotide Sugar Transporters .................... The Subcelluar Distribution of Nucleotide Sugar Transporters ..... Molecular Defects that Cause Inactive UDP-Galactose and CMP-Sialic Acid Transporters ....................................... Association Between Defects in Nucleotide Sugar Transporters and Diseases ............................................................... Involvement of Nucleotide Sugar Transporters in the Regulation of Glycosylation ......................................................... Future Perspectives ................................................... Acknowledgements ................................................... References ............................................................
19
Biosynthesis of Oligosaccharyl Dolichol .............................. Sharon S. Krag General Overview .................................................... Oligosaccharyl Dolichol .............................................. Key Enzymatic Steps in the Assembly Process ...................... Topology of the Assembly Process................................... Utilization of Oligosaccharyl Dolichol .............................. Acknowledgment ..................................................... References ............................................................
19 20
20 22 25 27 28 29 29 30 31 32 37 37 38 39 42 42 43 43
Contents XXIX 4
4.1 4.2 4.2.1 4.2.2 4.2.3 4.2.4 4.3 4.4 4.4.1 4.4.2 4.4.3 4.4.4 4.4.5 4.4.6 4.4.7 4.4.8 4.5
5
5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8
6
6.1 6.2
Biochemistry and Molecular Biology of the N-Oligosaccharyltransferase Complex................................................... Roland Knauer and Ludwig Lehle Introduction ........................................................... Biochemistry of OST ................................................. Lipid-Saccharide Donor ............................................. Acceptor Specificity of OST .......................................... Catalytic Mechanism of OST ........................................ Regulation of OST Activity .......................................... Isolation of OST Complexes from Different Sources ................ Molecular Biology of OST ........................................... WBPl/OST48 ......................................................... SWPl/Ribophorin I1 ................................................. OSTl/Ribophorin I ................................................... OST3/OST6 ........................................................... OST5 .................................................................. OST4 .................................................................. OST2/DAD1 .......................................................... STT3 .................................................................. Structural Organization of the OST Complex ....................... Acknowledgments .................................................... References ............................................................. Processing Enzymes Involved in the Deglucosylation of N-Linked Oligosaccharides of Glycoproteins: Glucosidases I and 11 and Endomannosidase ..................................................... Robert G. Spiro Introduction ........................................................... Glucosidase I .......................................................... Glucosidase 11......................................................... Endo-a-mannosidase .................................................. Concerted Action of Deglucosylation Enzymes ...................... Mutants ............................................................... Role of Monoglucosylated N-Linked Oligosaccharides and Glucose Trimming Enzymes in Regulating Quality Control of Glycoproteins ......................................................... Effect of Glucosidase Inhibitors on Viral Proliferation .............. Acknowledgments .................................................... References............................................................. a-Mannosidases in Asparagine-linked Oligosaccharide Processing and Catabolism ........................................................ Kelley W. Moremen Overview .............................................................. Introduction ...........................................................
45 45 46 47 48 49 51 51 52 54 54 55 55 56 56 57 58 59 60 60
65 65 66 68 70 72 74
75 77 78 78
81 81 82
XXX 6.2.1 6.2.2 6.2.3 6.2.4 6.2.5 6.3 6.3.1 6.3.2
6.3.3
6.4
7 7.1 7.2 7.3 7.4 7.5
8 8.1 8.2
Contents
Roles of N - and O-Linked Glycans and Compartmentalization of Biosynthetic and Catabolic Reactions ............................... 82 82 Processing of Asn-Linked Oligosaccharides ......................... Early Trimming Events: importance for quality control glycoprotein degradation and anteriograde transport ..................... 85 Glycoprotein Catabolism: multiple routes for glycoprotein 87 breakdown ............................................................ Consequences of Genetic Defects in Oligosaccharide Biosynthesis and Catabolism ....................................................... 88 Mannosidases in Glycoprotein Processing and Catabolism ......... 89 89 Classification of Mannosidases ....................................... Class 1 Mannosidases: enzymes of the ER and Golgi ............... 93 ER mannosidase I subfamily ......................................... 93 95 Golgi mannosidase I sub-family ..................................... Fungal secreted mannosidases ....................................... 97 New genes with unknown functions ................................. 98 Class 2 Mannosidases: enzymes of the cytosol, ER, Golgi, and Lysosomes ............................................................ 98 99 Golgi mannosidase I1................................................. Lysosomal mannosidase ............................................. 101 Epididymal/sperm mannosidase .................................... 103 Heterogeneous cluster of mannosidase homologs among eukary a, 104 eubacteria, and archaea ............................................. Conclusions and Future Prospects .................................. 106 107 Acknowledgments ................................................... 107 References ........................................................... The Role of UDP-Glcyglycoprotein Glucosyltransferase as a Sensor of Glycoprotein Conformations ...................................... Armando J. Parodi Introduction ......................................................... General Properties ................................................... GT Recognizes Glycoprotein Conformations ...................... The Primary Structure of the UDP-Glcyglycoprotein Glucosyltransferase .................................................. The Role of Monoglucosylated Oligosaccharides in Glycoprotein Folding ............................................................... Acknowledgments ................................................... References ........................................................... Mannosyltransferases................................................ Peter Orlean Introduction ......................................................... Occurrence of Covalently-linked Mannose .........................
119 119 120 121 122 123 126 127 129 129 130
Contents XXXI
8.2.1 8.2.2 8.2.3 8.2.4 8.3 8.3.1 8.3.2 8.3.3 8.3.4 8.4 8.4.1
8.4.2
8.4.3 8.4.4 8.5 8.6
9
9.1 9.2 9.3 9.3.1 9.3.2 9.4 9.5 9.6
Eukaryotic Secretory Glycoproteins ................................ Glycophospholipids .................................................. Eubacterial and Archaeal Mannose-containing Molecules ......... C-linked Mannose ................................................... Biochemistry of Mannosyl Transfer ................................. Many Linkages, Two Donors ....................................... Donor Specificity .................................................... Acceptor Specificity.................................................. Structural Features of Man-T ....................................... Man-T Families and the Pathways They Participate in ............ Man-Ts of the ER [l-51 ............................................. Alglp ................................................................. Alg2p/Algl l p ........................................................ Dpmlp ............................................................... Alg3p ................................................................. Alg9p/PIG-Bp family ................................................ Pmtlp family ......................................................... Golgi Man-Ts and Fungal Mannan Synthesis ...................... Ochlp family ......................................................... ................................. Mnn9p family ........... MnnlOp/Mnnl l p family ............................................ Mnnlp family ........................................................ Ktrlp family ......................................................... “Missing” Eukaryotic Man-T ....................................... Eubacterial and Archaeal Man-T ................................... Coordinating Man Transfer with the Cell Cycle and Morphogenesis ....................................................... Concluding Remarks ................................................ Acknowledgments ................................................... References............................................................
130 130 130 130 131 131 131 132 132 133 134 134 134 135 135 135 136 136 137 137 138 138 138 138 139
Branching of N-Glycans: N-Acetylglucosaminyltransferases........ Hurry Schuchter Introduction .......................................................... Processing of N-Glycans within the Endomembrane Assembly Line .................................................................. General Properties of the N-Acetylglucosaminyltransferases....... Domain Structure .................................................... Targeting to the Golgi Apparatus ................................... UDP-GlcNAc:Manal-3R [GlcNAc to Manwl-31 p.1,2. N . Acetylglucosaminyltransferase I (GnT I, EC 2.4.1.101) ............ UDP-GlcNAc:Manal-6R [GlcNAc to Manal-61 p.1,2. N . Acetylglucosaminyltransferase 11 (GnT 11, E.C. 2.4.1.143). ........ The Role of GnT I and I1 in Mammalian Development ...........
145
139 140 140 140
145 146 148 148 150 150 152 153
XXXII 9.7 9.7.1 9.7.2 9.8 9.9 9.9.1 9.10 9.11
10 10.1 10.2 10.2.1 10.3 10.3.1 10.3.2 10.3.3 10.3.4 10.3.5 10.4 10.4.1 10.4.2 10.4.3 10.4.4 10.5 10.5.1 10.5.2 10.5.3 10.5.4
Contents UDP-G1cNAc:RI-Manal-6[GlcNAc~l-2Manul-3]Man~l-4R~ [GlcNAc to ManP 1-41 P- 1,4. N.Acetylglucosaminyltransferase 111 155 (GnT 111. E.C. 2.4.1.144). ........................................... 156 Overexpression of GnT 111 Activity ................................. 157 GnT 111 Activity and Cancer ........................................ UDP-GlcNAc:R1Manal-3R2 [GlcNAc to Manal-31 (3.1,4. N . Acetylglucosaminyltransferase IV (GnT IV, E.C. 2.4.1.145). ...... 157 UDP-GlcNAc:Rl Manal-6R2 [GlcNAc to Manal-61 P-1,6-NAcetylglucosaminyltransferase V (GnT V, E.C.2.4.1.155) ......... 158 159 GnT V Activity and Cancer ......................................... UDP-GlcNAc:Rl (R2)Manal-6R3 [GlcNAc to Manul-61 P-1, 4-Nl -Acetylglucosaminyltransferase VI (GnT VI) ............. 161 161 GnT VII and GnT VIII ............................................. 162 References ........................................................... The Galactosyltransferases .......................................... Nancy L . Shaper. Martin Charron. Neng- Wen Lo. Jane R. Scocca. and Joel H . Shuper Introduction ......................................................... Using the Databanks to Obtain Information on the Galactosyltransferases ............................................... Nomenclature ........................................................ The Dual Role of P4-Galactosyltransferase-I (P4GalT-I) in Oligosaccharide and Lactose Biosynthesis: The Early Days ....... p4GalT-I: Isolation and Characterization of cDNA Clones ....... The Murine NGalT-I Gene: Genomic Organization and Structure of the 5'-End ......................................................... P4GalT-I and Lactose Biosynthesis ................................. P4GalT-I and the Vertebrate P4GalT Gene Family ................ Evolution of the 04-Galactosyltransferase Gene Family ........... The Vertebrate P3Galactosyltransferase (P3GalT) Gene Family . . General Characteristics of the P3-Galactosyltransferase Gene Family Members .................................................... P3GalT-IV: UDP-ga1actose:GM 1 P3-galactosyltransferase (GM1 Synthase; GalT-3) ................................................... Other Vertebrate P-Galactosyltransferase Activities................ UDP-Ga1actose:Ceramide P-Galactosyltransferase (CGalT; EC 2.4.1.45). ............................................................. The Vertebrate u3-Galactosyltransferase Gene Family ............ u3-Galactosyltransferase (a3GalT: UDP-Gal:Galp4GlcNAca3Galactosyltransferase; EC 2.4.1.87) ................................. The Blood Group B a3-Galactosyltransferase (EC 2.4.1.37). ...... The Forssman Glycolipid Synthetase (EC 2.4.1.88). ............... Evolution of the u3GalT Gene Family .............................
175 175 177 177 178 181 181 182 182 184 185 186 187 187 187 188 188 190 191 191
Contents XXXIII
10.6
A UDP-Gal:Gal@3GalNAca4Galactosyltransferase Activity ..... 192 192 Acknowledgments ................................................... References............................................................ 192
11
Fucosyltransferases ................................................... 197 Ernest0 T. A . Marques. Jr. Introduction .......................................................... 197 198 General Characteristics .............................................. Nomenclature ........................................................ 198 199 Gene Structure ....................................................... Sequence Peptide Motifs ............................................. 199 Specificity ............................................................ 199 200 Protein Structure and Topology ..................................... Enzymatic Reaction Mechanism .................................... 201 203 Inhibitors ............................................................. 203 Specific Fucosyltransferases ......................................... GDP-Fucose: Fucal (Fucal,2Fuc)a2-fucosyltransferase ............ 204 GDP-Fucose: GalPl (Fucal,2Gal)a2-fucosyltransferase ............ 204 GDP-Fucose: Gal~l,4/3GlcNAc(Fucal,3/4GlcNAc)a3/ 4204 fucosyltransferases ................................................... 204 Blood group Lewis: FucT 111, V and VI ............................ Myeloid enzyme: FucT IV ........................................... 205 Leukocyte enzyme: FucT VII ....................................... 206 206 Neuronal enzyme: FucT IX ......................................... GDP-Fucose: Gal~l,3GlcNAc(Fuca1, 3GlcNAc) bacterial (Helicobacter pylori) a3-fucosyltransferase ......................... 207 GDP-Fucose: GlcNAc-N(Fuca1,6GlcNAc)a6fucosyltransferases . 207 GDP-Fucose: O-Ser(Fuca1-O-Ser)GlcNAc polypeptide 207 fucosyltransferases ................................................... Unconventional Types of Fucosylation: FucPl -P-Ser and cytoplasmic Fucal,2-GalP1,3-GlcNAc-Pro (Dictyostelium discoideum) 207 FucP 1-P-Ser .......................................................... 207 208 Fucal,2-GalP1,3-GlcNAc-Pro ...................................... Acknowledgments ................................................... 208 208 References............................................................
11.1 11.2 11.2.1 11.2.2 11.2.3 11.2.4 11.2.5 11.2.6 11.2.7 11.3 11.3.1 11.3.2 11.3.3
11.3.4 11.3.5 11.3.6 11.3.7
12
12.1 12.2 12.3 12.4 12.5 12.6 12.7
Sialyltransferases..................................................... Joseph T. Y. Lau and Sherry A . Wuensch Introduction .......................................................... General Features of Sialyltransferases .............................. Cloning and ldentification Strategies for Sialyltransferases ........ Sialyltransferase Classification and Nomenclature .................. The a2, 3-ST Family ................................................. The 1x2,6-ST Family ................................................. The 1x2.8.ST Family .................................................
213 213 213 215 216 216 217 218
XXXIV Contents 12.8
Regulation and Functionality of Sialyltransferases................. 219 221 References ...........................................................
13
Biochemistry of Sialic Acid Diversity................................ Roland Schauer Introduction ......................................................... Occurrence and Biosynthesis ........................................ General Biological Functions ....................................... N-Glycolylneuraminic Acid ......................................... 0-Acetylated Sialic Acids ........................................... 0-Methylated and 0-Sulfated Sialic Acids ......................... Acknowledgments ................................................... References ...........................................................
13.1 13.2 13.3 13.4 13.5 13.6
14 14.1 14.2 14.3 14.4 14.5 14.6 14.7
15
15.1 15.2 15.3 15.4 15.5 15.6 15.7 15.8 15.9 15.10
Carbohydrate Sulfotransferases...................................... Steven D. Rosen. Annette Bistrup. and Stefan Hemmerich Introduction ......................................................... Basic Features of Sulfotransferase Reactions ....................... Tyrosine Sulfation ................................................... Diversity of Carbohydrate Sulfation ................................ Biochemical Demonstration of Carbohydrate Sulfotransferases ... Molecular Cloning of Carbohydrate Sulfotransferases ............. Primary Structures of Carbohydrate Sulfotransferases ............. Acknowledgments ................................................... References ........................................................... Novel Variant Pathways in Complex-type Oligosaccharide Synthesis ............................................................. Dirk H . van den Eijnden Introduction ......................................................... The lacNAc Pathway of Complex-type Oligosaccharide Synthesis Occurrence and Biology of 1acdiNAc-based Complex-type Oligosaccharides ..................................................... Biosynthesis of lacdiNAc Backbone Units .......................... The lacdiNAc Pathway of Complex-type Oligosaccharide Synthesis ............................................................. Other Shared Properties of 04-GalT and 04-GalNAcT ............ Cloning of a snail UDP-G1cNAc:GlcNAcP-R 04-Nacetylglucosaminyltransferase ....................................... The Chitobio Pathway of Complex-type Oligosaccharide Synthesis ............................................................. Competition Between Pathways ..................................... Concluding Remarks ................................................ References ...........................................................
227 227 227 229 231 234 238 239 239 245 245 245 246 246 249 250 252 256 256
261 261 261 262 263 264 266 266 267 267 269 269
Contents XXXV 16
16.1 16.2 16.3 16.4 16.4.1 16.4.2
16.5 16.6 16.7
17
17.1 17.2 17.2.1 17.2.2 17.2.3 17.2.4 17.2.5 17.3
Control of Mucin-Type 0-Glycosylation: 0-Glycan Occupancy is Directed by Substrate Specificities of Polypeptide GalNAcTransferases .......................................................... 273 Helle Hassan. Eric P. Bennett. Ulla Mandel. Michael A . Hollingsworth. and Henvik Cluusen Introduction .......................................................... 273 The Mammalian UDP-GalNAc: Polypeptide GalNAc-Transferase Gene Family ......................................................... 274 The GalNAc-Transferase Gene Family is Evolutionarily Old ..... 276 The Kinetic Properties of GalNAc-Transferase Isoforms are Different.............................................................. 278 Lessons from in vivo Analysis of GalNAc-transferase Substrate Specificities ........................................................... 219 Lessons from in vitro Analysis of the Acceptor Substrate Specificities of GalNAc-transferase Isoforms ....................... 280 Isoforms may have distinct acceptor substrate specificities ......... 281 Isoforms may have overlapping substrate specificities .............. 283 Isoforms may act in different order on substrates with multiple 283 acceptor sites ......................................................... Isoforms may require prior (GalNAc) glycosylation ............... 283 Expression of the GalNAc-Transferase Genes are Differentially Regulated ............................................................ 285 288 Predictive Value of in vitro 0-glycosylation? ....................... Conclusions and Future Perspectives................................ 288 References ............................................................ 289 Glycosyltransferase Inhibitors ........................................ Xianyping Qian and Monica M . Palcic Introduction .......................................................... Inhibitors of Glycosyltransferases................................... Inhibitors of Galactosyltransferases ................................. Inhibitors of pl, 4-galactosyltransferase ............................. Inhibitors of cr.l,3-galactosyltransferase............................. Inhibitors of Fucosyltransferases .................................... Inhibitors of 1x1,2-fucosyltransferases ............................... Inhibitors of a1,3-fucosyltransferases ............................... Inhibitors of Sialyltransferases ...................................... Inhibitors of a2, 6-sialyltransferase .................................. Inhibitors of a2, 3.sialyltransferase .................................. Inhibitors of N-Acetylglucosaminyltransferases .................... Inhibitors of Human Blood Group A and B Glycosyltransferases. Summary ............................................................. Acknowledgments ................................................... References............................................................
293 293 296 296 296 297 298 300 300 301 302 304 305 306 309 309 309
XXXVI Contents 18
18.1 18.2 18.3 18.4 18.5 18.6 18.7 18.8 18.9 18.10 18.11 18.12
19
19.1 19.2 19.3 19.4 19.5 19.6 19.7
20
20.1 20.2 20.3 20.3.1 20.3.2 20.3.3 20.3.4 20.3.5 20.4 20.4.1 20.4.2 20.4.3 20.5
Biosynthesis of the O-Glycan Chains of Mucins and Mucin Type Glycoproteins ........................................................ Inka Brockhausen Summary ............................................................. Introduction ......................................................... Structures of O-Glycans ............................................. Functions of Much Type O-Glycans ............................... Primary O-Glycosylation ............................................ Synthesis of O-Glycan Core 1....................................... Synthesis of O-Glycan Core 2 ....................................... Synthesis of O-Glycan Core 3 ....................................... Synthesis of O-Glycan Core 4 ....................................... Synthesis of O-Glycan Cores 5-8 ................................... Elongation and Branching Reactions ............................... Synthesis of Terminal Structures .................................... Acknowledgments ................................................... References ........................................................... Glycosyltransferases in Glycosphingolipid Biosynthesis ............. Subhash Basu. Kamal Das. and Manju Basu Introduction ......................................................... Fucosyltransferases in Glycolipid Biosynthesis ..................... Galactosyltransferases in Glycolipid Biosynthesis .................. N-Acetylgalactosaminyltransferases in Glycolipid Biosynthesis.... N-Acetylglucosaminyltransferases in Glycolipid Biosynthesis...... Sialyltransferases in Glycolipid Biosynthesis ....................... Glucuronyltransferases in Glycolipid Biosynthesis ................. Acknowledgments ................................................... References ........................................................... Biosynthesis of Glycogen ............................................ Peter J . Roach Summary ............................................................. Introduction ......................................................... Glycogenin and the Initiation of Glycogen Synthesis .............. History ............................................................... Properties ............................................................ Reaction Mechanism ................................................ Domain Structure ................................................... Function ............................................................. Glycogen Synthase and the Bulk Synthesis of Glycogen ........... Properties ............................................................ Structure of Glycogen Synthase ..................................... Branching Enzyme .................................................. Intermediates in the Biosynthesis of Glycogen .....................
313 313 313 314 314 315 315 317 319 319 319 320 321 324 324 329 329 329 332 334 336 337 340 342 342 349 349 350 351 351 351 352 352 354 354 354 355 356 357
Contents XXXVII 20.6
Conclusion ........................................................... Acknowledgments ................................................... References ............................................................
358 359 359
21
Biosynthesis of Hyaluronan .......................................... Paraskevi Heldin and Torvard C. Laurent Introduction .......................................................... Site of Biosynthesis .................................................. Biosynthetic Precursors .............................................. Hyaluronan Synthases ............................................... Microbial Enzymes .................................................. Vertebrate Synthases ................................................. Mechanism of Synthesis ............................................. Chain Elongation .................................................... Translocation ........................................................ Shedding ............................................................. Regulation of HA Synthesis ......................................... Concluding Remarks ................................................ Acknowledgments ................................................... References ............................................................
363
21.1 21.2 21.3 21.4 21.4.1 21.4.2 21.5 21.5.1 21.5.2 21.5.3 21.6 21.7
22
22.1 22.2 22.2.1 22.2.2 22.2.3 22.2.4 22.3 22.3.1 22.3.2 22.3.3
22.3.4
Biosynthesis of Chondroitin Sulfate and Dermatan Sulfate Proteoglycans ........................................................ Geetha Sugumaran and Barbara A4. Vertel Introduction .......................................................... Proteoglycan Structure .............................................. Proteoglycans and Their Core Proteins ............................. What Initiates GAG Chain Addition? .............................. The Linkage Region ................................................. .................................... CS and DS Chains ........... Biosynthesis of CS and DS Proteoglycans .......................... Biosynthesis of the Core Protein .................................... Origin of Sugar and Sulfate Precursors ............................. Addition of the Linkage Oligosaccharides .......................... Xylosylation .......................................................... Galactosylation ...................................................... Addition of GlcA and completion of the common tetrasaccharide linkage region ........................................................ Initiation of CS/DS chains by addition of the first GalNAc ....... Formation of the CS/DS Chains .................................... Addition of the repeating disaccharides ............................. Epimerization of GlcA to IdoA to form DS ........................ Sulfation of GalNAc................................................. Sulfation of uronic acid ..............................................
363 364 364 365 365 366 367 368 369 369 370 371 372 372
375 375 379 379 381 381 382 383 383 384 385 385 386 387 388 388 388 389 390 391
XXXVIII Contents 22.4
Concluding Remarks/Perspectives .................................. Acknowledgments ................................................... References ...........................................................
391 392 392
23
Biosynthesis of Heparin and Heparan Sulfate Proteoglycans ....... Lena Kjellin and U r Lindahl Introduction ......................................................... The Proteoglycans: Structure. Location and Functions ............ Biosynthesis of the Polysaccharide Backbone ...................... Outline of Polymer-Modification Reactions ........................ The N.Deacetylase/N.Sulfotransferases ............................. The C5-Epimerase ................................................... The 2-O-Sulfotransferase............................................ The 6-O-Sulfotransferases........................................... The 3 - 0 Sulfotransferases........................................... The Products. Heparin and Heparan Sulfate ....................... Interactions with Proteins ........................................... Regulation of HS Biosynthesis ...................................... References ...........................................................
395
23.1 23.2 23.3 23.4 23.4.1 23.4.2 23.4.3 23.4.4 23.4.5 23.5 23.6 23.7
24 24.1 24.2 24.2.1 24.2.2 24.2.3 24.2.4 24.3 24.3.1 24.3.2 24.3.3 24.3.4 24.4 24.5
25
25.1 25.2 25.2.1
395 396 396 397 399 399 399 400 400 400 401 402 403
Biosynthesis of Proteoglycans with Keratan Sulfates................ James L . Funderburgh Introduction: Keratan Sulfate Renaissance ......................... Keratan Sulfate Structure and Distribution ........................ Corneal KS .......................................................... Non-corneal KSI .................................................... KSII .................................................................. KSIII ................................................................. Keratan Sulfate Proteoglycans ...................................... SLRPs ................................................................ Aggrecan ............................................................. Cell-Associated KS .................................................. Brain ................................................................. Enzymatic Reactions of KS Biosynthesis ........................... Metabolic Control of KS Synthesis ................................. Acknowledgments ................................................... References ...........................................................
407
The Biosynthesis of GPI Anchors .................................... Yasu S. Morita, Alvaro Acosta.Serrano. and Paul T. Englund Introduction ......................................................... Structure of GPI Anchors ........................................... Glycan Core Modifications .........................................
417
407 407 408 409 409 410 410 410 411 411 412 412 413 414 414
417 417 417
Contents XXXIX 25.2.2 25.3 25.3.1 25.3.2 25.3.3 25.3.4 25.3.5 25.3.6 25.3.7 25.3.8 25.4 25.4.1 25.4.2 25.4.3 25.5 25.6
Variations in Anchor Lipid Structure ............................... GPl Precursor Synthesis ............................................. GlcNAc-PI Synthesis ................................................ GlcNAc-PI Deacetylation ........................................... Inositol Acylation .................................................... GPI Mannosylation .................................................. Transfer of EtN-P ................................................... Lipid Remodeling.................................................... Addition of Carbohydrate Side Chains ............................. Topology of Biosynthetic Pathways ................................. Attachment of the GPI Precursor to a Protein ..................... Basic Features ....................................................... Protein Machinery for GPI Addition ............................... Signal Sequence for GPI Addition. ................................. Evolution of GPI Biosynthesis ...................................... Future Studies ....................................................... Acknowledgments ................................................... References............................................................
419 419 420 421 421 422 423 423 424 424 425 425 426 426 426 427 427 427
26
435
26.1 26.2 26.3 26.3.1 26.3.2 26.3.3 26.3.4 26.3.5 26.3.6 26.4 26.5
Escherichiu coli Lipid A: A Potent Activator of Innate Immunity ... Teresa A . Garrett and Christian R . H . Raetz Introduction .......................................................... Structure of Lipopolysaccharide..................................... Lipid A Biosynthesis in E. coli ...................................... Acylation of UDP-GlcNAc ......................................... Disaccharide Formation ............................................. Phosphorylation by the Lipid A 4' Kinase .......................... Kdo Addition and the Late Acyltransferases ....................... Other Acyltransferases............................................... Transport of Lipid A and the Role of MsbA ....................... Lipid A Activation of Signal Transduction in Animal Cells ....... Summary ............................................................. Acknowledgments ................................................... References............................................................
435 435 437 439 440 440 441 442 442 444 447 447 447
11
Glycosidases ...........................................................
453
27
Lysosomal Degradation of Glycolipids............................... 455 Thomas Kolter and Konrad Sandhof Summary ............................................................. 455 Introduction .......................................................... 455 Mechanisms of Lysosonial Glycolipid Degradation ................ 456 Glycosidases ......................................................... 456
27.1 27.2 27.3 27.3.1
XL 27.3.2 27.3.3 27.3.4 27.3.5 27.3.6 27.4 27.4.1 27.4.2 27.4.3 27.4.4 27.4.5 27.4.6 27.4.7 27.5 27.5.1 27.5.2 27.6
28 28.1 28.2 28.3 28.4 28.4.1 28.4.2 28.4.3 28.5
28.5.1 28.5.2 28.5.3 28.6
29 29.1 29.2
Contents Topology of Endocytosis and Lysosomal Glycolipid Degradation Sphingolipid Activator Proteins ..................................... The GM2-activator and its role in lysosomal digestion ............ SAP-A to SAP-D .................................................... Lateral Pressure ...................................................... Lipid Composition .................................................. Membrane Curvature ............................................... Degradation of Selected Lipids ....... ........................... Ganglioside GM2 ................................................... Lactosylceramide .................................................... Glucosylceramide .................................................... Ceramide ............................................................. Sphingomyelin ....................................................... Sulfatide ............................................................. Galactosylceramide .................................................. Pathobiochemistry ................................................... Animal Models for Sphingolipidoses ............................... Therapy .............................................................. Future Directions .................................................... References ...........................................................
457 458 459 460 460 461 462 462 462 464 464 465 465 465 466 466 467 469 470 470
Lysosomal Degradation of Glycoproteins ........................... Nathan N . Aronson. Jr. Summary ............................................................. Introduction ......................................................... Roles of Lysosomes ................................................. Lysosomal Degradation of N-Linked Glycoproteins ............... General Features .................................................... Carbohydrate Digestion ............................................. Protein and Linkage Hydrolysis .................................... Formation of Thyroid Hormone via Lysosomal Degradation of Thyroglobulin ....................................................... Synthesis of Thyroid Hormone ..................................... Carbohydrate Degradation .......................................... Proteolysis ........................................................... Degradation of Free Polymannose-Type Oligosaccharides Derived from N-Linked Glycoproteins During Biosynthesis ................ References ...........................................................
473
Sialidases............................................................. Garry Taylor. Susan Crennell. Carl Thompson. and Marina Chuenkova Abstract .............................................................. Introduction .........................................................
473 473 474 475 475 476 476 477 477 478 479 479 481 485
485 485
Contents
XLI
29.3 29.4 29.5 29.6 29.7 29.8 29.9
Influenza Virus Neuraminidase ...................................... Paramyxovirus Hemagglutinin-Neuraminidase (HN) .............. Non-Viral Sialidases ................................................. Small Sialidases ...................................................... Large Sialidases ...................................................... T . cruzi Trans-Sialidase (TS) ........................................ Conclusion ........................................................... Acknowledgments ................................................... References ............................................................
486 487 487 490 491 491 493 494 494
30
Microbial Glycosidases............................................... Kenji Yamamoto. Su-Chen Li. and Yu-Teh L i Exo-Glycosidases .................................................... u-Glucosidase ........................................................ P-Glucosidase ........................................................ a-Galactosidase ...................................................... P-Galactosidase ...................................................... u.Mannosidase ....................................................... 0.Mannosidase ....................................................... P-N-Acetylhexosaminidase .......................................... u.N.Acetylga1actosaminidase ........................................ u-L-Fucosidase ....................................................... P.D.Fucosidase ....................................................... Sialidase .............................................................. KDNase .............................................................. u-L-Rhamnosidase ................................................... p-Xylosidase ......................................................... Endo.Glycosidases ...................................................
497
30.1 30.1.1 30.1.2 30.1.3 30.1.4 30.1.5 30.1.6 30.1.7 30.1.8 30.1.9 30.1.10 30.1.11 30.1.12 30.1.13 30.1.14 30.2 30.2.1 30.2.2 30.2.3 30.2.4 30.2.5 31
31.1 31.2 31.3 31.4 31.5 31.6 31.7 31.8
Endo.P.N.acetylg1ucosaminidase .................................... Peptide-N-glycanase F ............................................... Endo.u.N.acetylga1actosaminidase .................................. Endo-P-galactosidase ................................................ Endoglycoceramidase ................................................ References. ........................................................... Glycoprotein Processing Inhibitors ................................... Magid Osser and Alan D . Elbein Introduction ........................................................... Structural Classification ............................................. Distribution of Glycosidase Inhibitors in the Plant Kingdom ...... Isolation and Structural Determination ............................. Glycosidase Inhibitory Activity ..................................... Structure-Activity Relationships .................................... N-Linked Oligosaccharide Processing ............................... Inhibitors of N-Linked Oligosaccharide Processing.................
497 497 498 498 499 500 500 501 501 502 503 503 504 504 505 505 505 506 506 507 507 508 513 513 515 515 516 517 518 519 522
XLII 31.8.1 31.8.2 31.9
Contents Glucosidase Inhibitors ............................................... Mannosidase Inhibitors ............................................. Summary and Future Prospects ..................................... References ...........................................................
522 525 528 529
Index ...................................................................
11
Part I1
Biology of Saccharides
Vol. 4
Lectins and Saccharides Biology
Ill
Lectins ...............................................................
533
32
Plant Lectins ......................................................... Marilynn E. Etzler Summary ............................................................. Introduction ......................................................... Carbohydrate Specificity ............................................ Other Activities ...................................................... Structure ............................................................. Biological Roles ..................................................... Acknowledgments ................................................... References ...........................................................
535
32.1 32.2 32.3 32.4 32.5 32.6
33 33.1 33.2 33.3 33.4 33.5 33.6 33.7 33.8
34
34.1 34.2 34.3
Interactions of Oligosaccharides and Glycopeptides with Hepatic Carbohydrate Receptors ............................................. Yuan C. Lee and Reiko T. Lee Summary ............................................................. Introduction ......................................................... Molecular Characteristics of Hepatic Lectins ...................... Cellular Aspects of HL .............................................. Binding Specificity................................................... Photoaffinity Labeling ............................................... Subunit Organization on Rat Hepatocyte Surface ................. Applications ......................................................... References ........................................................... P-Type Lectins and Lysosomal Enzyme Trafficking ................ Patricia G. Marron-Teradu and Nancy M . Dahms Introduction ......................................................... Intracellular Trafficking of the MPRs .............................. Primary Structure and Biosynthesis of the MPRs ..................
535 535 536 539 540 543 546 547
549 549 550 551 552 553 557 558 559 560 563 563 564 566
Contents
XLIII
34.3.1 34.3.2 34.4 34.5 34.5.1 34.5.2 34.6
CI-MPR .............................................................. CD-MPR ............................................................. Lysosomal Enzyme Recognition by the MPRs ..................... Structural Determinants of Man-6-P Recognition .................. Expression of Mutant Forms of the MPRs ......................... Crystal Structure of the CD-MPR .................................. Conclusions .......................................................... Acknowledgments ................................................... References............................................................
35
The Siglec Family of I-Type Lectins ................................. 579 Paul R. Crocker and Soerye Kelm Introduction .......................................................... 579 The Immunoglobulin Superfamily and Carbohydrate Recognition 579 Siglecs as a Family of Sialic Acid Binding Proteins ................ 580 Biology of Siglecs .................................................... 581 Sialic Acids in Cellular Recognition ................................ 583 Mode of Carbohydrate Recognition by Siglecs ..................... 584 Importance of Multivalent Binding ................................. 588 Sialic Acid Recognition by the Immunoglobulin FoldEvolutionary Considerations ........................................ 588 Role of cis Interactions in Modulating Adhesion to Other Cells in trans ............................................................... 589 Sialoadhesin as a Macrophage Adhesion Molecule................. 590 Signalling Versus Adhesion Mediated by Siglecs ................... 591 Conclusions .......................................................... 592 Acknowledgments ................................................... 592 References............................................................ 592
35.1 35.2 35.3 35.4 35.5 35.6 35.7 35.8 35.9 35.10 35.11 35.12
36
36.1 36.2 36.2.1 36.2.2 36.3 36.3.1 36.3.2 36.3.3 36.3.4 36.3.5 36.3.6 36.3.7 36.4
C-Type Lectins and Collectins ....................................... Russell Wallis Summary ............................................................. Structure and Function of C-Type Animal Lectins ................. The Carbohydrate-Recognition Domain ............................ Ligand Binding ...................................................... Mannose-Binding Protein and Collectins ........................... Domain Organization ............................................... MBPs as Prototype Collectins ....................................... Ligand Binding by Serum MBP ..................................... MBP and Innate Immunity .......................................... Liver MBP ........................................................... Pulmonary Surfactant Proteins ...................................... Conglutinin and CL-43 .............................................. Conclusions ..........................................................
566 567 569 571 571 572 574 574 575
597 597 598 599 600 601 601 603 603 604 607 608 608 609
XLIV
Contents
Acknowledgments ................................................... References ...........................................................
609 609
Selectins .............................................................. Rodger P. McEver Introduction ......................................................... Structure of Selectins ................................................ Selectin Ligands ..................................................... Requirements for Selectins to Mediate Tethering and Rolling of Leukocytes under Hydrodynamic Flow ............................ Functions of Selectins and their Ligands in vivo ................... Conclusions .......................................................... References ...........................................................
613
Galectins ............................................................. Douglas N . W. Cooper and Samuel H . Barondes Introduction ......................................................... Galectin Structure ................................................... Novel Candidate Galectins .......................................... Unorthodox Subcellular Targeting .................................. Regulation of Galectin Expression .................................. Galectin Binding Specificity and Identified Ligands ................ Physiological Functions ............................................. Summary ............................................................. References ...........................................................
625 625 626 631 635 637 639 640 642 642
IV
Saccharide Biology ...................................................
649
39
Structures and Functions of Nuclear and Cytoplasmic Glycoproteins Robert S. Haltiwanger Introduction ......................................................... O-Linked N-Acetylglucosamine (0.GlcNAc) ....................... O-GlcNAc Appears to be a Regulatory Modification much like Phosphorylation ..................................................... Modulation of Protein Stability and Function by O-GlcNAc ..... Other Forms of Nuclear and Cytoplasmic Glycosylation .......... Unique Cytoplasmic Forms of Glycosylation ...................... Conventional Forms of Glycosylation in the Nucleus and Cytoplasm ........................................................... Nuclear and Cytoplasmic Lectins ................................... Conclusions .......................................................... Acknowledgments ................................................... References ...........................................................
65 1
37 37.1 37.2 37.3 37.4 37.5 37.6
38
38.1 38.2 38.3 38.4 38.5 38.6 38.7 38.8
39.1 39.2 39.2.1 39.2.2 39.3 39.3.1 39.3.2 39.3.3 39.4
613 613 614 619 621 621 622
651 652 653 655 658 658 660 661 662 662 662
Contents 40
40.1 40.2 40.3 40.3.1 40.3.2 40.3.3 40.4 40.5 40.6 40.7 40.7.1
40.7.2 40.7.3 40.7.4 40.7.5 40.8
41
41.1 41.2 41.3 41.3.1 41.3.2 41.3.3 41.3.4 41.3.5 41.4 41.4.1 41.4.2
XLV
Structure and Functions of Mucins .................................. 669 Joyce Taylor-Papadimitriou and Joy M . Burchell Classification of Mucins ............................................. 669 670 The Epithelial Mucins ............................................... Mucin Type O-Glycosylation Pathways ............................ 670 671 Initiation of O-Glycosylation ........................................ Chain Extension ..................................................... 671 Chain Termination ................................................... 671 Expression of Epithelial Mucins ..................................... 672 The Complex Gel-Forming Mucins: Processing and Function ..... 672 674 Epithelial Membrane Mucins ....................................... Studies Related to the MUCl Much ............................... 675 Changes in the Patterns of O-Glycosylation of MUCl in Breast 675 Cancer ................................................................ Differences in sites of glycosylation ................................. 675 Changes in the composition of O-glycans added to MUCl in 676 Breast Cancer ........................................................ Correlation in changes of Glycosyltransferase activities with changes in O-glycan structure in Breast Cancer .................... 676 Changes in Glycosylation of MUCl in other Cancers ............. 677 Effects of MUCl Expression on the Behavioral Properties of Cancer Cells.......................................................... 677 Effects on cell interactions and tumourogenicity .................... 677 678 MUCl Expression and Immune Responses ......................... Active Specific Immunotherapy Based on MUCl .................. 679 679 Animal models ....................................................... 680 Clinical studies ....................................................... Comments ............................................................ 681 References............................................................ 681 Biological Roles of Hyaluronan...................................... Bryan P . Toole Introduction .......................................................... Hyaluronan is a Biopolymer with Unusual Physical Properties .... Hyaluronan Binds to Several Types of Proteins (Hyaladherins) ... General Properties of Hyaladherins ................................. Structural Hyaluronan-Binding Proteins ............................ Hyaluronan Receptors ............................................... Intracellular Hyaluronan-Binding Proteins ......................... Inter-a-Trypsin Inhibitor ............................................ Hyaluronan-Dependent Pericellular Matrices Assemble Around Several Cell Types ................................................... Hyaluronan-Dependent Cellular “Coats” .......................... Assembly of Chondrocyte Pericellular Matrix ......................
685 685 685 687 687 688 688 689 689 690 690 691
XLVI 41.4.3 41.5 41.5.1 41.5.2 41.5.3 41.5.4 41.5.5 41.6
42 42.1 42.2 42.3 42.4 42.5 42.5.1 42.6 42.6.1 42.6.2 42.7 42.7.1 42.7.2 42.8 42.8.1 42.8.2 42.8.3 42.9 42.9.1 42.9.2 42.9.3 42.10
43 43.1 43.2
Contents
Tethering of Cell Surface Hyaluronan to Hyaluronan Synthase ... Hyaluronan Influences Cell Behavior During Morphogenesis and Tissue Remodeling .................................................. Migratory and Proliferating Cells are Surrounded by Hyaluronanenriched Matrices .................................................... Hydrated Pericellular Milieux Provide Cellular Pathways ......... Receptors Mediate Effects of Hyaluronan .......................... Hyaluronan-Cell Interactions in Limb Development ............... Hyaluronan-Cell Interactions in Other Physiological and Developmental Systems ............................................. Hyaluronan Plays a Crucial Role in Cancer ........................ References ...........................................................
691 693 693 693 693 694 695 696 696
Biological Roles of Heparan Sulfate Proteoglycans................. 701 Ofer Reizes. Pyong Woo Park. and Merton BernJeld Introduction ......................................................... 701 Heparan Sulfate Biosynthesis ....................................... 701 Functions of Heparan Sulfate ....................................... 702 703 Proteoglycans ........................................................ Intracellular Proteoglycans .......................................... 703 Serglycin and Heparin ............................................... 704 Cell Surface Heparan Sulfate Proteoglycans ........................ 705 Syndecans ............................................................ 705 Glypicans ............................................................ 706 Part-time Cell Surface Heparan Sulfate Proteoglycans ............. 707 Betaglycan ........................................................... 707 CD44 ................................................................. 708 Functions of Cell Surface Heparan Sulfate Proteoglycans ......... 708 Ligand Receptors .................................................... 708 709 Ligand Coreceptors .................................................. Shed Effectors ....................................................... 709 Extracellular Matrix Heparan Sulfate Proteoglycans and Their 710 Functions ............................................................ 710 Perlecan .............................................................. Agrin ................................................................. 712 Other Extracellular HSPGs ......................................... 713 713 Conclusions .......................................................... References ........................................................... 713 Biological Roles of Keratan Sulfate Proteoglycans.................. 717 Gary W. Conrad Introduction ......................................................... 717 718 Corneal Transparency ...............................................
Contents XLVII 43.3 43.4 43.5
Nerve Growth Cone Guidance ...................................... Cell Adhesion ........................................................ Other Possible Roles of KSPGs ..................................... Acknowledgment .................................................... References ............................................................
44
Developmental and Aging Changes of Chondroitin/Dermatan Sulfate Proteoglycans ........................................................ 729 J . Michael Sorrell. David A . Carrino. and Arnold I. Caplan Proteoglycans ........................................................ 729 Glycosaminoglycans ................................................. 729 Core Proteins ........................................................ 731 Hyalectans ........................................................... 731 Small Leucine-rich Proteoglycans ................................... 733 Chondrotin/Dennatan Sulfate Proteoglycans in Development and Aging ................................................................. 735 Core Proteins in Development. Aging. and Pathologies ............ 735 Chondroitin/Dermatan Sulfate Glycosaminoglycan Chains in Development. Aging. and Pathologies .............................. 736 Summary ............................................................. 740 References ............................................................ 740
44.1 44.2 44.3 44.3.1 44.3.2 44.4 44.4.1 44.4.2 44.5
45
45.1 45.2 45.3 45.4 45.5 45.6 45.7
46 46.1 46.2 46.3 46.4 46.5 46.6
719 721 722 723 723
Proteoglycans and Hyaluronan in Vascular Disease ................. Thomas N . Wight Introduction .......................................................... Proteoglycans and Hyaluronan ...................................... Versican (CSPGs) .................................................... Hyaluronan .......................................................... Decorin/Biglycan (DSPGs) .......................................... Perlecan/Syndecans (HSPGs)........................................ Summary ............................................................. Acknowledgments ................................................... References ............................................................
743
Functions of Glycosyl Phosphatidylinositols ......................... Nikola A . Baumann. Anant K. Menon. and David M . Rancour Introduction ........................................................... Parasite Coats: Extreme GPI-Anchoring ............................ Yeast GPIs and the Cell Wall ....................................... Paroxysmal Nocturnal Hemoglobinuria (PNH): Disease and Defects in GPI-Anchoring of Proteins .............................. GPIs in the Secretory and Endocytic Pathways ..................... Organization of GPI Proteins in the Plasma Membrane ...........
757
743 744 745 747 748 749 750 750 750
757 758 758 759 760 762
XLVIII
Contents
46.7 46.8 46.9 46.10 46.11
Association of GPI-Anchored Proteins with Caveolae ............. Detergent Insolubility and Signaling via GPI-Proteins ............. Membrane Release of GPI-Anchored Proteins ..................... GPIs as Second Messenger Signaling Molecules ................... Summary ............................................................. Acknowledgments ................................................... References ...........................................................
47
Glycosphingolipid Microdomains in Signal Transduction. Cancer. 771 and Development ..................................................... Sen-itiroh Hakomori and Kuzuko Handu Clustered GSLs as Functional Units ................................ 771 GSL Clusters. Associated with Signal Transducers. are Functional Units Separable from Caveolae ..................................... 772 Cell Adhesion Coupled with Signal Transduction Initiated by GSL Microdomain: Concept of Glycosignaling Domain (GSD) ........ 773 Role of GSLs in Control of Growth Factor and Hormone Receptors: Possible Relationship with GSL Microdomain ......... 774 Functional Role of Developmentally-Regulated and Tumor776 Associated GSLs ..................................................... References ........................................................... 778
47.1 47.2 47.3 47.4 47.5
48
48.1 48.2 48.3 48.3.1 48.3.2 48.4 48.5 48.6 48.7 48.8 48.9 48.10 48.10.1 48.10.2 48.10.3 48.10.4 48.11 48.12
The Primary Cell Walls of Higher Plants ........................... Jocelyn K. C. Rose, Mulcolm A . O’Neill, Peter Albersheim. and Alan Darvill Introduction (What is a Cell Wall?)................................. Purification of Cell Walls and Isolation of Wall Components ..... The Structural Components of the Primary Cell Wall ............. Cell Walls and the Diversity of Flowering Plants .................. The Structural Components of the Primary Wall .................. Biosynthesis of Wall Components .................................. Organization of the Plant Primary Cell Wall ....................... Cellulose-Xyloglucan Interactions .................................. Interactions Between Pectins and Other Cell Wall Components ... Glycoproteins in the Cell Wall ...................................... Heterogeneity in the Primary Cell Wall ............................ Function and Metabolism of Plant Primary Cell Walls ............ Mechanical Support ................................................. Regulation of Cell Expansion ....................................... Morphogenesis and Differentiation ................................. Plant Cell Wall Oligosaccharides in Defense and Cell Signalling .. Intercellular Transport and Storage ................................. Biotechnology and Future Directions in the Commercial Applications of Plant Primary Cell Walls ..........................
764 764 765 766 767 767 768
783 783 784 786 786 786 791 793 793 794 796 797 798 798 798 800 801 803 803
Contents
49
49.1 49.2 49.3 49.4 49.5 49.6
50
50.1 50.2 50.2.1 50.2.2 50.2.3 50.2.4 50.3 50.3.1 50.3.2 50.3.3
51
51.1 51.2 51.2.1 51.2.2 51.3 51.3.1 51.4
XLIX
Acknowledgment .................................................... References ............................................................
804 804
Glycolipids and Bacterial Pathogenesis .............................. ClifSord A . Lingwood Introduction .......................................................... Modulation of Glycolipid Receptor Function ...................... Stress Response and Glycolipid Receptors .......................... Subcellular Gb3 Trafficking ......................................... Model for Lipid Sorting Based on Chain Length ................... Glycosphingolipids and Signal Transduction ....................... Acknowledgments ................................................... References............................................................
809
Glycobiology of Viruses .............................................. Hildegard Geyer and Rudolf Geyer Summary ............................................................. General Aspects ...................................................... Functions of Viral Surface Glycoproteins ........................... Biosynthesis .......................................................... Function of Carbohydrate Substituents ............................. Oligosaccharide Diversity ........................................... Examples ............................................................. Friend Murine Leukemia Virus Complex ........................... Marburg Virus (MBGV) ............................................ Hepatitis B Virus (HBV) ............................................ Acknowledgments ................................................... References ............................................................
821
809 810 812 813 815 815 817 817
821 821 825 826 826 827 830 830 832 833 836 836
The Glycobiology of Influenza Viruses............................... 839 Stephen J. Stray and Gillian M . Air Introduction .......................................................... 839 Receptor Binding Proteins: Influenza A HAg and Influenza C HEF .................................................................. 840 Structure of Receptor Binding Domain and Mechanism of 843 Sialic Acid Recognition .............................................. HEF Esterase Domain and Mechanism of Cleavage ............... 844 844 Influenza NAm (types A and B) .................................... Mechanism of Sialic Acid Cleavage ................................. 845 Function of Viral Receptor Destroying Enzymes ................... 847 847 Acknowledgments ................................................... 848 References............................................................
L 52
52.1 52.2 52.3 52.4 52.5 52.6 52.7 52.7.1 52.7.2 52.7.3 52.7.4 52.7.5 52.8 52.9
53
53.1 53.2 53.3 53.3.1 53.3.2 53.3.3 53.4 53.4.1 53.4.2 53.4.3 53.4.4 53.4.5 53.4.6 53.4.7 53.5 53.6
Contents Glycobiology of Aids ................................................. Ten Feizi Abstract .............................................................. Introduction ......................................................... The Repertoire of N-Glycans on the Envelope Glycoprotein of HIV of Human Immunodeficiency Virus Produced in Different Cell Types............................................................ Evidence for the Occurrence of O-Glycans on the Envelope Glycoproteins of HIV-1 Produced in Certain Cell Lines ........... Oligosaccharides of gp 120 and gp 41 at N-Glycosylation Sites and Their Possible Influence on Viral Infectivity ................... gp 120 Glycosylation Can Influence Antigenicity and .. Immunogenicity ..................................................... Saccharides Recognized by Carbohydrate-binding Proteins and Antibodies as Potential Neutralization Epitopes on the Envelope Glycoprotein of HIV-1 .............................................. Lectins and Antibodies with Mannose-related Specificities ........ Antibodies to O-Glycan Sequences ................................. Antibodies to Blood Group A ...................................... Xeno-antibodies to Gala1-3Gal Sequence .......................... Potential Medical Relevance ........................................ Does Viral Oligosaccharide Display Influence Tissue Tropism? ... Concluding Remarks ................................................ Acknowledgment .................................................... References ...........................................................
851
Glycobiology of Protozoan and Helminthic Parasites ............... Richard D . Cummings. and A . Kwame Nyame Introduction ......................................................... General Classification of Parasites .................................. The Major Protozoan Parasites ..................................... Malaria .............................................................. . . ..................................................... Trypanosomiasis Leishmaniasis........................................................ Other Protozoan Parasites........................................... Entamoeba histolytica ............................................... Acanthamoeba ....................................................... Giardia lumblia ...................................................... Cryotosporidium parvum ............................................. Sarcocystis spp ....................................................... Toxoplusmu gondii., ................................................. Pneumocystis carinii ................................................. Helminthic Parasites ................................................. Carbohydrate-Binding Proteins in Parasitic Helminths ............
867
851 851 853 854 855 856 857 857 858 858 859 859 860 862 862 863
867 867 868 868 873 874 878 818 878 878 878 879 879 879 879 883
Contents 53.7 53.8
Unusual Glycans in Other Helminthic Parasites .................... Future Directions .................................................... Acknowledgments ................................................... References ............................................................
54
The Involvement of the Oligosaccharide Chains of Glycoproteins in Gamete Interactions at Fertilization ................................. Noritaka Hirohashi and William J . Lennarz Introduction .......................................................... Advantages of Marine Invertebrates as an Experimental System . . Induction of the Acrosome Reaction ................................ Studies in Sea Urchins ............................................... Studies in Starfish .................................................... Sperm-Egg Coat Binding ........................................... Studies in Mammals ................................................. Studies in Frog ....................................................... Studies in Ascidians .................................................. Studies in Sea Urchins ......... ................................... Carbohydrate as a Species-Specific Determinant ................... References ............................................................
54.1 54.2 54.3 54.3.1 54.3.2 54.4 54.4.1 54.4.2 54.4.3 54.4.4 54.5
55
55.1 55.2 55.3 55.4 55.4.1 55.4.2 55.5 55.6 55.6.1 55.6.2 55.6.3 55.7
56
56.1 56.2 56.3
LI 883 885 885 886
895 895 895 896 896 899 899 900 900 902 904 906 907
Glycosylation and Development ...................................... 909 Michde Aubery and Christian Derappe Summary ............................................................. 909 Introduction .......................................................... 911 Lectins as Tools to Analyze Changes in Cell-surface 911 Glycoconjugates During Development .............................. Cell-adhesion Molecules ............................................. 912 Neural Cell-adhesion Molecule ...................................... 912 The Adhesion Molecule L1 .......................................... 914 Glycosyltransferases ................................................. 914 Altered Expression of Endogenous Lectins During Development . 915 Galectins ............................................................. 915 Selectins .............................................................. 917 Other Endogenous Lectins .......................................... 917 Conclusion ........................................................... 918 References ............................................................ 918 Protein Glycosylation and Cancer ................................... James W. Dennis and Maria Granovskjj Introduction .......................................................... Protein Glycosylation Generates Molecular Diversity .............. Cancer Initiation and Progression ...................................
923 923 923 926
LII
Contents
56.4 56.5 56.6 56.7 56.8 56.9
Tumor Cell Proliferation ............................................ Cell Migration ....................................................... Sialylation and Metastasis........................................... Endogenous Lectins and Tumor Cell Adhesion .................... Carbohydrate Processing Inhibitors as Anti-Cancer Agents ....... Other Considerations ................................................ Acknowledgments ................................................... References ...........................................................
927 930 933 934 935 936 937 937
57
Lysosomal Storage Diseases ......................................... Nathan N . Aronson. Jr. Summary............................................................. Introduction ......................................................... Animal Models ...................................................... Mucopolysaccharidoses ............................................. Cathepsin K Deficiency and Pycnodysostosis ...................... Mouse Models for Tay-Sachs and Sandhoff Diseases .............. Impact of Lysosomal Diseases and Their Study .................... References ...........................................................
945
Genetic Diseases of Glycosylation ................................... Tomoya Akama and Michiko N . Fukuda Introduction ......................................................... CDGS ................................................................ CDGS Type I ........................................................ CDGS Type I1 ....................................................... HEMPAS ............................................................ References ...........................................................
959
57.1 57.2 57.3 57.4 57.5 57.6 57.7
58
58.1 58.2 58.2.1 58.2.2 58.3
59
59.1 59.2 59.3 59.3.1 59.3.2 59.3.3 59.3.4 59.3.5 59.3.6 59.4 59.5
Glycobiology of Helicobacter pylori and Gastric Disease ........... Karl-Anders Karlsson Introduction ......................................................... The Bacterial Surface and Molecular Mimicry ..................... Host Surfaces and H . pylori Recognition of Glycoconjugates: Unique Complexity.................................................. Sialic Acid ........................................................... Sulfatide ............................................................. Heparan Sulfate ..................................................... Fucose-Dependent Binding (H-1 and Lewis b) ..................... Gangliotetraosylceramide ........................................... Lactosylceramide .................................................... The Meaning of Multiple Binding Specificities ..................... Aspects for the Future ............................................... References ...........................................................
945 946 947 947 949 951 953 954
959 959 959 961 963 964 967 967 968 968 969 970 970 971 971 972 972 973 973
Contents
60 60.1 60.2 60.3 60.4 60.5 60.6 60.7 60.7.1 60.7.2 60.8 60.9 60.10 60.1 1 60.12 60.13 60.14 60.15
61
61.1 61.2 61.3 61.4 61.5
62 62.1 62.2 62.3 62.3.1
LIII
Immunoglobulin G Glycosylation and Galactosyltransferase Changes in Rheumatoid Arthritis .................................... 977 John S. Axford Introduction .......................................................... 977 977 Oligosaccharide Synthesis ........................................... Galactosyltransferase ................................................ 978 978 Immunoglobulin G .................................................. Rheumatoid Arthritis ................................................ 979 979 Quantification of IgG sugars in RAr ................................ RAr and Pregnancy .................................................. 980 980 Galactosylation of IgG .............................................. a3-Fucosylation of a1-Acid Glycoprotein .......................... 981 Agalactosyl-IgG and Rheumatoid Factor Binding ................. 982 Tissue-specific Galactosyltransferase Abnormalities in an Experimental Model of Rheumatoid Arthritis ...................... 983 Glycosylation Homeostasis within RAr Lymphocytes is Abnormal ............................................................ 985 Are the Rheumatoid Arthritis Associated Glycosylation Abnormalities Unique? .............................................. 986 Sugar Printing Rheumatic Disease is Possible ...................... 990 Rapid Profiling of IgG N-Glycans by Fluorophore-coupled Oligosaccharide Electrophoresis has the Potential of Differentiating 992 Rheumatic Diseases.................................................. In What Way could GTase Enzymatic Control be Abnormal? . . . . 992 Conclusion ........................................................... 993 References ............................................................ 994
Calnexin. Calreticulin and Glycoprotein Folding Within the Endoplasmic Reticulum .............................................. 997 Michael R. Leach and David B. Williams Structure and Properties of Calnexin and Calreticulin ............. 997 1000 Biological Functions ............................................... Mechanism of Action .............................................. 1002 Functional Relationship Between Calnexin and Calreticulin ..... 1005 Relationship with other ER Chaperones and Folding Catalysts . 1007 1008 References .......................................................... Glycobiology of The Nervous System .............................. Ronald L . Schnaar Introduction ........................................................ Nervous System Glycoconjugates-Overview .................... Nervous System Glycolipids ....................................... Galactosylceramides ...............................................
1013 1013 1013 1014 1014
LIV
Contents
62.3.2 62.4 62.4.1 62.4.2 62.5 62.6 62.6.1 62.6.2 62.7
Gangliosides and Related Anionic Glycosphingolipids ........... Nervous System Glycoproteins ..................................... Polysialic Acid ...................................................... The HNK-I Determinant .......................................... Nervous System Glycosaminoglycans ............................. Lectins in the Brain ................................................. Myelin-Associated Glycoprotein ................................... Other Nervous System Lectins ..................................... Concluding Remarks ............................................... References ..........................................................
1016 1019 1020 1020 1020 1021 1021 1022 1023 1023
63
Glycobiology of the Immune System ............................... Elizabeth F. Hounsell Infection and Pathogenesis ......................................... Control of the Immune Response .................................. Bacterial and Tumor Antigens. Mucins and Mucin-like Molecules ........................................................... Immunoglobulins and Pathology .................................. References ..........................................................
1029
63.1 63.2 63.3 63.4
64
64.1 64.2 64.2.1 64.2.2 64.2.3 64.3 64.3.1
64.3.2
Metabolic Engineering Glycosylation: Biotechnology’s Challenge to the Glycobiologist in the Next Millennium ...................... Thomas G. Warner Introduction ........................................................ Recent Developments in Carbohydrate Biosynthesis.............. Optimizing Sialylation of Recombinant Proteins by Metabolic Engineering Sialic Acid Biosynthesis............................... Optimizing Galactosylation of Recombinant Proteins by Metabolically Engineering Galactose Biosynthesis ................ Mannose Biosynthesis and Mannosylation of Recombinant Proteins ............................................................. Glycosylation Engineering Alternate Expression Hosts For Recombinant Protein Therapeutic Production .................... Engineering Glycosylation of Recombinant Proteins Expressed in Baculovirus-Insect Cells ......................................... Genes needed to supplement glycosylation of recombinant proteins in insect cells .............................................. Deleterious genes may need to be deleted or inhibited to enhance recombinant glycoprotein biosynthesis in insect cells.............. Engineering Glycosylation of Recombinant Proteins Expressed in Plants ............................................................. Genetic addition and supplementation needed to improve plant recombinant protein glycosylation ................................. Inhibition or deletion of plant glycosylation genes ................
1029 1033 1034 1036 1038
1043 1043 1044 1044 1049 1052 1053 1053 1054 1054 1056 1058 1059
Contents
64.4
Summary ........................................................... Acknowledgments ................................................. References ..........................................................
Index ...................................................................
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1059 1060 1060 11
List of Contributors (only main authors are listed)
Gillian Air Dept. of Biochemistry and Molecular Biology University of Oklahoma, Health Center BMSB 840, P. 0. Box 26901 Oklahoma City, OK 73190 USA Nathan N . Aronson Department of Biochemistry University of South Alabama MSB 2152 Mobile, AL 36688 USA Michele Aubery Ul80-INSERM U180 45 Rue Des Saints Peres 75006 Paris France Claudine Auge Laboratoire de Chimie Organique Multifonctionelle DR2 CNRS 462 Universite de Paris-Sud Inst. de Chimie Moleculaire (ICMO) 91405 Orsay Cedex France John S. Axford St. George’s Hospital Med. Ctr. Cranmer Terrace London SW17 ORE England
Samuel H. Barondes Langley Porter Psych. Inst. 401 Parnassus Avenue San Francisco, CA 94143-0984 USA Subhash Basu University of Notre Dame Stephan Chemistry Hall, Rm 443 Notre Dame, IN 46556 USA Jean-Marie Beau Univ. Paris-Sud Lab. de Synthese de Biomolecules URA CNRS 462, Inst. de Chimie 91405 Orsay France Merton Bernfield Harvard Medical School Enders Building, Room 961 300 Longwood Avenue Boston, MA 021 15 USA Mikael Bols Department of Organic Chemistry Aarhus University Langelandsgade 140 8000 Aarhus C Denmark
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List of Contributors
Inka Brockhausen Department of Medicine Queen’s University Etherington Hall, room 1021 Kingston, Ontario K7L 3N6 Canada Max Burger Friedrich Miescher Institut 4002 Basel Switzerland Arnold I. Caplan Case-Western Reserve University Cleveland, OH 44106 USA Henrik Clausen Department of Oral Diagnostics University of Copenhagen Norre Alle 20 DK-2200 Copenhagen N. Denmark Gary W. Conrad Kansas State University Ackert Hall Manhattan, KS 55606 USA Paul Crocker Department of Biochemistry The Welcome Trust Building University of Dundee Dundee, DDl 5HN Scotland Richard D. Cummings Department of Biochemistry and Molecular Genetics University of Oklahoma HSC 975 NE lofhSt., BRC 417 Oklahoma, OK 73104 USA Nancy Dahms Medical College of Wisconsin P. 0. Box 26509 8701 Watertown Plank Road Milwaukee, WI 53226-0509 USA Samuel J. Danishefsky Department of Chemistry Columbia University Havemeyer Hall
New York, NY 10027 and Laboratory for Bioorganic Chemistry The Sloan-Kettering Institute for Cancer Research 1275 York Avenue New York, NY 10021 USA Alan Darvill University of Georgia 220 Riverbend Road Athens, GA 30602 USA James W. Dennis Div. of Cancer and Cell Biology Mount Sinai Hosp., Res. Inst. 600 University Ave. Toronto, ON M5G 1x5 Canada Anne Dell Wolfson Laboratories Department of Biochemistry Imperial College of Science London SW7 2AY England Hugues Driguez CERMAV-CNRS B. P. 53 38041 Grenoble cedex France Dirk H. van den Eijnden Department of Medicinal Chemistry Vrije Universiteit Van der Boechorststraat 7 1081 BT Amsterdam The Netherlands Alan D. Elbein Dep. of Biochemistry and Molecular Biology University of Arkansas, Slot 516 4301 W. Markham Street Little Rock, AR 72205-7199 USA Paul Englund Johns Hopkins University School of Medicine 725 North Wolfe St. Baltimore, MD 21205 USA
List of Contributors Beat Ernst Institute of Molecular Pharmacy Pharmacenter Universitat Basel Klingenbergstrasse 50 4051 Basel Switzerland Mail:
[email protected] Marilynn E. Etzler University of California Davis, CA 95616 USA Ten Feizi Clinical Research Centre Watford Road Harrow, MDX HA1 3UJ England Bert Fraser-Reid Natural Products and Glycotechnology Research Institute Inc. 41 18 Swarthmore Road Durham, North Carolina 27707 USA Hudson H. Freeze The Burnham Institute 10901 N. Torrey Pines Road La Jolla, CA 92093 USA Michiko Fukuda Burnham Institute (formerly La Jolla Cancer Res. Fndm.) 10901 North Torrey Pines Road La Jolla, CA 92037 USA James L. Funderburgh Kansas State University Ackert Hall Manhattan, KS 66506 USA Robert M. Garbaccio Laboratory for Bioorganic Chemistry The Sloan-Kettering Institute for Cancer Research 1275 York Avenue New York, NY 10021 USA
Teresa Garrett Duke University Medical Center c/o Dr. C. Raetz P. 0. Box 3711 Durham, N C 27710 England Rita Gerardy-Schahn Institut fur Medizinische Mikrobiologie Medizinische Hochschule Hannover Carl-Neuberg-Strak 1 30625 Hannover Germany Roberto A. Geremia Centre de Recherches sur les M acromolkcules Vegttales Universite Joseph Fourier Cermav-CNRS, BP 53 38041 Grenoble Cedex 9 France Rudolph Geyer Biochemisches Institut Universitat Giessen Friedrichstr. 24 35392 Giessen Germany Gary R. Gray Department of Chemistry Institute of Technology University of Minnesota 207 Pleasant Street S. E. Minneapolis, MN 55455-0431 USA Sen-Itiroh Hakomori Pacific Northwest Research Foundation 720 Broadway Seattle, WA 98122 USA Robert S. Haltiwanger State University of New York Stony Brook, NY 11794-5215 USA Gerald W. Hart Department of Biological Chemistry John Hopkins University 401 Hunterian Building 725 N. Wolfe Street Baltimore, M D 21205-2185 USA
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LX
List of Contributors
Steve W. Homans School of Biochemistry and Molecular Biology University of Yeeds LSZ 9ST United Kingdom
Horst Kunz Institut fur Organische Chemie Universitat Mainz J.-Joachim-Becher-Weg 18-20 55099 Mainz Germany
Hideharu Ishida Department of Applied Bio-organic Chemistry Gifu University Gifu 501-11 Japan
Joseph T. Y. Lau Roswell Park Memorial Institute 666 Elm Street Buffalo, NY 14263 USA
Wolfgang Jager Biacore AB Jechtinger Strasse 8 79 111 Freiburg Germany Osamu Kanie Glycoscience Laboratory Mitsubishi Kasei Institute of Life Sciences (MILS) Machida-shi Tokyo 194-8511 Japan Karl-Anders Karlsson Institute of Medical Biochemistry G~teborgUniversity Medicinaraegatan 9A P. 0. Box 440 405 30 Gateborg Sweden Makoto Kiso Department of Applied Bio-organic Chemistry Gifu University Gifu, 50 1-11 Japan Sharon S. Krag Department of Biochemistry Johns Hopkins University 615 North Wolfe Street Baltimore, MD 21205 USA J. J. Krepinsky Department of Medical Genetics and Microbiology University of Toronto Toronto, ON M5S 1A8 Canada
Torvard C. Laurent Department of Medical and Physiological Chemistry University of Uppsala Avd. for Med. Kemi, Box 575 751 23 Uppsala Sweden Yuan C. Lee Department of Biology Johns Hopkins University 3400 North Charles Street Baltimore, MD 21218 USA Ludwig Lehle Lehrstuhl fur Zellbiologie und Pflanzenphysiologie Universitat Regensburg 93040 Regensburg Germany William J. Lennarz Department of Biochemistry and Cell Biology SUNY Stony Brook Room 450, Life Sciences Bldg. Stony Brook, NY 11794-5215 USA Steven V. Ley Chemical Laboratory University of Cambridge Lensfield Road Cambridge CB2 1EW England Yu-Teh Li Department of Biochemistry, SL43 Tulane University School of Medicine 1430 Tulane Avenue New Orleans, LA 701 12 USA
List of Contributors Ulf Lindahl Medical and Physiological Chemistry Uppsala Universitet P. 0. Box 575 75 12 Uppsala Sweden
Kelly L. Moremen Complex Carbohydrate Research Center University of Georgia 220 River Bend Road Athens, GA 30602-7229 USA
Clifford A. Lingwood Department of Microbiology Hospital for Sick Children 555 University Avenue Toronto, OT M5G 1x8 Canada
Heinz E. Moser GeneSoft Inc Two Corporate Drive South San Francisco, CA 9080 USA
Jean-Maurice Mallet Department de Chimie, URA 1686 Ecole Normale Superieure 24 rue Lhormond 75231 Paris Cedex 05 France Ernesto Marques Department of Pharmacology and MoleculaI Science John Hopkins University School of Medicine 725 North Wolfe Street Baltimore, M D 21205-2185 USA Jamey Marth Howard Hughes Medical Institute University of California, San Diego 9500 Gillman Drive, 0625 La Jolla, CA 92093-0625 USA Rodger McEver Oklahoma Medical Research Ed. 825 NE 13'h Street Oklahoma City, OK 73 104 USA Anant K. Menon Department of Biochemistry University of Wisconsin-Madison 420 Henry Mall Madison, WI 53706-1569 USA
LXI
Reinhold Ohrlein Ciba Speciality Chemicals Inc. K-420.2.19 4002 Base1 Switzerland Peter Orlean 309 Roger A d a m Laboratory University of Illinois at Urbana 600 South Mathews Avenue Urbana, IL 61801 USA Stefan Oscarson Department of Organic Chemistry Stockholm University Arrhenius Laboratory Svante Arrhenius vag 12 10691 Stockholm Sweden Monica M. Palcic Department of Chemistry University of Alberta Edmonton, AB, T6G 2G2 Canada Luigi Panza Dipartimento di Chimica Organica e Industriale Via Venezian 2 1 20133 Milano Italy Armando J. Parodi Biquimicas-Fundaction Campomar University of Buenos Aires Antonio Machado 151 Buenos Aires, 1405 Argentina
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List of Contributors
Serge Perez Centre de Recherches sur les MacromolCcules Vegetales UniversitC Joseph Fourier Cermav-CNRS, BP 53 38041 Grenoble Cedex 9 France
Konrad Sandhoff Institut fur Organische Chemie und Biochemie Universitat Bonn Gerhard-Domagk-Str. 1 53121 Bonn Germany
Thomas Peters Medizinische Universitat Zu Lubeck Institut fur Chemie Ratzeburger Allee 160 23538 Lubeck Germany
Harry Schachter Department of Biochemistry Hospital for Sick Children 555 University Avenue Toronto, ON M5G 1x8 Canada
Vince Pozsgay National Institute of Health 6 Center Dr. MSC 2720 Bldg. 6, Rm. 2A02 Bethesda, MD 20892-2720 USA
Roland Schauer Christian-Albrechts Universitat zu Kiel Olshausenstr. 40 24098 Kiel Germany
Christian Raetz Department of Biochemistry Duke University Medical Center P.O. Box 371 1 Durham, NC 27710 USA
Ronald L. Schnaar Department of Pharmacology and Molecular Science Johns Hopkins School of Medicine 725 North Wolfe Street Baltimore, MD 21205 USA
Ossi Renkonen University of Helsinki Institute of Biotechnology P. 0. Box 56 00014 University of Helsinki Finland
Richard R. Schmidt Fakultat fur Chemie Universitat Konstanz Postf. 5560 M 125 78457 Konstanz Germany
Peter J. Roach Department of Biochemistry Indiana University School of Medicine 635 Barnhill Dr. Indianapolis, IN 46202-5122 USA
Joel Shaper Oncology Center Johns Hopkins Hospital 600 North Wolfe Street Baltimore, MD 21287 USA
Steven D. Rosen Department of Anatomy University of California, San Francisco Box 0452 San Francisco, CA 94143 USA
Pierre Sinay Departement de Chimie Ecole Normale Superieure 24 rue Lhomond 75231 Paris Cedex 50 France
List of Contributors Dorothe Spillmann Department of Medical Biochemistry and Microbiology University of Uppsala The Biomedical Centre P. 0. Box 575 751 23 Uppsala Sweden Robert C. Spiro Department of Biological Chemistry Haward Medical School J o s h Research Laboratory 1 J o s h Place Boston, MA 02215 USA Jun-ichi Tamura Faculty of Education Tottori University Tottori 680-0945 Japan Garry Taylor Department of Biology and Biochemistry University of Bath Calverton Down Bath, BA2 7AY England Joyce Taylor-Papadimitriou Epithelial Cell Biology Laboratory Imperial Cancer Research Fund 44 Lincoln's Inn Field London, WC2A 3PX England Bryan P. Toole Department of Anatomy Tufts University HSC 136 Harrison Avenue Boston, MA 021 11 USA Eric J. Toone Department of Chemistry Duke University Durham, NC 27708-0346 USA Andrea T. Vasella Laboratorium fur Organische Chemie Eidgenossische Technische Hochschule Universitatsstrasse 19 8092 Zurich Switzerland
LXIII
Alain Veyrieres Ecole Nationale Superieure de Chimie de Rennes Campus du Beaulieu Avenue du General Leclerc 35700 Rennes France Barbara Vertel Department of Cell Biology and Anatomy Chicago Medical School 3333 Green Bay Road North Chicago, IL 60064-3095 USA Johannes F. G. Vliegenhart Bijvoet Center for Biomolecular Research Utrecht University P. 0. Box 80075 3508 TB Utrecht The Netherlands David J. Vocadlo Department of Chemistry University of British Columbia Vancouver B.C. V6T 1Zl Canada Russell Wallis Department of Biochemistry Glycobiology Institute University of Oxford South Parks Road Oxford, OX1 3QU England Thomas Warner 541 Wellington Drive San Carlos, CA 94070 USA Hans Peter Wessel F. Hoffmann-La Roche AG Pharma Research Discovery Chemistry PRPV-D Building 15/110 4070 Basel Switzerland Thomas N. Wight Department of Pathology SM-30 University of Washington School of Medicine Seattle, WA 98 195 USA
LXIV
List of Contributors
David B. Williams Department of Biochemistry University of Toronto Toronto, ON M5S 1A8 Canada Lawrence J. Williams Laboratory for Bioorganic Chemistry The Sloan-Kettering Institute for Cancer Research 1275 York Avenue New York, NY 10021 USA
Stephen G. Withers Department of Chemistry University of British Columbia 2036 Main Mall Vancouver, B.C. V6T 1Zl Canada Chi-Huey Wong Department of Chemistry Scripps Research Institute 10550 N. Torrey Pines Road La Jolla, CA 92037 USA
Abbreviations Used in Volumes 1 and 2
a1,3-GalT a2,3-SialT a2,6-SialT aGalF aGlcF aMuNeuAc apNPFuc apNPGal apNPGalNAc apNPGlc apNPGlcNAc apNPMan apNPNeuAc
A aa Ac AcHmb ACP AF AFM AgOTf AGP 019.
All AllNAc Aloc APC APP APS APS
a 1,3-GaIactosyltransferase a2,3-Sialyltransferase a2,6-sialyltransferase a-Galactosyl fluoride a-Glucosyl fluoride 4-Methylumbelliferone a-D-N-acetylneuraminic acid para-Nitrophenyl a-L-fucopyranoside para-Nitrophenyl a-D-galactopyranoside para-Nitrophenyl 2-acetamido-2-deoxy-a-~-galactopyranoside para-Nitrophenyl a-D-glucopyranoside para-Nitrophenyl 2-acetamido-2-deoxy-a-~-glucopyranoside para-Nitrophenyl a-D-mannopyranoside para-Nitrophenyl-a-D-N-acetyheuraminic acid Angstrom amino acid acetyl
N-2-acetoxy-4-methoxybenzyl acyl carrier protein aggregation factor atomic force microscopy silver trifluoromethanesulfonate (silver triflate) arabinogalactan protein asparagine-linked glycosylation ally1 N-Acetylallosamine Ally loxycarbony1 antigen-presenting cell acute phase protein adenosine phosphosulfate ammonium persulfate
LXVI
Abbreviations Used in Volumes I and 2
AR ARIS ASGP-R Asialo-GM1 Asn Asn GT ASTF-GP j3 1,4-GalT PManF PMuGlc PMuGlcNAc PMuXyl PoNPGal PoNPGlc PpClPMan PPhGal PPhGlc ppNP2dGlc PpNPGal PpNPGalNAc PpNPGlc PpNPGlcNAc PpNPMan PPNPXYl PpOMePhGlc PVGal 9-BBN BDA BHK bFGF BLAST BM BMP Bn Boc BOP bP Bu BU4NSSTr Bz Bzl CAA CAM CAN
acrosome reaction acrosome reaction-inducing substance asialoglycoprotein receptor p-D-Gal( 1-3)-b-~-GalNAc(1-4)-P-D-Gal( 1-4)-P-~-Glc-O-Cer asparagine asparagine glucosyltransferase (GalG1cNAcMan)zManGlcNAcz Asn P 1,4-Galactosyltransferase P-Mannosyl fluoride 4-Methylumbelliferyl P-o-glucopyranoside 4-Methylumbelliferyl 2-acetamido-2-deoxy-~-~-glucopyranoside 4-Methylumbelliferyl P-D-xylopyranoside ortho-Nitrophenyl P-D-galactopyranoside ortho-Nitrophenyl P-D-glucopyranoside para-Chlorophenyl P-D-mannopyranoside Phenyl P-D-galactopyranoside Phenyl P-D-glucopyranoside para-Nitrophenyl2-deoxy-P-~-arabino-hexopyranoside para-Nitrophenyl P-D-galactopyranoside para-Nitrophenyl 2-acetamido-2-deoxy-P-~-galactopyranoside para-Nitrophenyl P-D-glucopyranoside para-Nitrophenyl 2-acetamido-2-deoxy-~-~-glucopyranoside para-Nitrophenyl P-D-mannopyranoside para-Nitrophenyl P-D-xylopyranoside para-Methoxyphenyl P-D-glucopyranoside Vinyl 0-D-galactopyranoside 9-Borabicyclo[3.3.llnonane butane diacetal baby hamster kidney basic fibroblast growth factor basic alignment search tool basement membrane bis(monoacylg1ycero)phosphate benzyl tert-butoxycarbonyl benzotriazol-1-yloxy-tris(dimethy1amino)phosphoniumhexafluorophosphate base pair butyl Tetrabutylammoniumsaltoltriphenylmethanethiol benzoyl benzyl carbohydrate amino acids cell adhesion molecule Cer(1V)ammoniumnitrate
Ahhreviutions Used in Volumes 1 and 2 LXVII CAT CBMIT CBS CBS Cbz CCCP CD CDA CDGS CDMT cDNA CDTA Cer CHO CgOse4 CGTase CI CI8 CIAP ClAc ClNP CMLN CMP CMP CMP-NeuAc/ CMP-NeuSAc CMP-Sia CNBr CNS CoA Coll CPG CRD
cs
CSA CSA
csc CSPG CST CTL CTP Da DAF DAG DBA DBMP
chloramphenicol acetyltransferase 1,l-carbonylbis(3-methylimidazolium triflate Gal(S03)Pl- 1Cer Cerebroside sulfate benzyloxycarbonyl carbonyl cyanide m-chlorophenylhydrazone cation dependent cyclohexane-l,2-diacetal carbohydrate-deficient glycoprotein syndrome 2-chloro-4,6-dimethoxy-l,3,5-triazine complementary DNA diaminocyclohexane tetraacetic acid ceramide Chinese hamster ovary fi-~-Gal( 1-3)-o-~-GalNAc( 1-4)-fi-~-Gal( 1-4)-p-~-Glc cyclodextrin glucosyltransferase cation independent Cycloisomalto-octaose calf intestine alkaline phosphatase [ EC.3.1.3.11 chloroacetyl 2-Chloro-4-nitrophenyl calf mesenteric lymph node cytosine monophosphate cytidine 5'-monophosphate cytidine 5'-monophosphate N-acetylneuraminic acid Cytidine 5'-monophosphate sialic acid cyanogen bromide central nervous system coenzyme A sym-collidine controlled pore glass carbohydrate-recognition domain chondroitin sulfate Campher sulfonic acid chondroitin-4-sulfate chondroitin-6-sulfate chondroitin sulfate proteoglycan castanospermine cytotoxic T cell 5 '-Cytidinetriphosphate Dalton decay accelerating factor diacylglucosamine ((strain D x strain B) x strain A) mice 2,6-di-tert-butyl-4-methylpyridine
LXVIII Abbreviations Used in Volumes 1 and 2 DBU DCC DCPhth Dde DDP DDQ DEPC DF 2dGlc DIBALH DiBz DIG DIM DIPEA DMA DMAP DME DMF DMJ DMM DMPU DMSO DMTST DMTST 2,4-DNP DNJ DNJ Do1 Dol-P Dol-PP DP DPPA DRM DS DTBMP DTBP DTBPI DTT E-CAD ECM EDCD/EDCI EDTA EGF eIF EM
1,8-diazabicyclo[5.4.01undec-7-ene N ,N’-Dicyclohexylcarbodiimide 4,s-dichlorophthaloyl N- 1- (4,4-dimethy1-2,6-dioxocyc1ohexy1idene) ethy 1 dialkyl N,N-diethylphosphoramidites 2,3-Dichloro-S,6-dicyano-1,4-benzoquinone diethylphosphoryl cyanide deactivation factor 2-Deoxy-arabino-hexose Diisobutylaluminumhydride Dibenzoyl detergent-insoluble glycolipid complex 1,4-dideoxy-1,4-imino-~-mannitol diisopropylethylamine N ,N-dimethylacetamide 4-Dimethylaminopyridine 1,Zdimethoxyethane N ,N-dimethylformamide 1-deoxymannojirimycin dimethylmaleoyl N ,N’-dimethylhexahydropyrimidin-2-one dimethyl sulfoxide dimethyl(methy1thio)sulfonium tetrafluoroborate (dimethyl (methy1thio)sulfoniumtriflate) Dimethyl(methy1thio)sulfoniumtetrafluoromethansulfonate 2,4-Dinitrophenyl deoxynojirimycin 1,5-Dideoxy-l,S-irnino-D-glucitol (deoxynojirimycin) dolichol dolichyl phosphate dolichyl pyrophosphate degree of polymerization diphenylphosphoryl azide detergent-resistant membrane dennatan sulfate 2,6-di-tert-butyl-4-methylpyridine 2,6-di-tert-butylpyridine 2,6-di-tert-butylpyridiniumiodide dithiothreitol Epithelial Cadherin I extracellular matrix 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ethylene diamine tetraacetic acid epidermal growth factor eukaryotic initiation factor electron microscopy
Abbreviations Used in Volumes I and 2 LXIX endo H EPGase ER ER ESI EST EtN EtN-P F FAK FCOE FGF Fmoc/FMOC FSP FT FTIR FTIR Fuc Fuc FucT GA GAG Gal Gal GalNAc GalT Gal- 1-P UT Gb3 Gb;l GC GC-MS GDI, GDP GDP GDP-Fuc GDP-Man GDPMP Gg3 GI GI1 Glc Glc GlcA GlcN GlcN
endo-0-N-acetylglucosaminidase H endopolygalacturonase endoplasmic reticulum endoplasmic reticulum electrospry ionization expressed sequence tag ethanolamine ethanolamine phosphate fusion protein focal adhesion kinase fluorophore-coupled oligosaccharide electrophoresis fibroblast growth factor 9-Fluorenylmethoxylcarbonyl(fluorenyl-9-methoxycarbonyl) fucose sulfate polysaccharide a-(1-3/4)-fucosyltransferase [ EC.2.4.1.651 Fourrier transform infra red Fourier transform infra-red (spectroscopy) fucose/fucosyl fucose fucosyltransferase Golgi apparatus glycosaminoglycan galactose galactose/galactosy1 N-acetylgalactosamine galactosyltransferase Gal- 1-P-uridyltransferase Gala+4Gal~1+4Glc~lA+lCer
GalNAcPl+3Gala1+4Gal~l+4Glc~l+lCer glucosyl cerdmide gas chromatography-mass spectrometrya-DNeuAc(2-3)-P-~-Gal(1-3)-p-~-GalNAc(1-4)-[a-DNeuAc(2-3)]-P-~-Gal(1-4)-p-~-Glc-o-CerGDP guanosine diphosphate guanosine 5'-diphosphate guanosine 5I-diphosphate fucose guanosine 5'-diphosphate mannose GDP-Man pyrophosphorylase
GalNAcP1+4Galpl+4Glcpl+lCer gastro-intestinal glucosidase I1 glucose/glucosyl glucose glucuronic acid 2-Amino-2-deoxy -glucopyr anose glucosamine
LXX
Abbreviations Used in Volumes I and 2
GkN3 Glc3NAc GlcNAc GlcNAc GlcNAc, GlcNAc-Oxazoline GlcNPht GlcNTeoc Glucal GLUT GM~ GMi
GM3 GM3 GMP GnT GP GPI GRIFIN GRP GSL GT GT GTA GTB H HA HAF HAg HAS HBP HBPyU hCG HEMPAS test HeP HEPES HEV HIV HIV
2-Azido-2-deoxy-~-glucopyranose 3-Acetamido-3-deoxy-~-glucopyranose N-acetylglucosamine 3,4,6-tri-O-acetyl-N-acetylglucosamine { p-~-GlcNAc( 1-4)) ( , - l ) - ~ - G l ~ N A ~ 2-Methyl-4,5-dihydro-(1,2-dideoxy-a-~-g~ucopyranoso) [2,1-d]-1,3-oxazole 2-Deoxy-2-phthalimido-~-glucopyranose 2-Deoxy-2-aminot~ethoxylcarbonyl-~-g~ucopyranose 1,5-Anhydr0-2-deoxy-~-arabino-hex1-enitol glucose transporter GalPl+3GalNAcf31 -+[NeuAcu2+3]Gal~l+4Glc~l+1Cer p-D-Gal(1-3)-p-~-GalNAc(1-4)-[u-~-NeuAc(2-3)1-p-~Gal( 1-4)-p-D-GlC-O-Cer P-D-GalNAc(1-4)-[u-~-NeuAc(2-3)]-p-~-Gal( 1-4)-p-~Glc-0-Cer NeuAca2+3Gal~l-+4G1c~l+ lCer a-~-NeuAc(2-3)-P-D-Gal(1-4)-p-~-Glc-O-Cer guanosine monophosphate N-acetylglucosaminyltransferase glycoprotein glycosyl phosphatidylinositol galectin-related interfiber protein glycine-rich protein glycosphingolipid p-(1-4)-galactosyltransferase [ EC.2.4.1.221 (glycosyltransferase) UDP-G1c:glycoprotein glucosyltransferase human blood group A glycosyltransferase human blood group B glycosyltransferase Fucul+2Galpl+3/4GlcNAcP hyaluronic acid, hyaluronate Hulicondriu aggregation factor hemagglutinin hyaluronate synthesizing enzyme hexosamine biosynthetic pathway 0-(benzotriazole-1-yl)-N,N,N,N-bis(tetramethy1ene)uronium hexafluorophosphate human chorionic gonadotropin hereditary erythroblastic multinuclearity with a positive acidified-serum-lysis (congenital dyserythropoietic anemia type 11) heparin N-(2-Hydroxyethylpiperazine)-N'-(2-ethanesulfon~c acid) high endothelial venules Human immunodeficiency virus human immunodeficiency virus
Abbreviations Used in Volumes 1 and 2 HL HMG HN HOAll HOAT HOBt HOMO HPLC HPLC HRGP HS HSD HSPG HUS HYCRON HYP ICAM IDCP IDCT IduA IGF Ig IgSF IIDQ IL IPG IU IV3NeuAc PGalNAcGbOse4 I V NeuAcII NeuAcCgOse4 K
kb kDa Kdn
Kdn/KDN Kdo Kdo K-4 Ki KIF Krn KS KSPG
LXXI
hepatic lectin high mobility group (proteins) hemagglutinin-neuraminidase Ally1 alcohol 1-hydroxy-7-azabenztriazole 1-hydroxybenzotriazole hydrate highest occupied molecular orbital high-performance liquid chromatography high performance liquid chromatography hydroxyproline-rich glycoprotein heparan sulfate highly sulfated domain heparan sulfate proteoglycan hemolytic uremic syndrome hydroxycrotyl-oligoethylene glycol-n-alkanoyl 4-hydroxyproline intercellular adhesion molecule iodonium dicollidine perchlorate iodonium dicollidine triflate iduronic acid insulin-like growth factor immunoglobulin immunoglobulin superfamily of proteins isobutoxyisobutyldihydroquinoline interleukin inositol phosphoglycan international unit p-D-GalNAc(1-3)-p-~-GalNAc(1-3)-u-~-Gal(1-4)-p-DGal(1-4)-~-Glc a-u-NeuAc(2-3)-P-~-Gal(1-3)-p-~-GalNAc(1-4)-[a-DNeuAc(2-3)]-P-~-Gal(1-4)-Dkinase enzyme activity coefficient kilobase (thousand base pairs) kiloDalton 2-keto-3-deoxy-nononicacid, formally 5-desamino-5hydroxyneuraminic acid 3-deoxy-~-y~ycero-D-gu~ucto-2-nonulosonic acid ketodeoxoctulosonic acid (3-deoxy-~-mannooctulosonic acid) 2-keto-3-deoxy-~-manno-octulosonic acid kinase enzyme activity coefficient, at equilibrium kinase enzyme activity coefficient, for inhibition kifunensine kinase enzyme activity coefficient, for structural modification(s) keratan sulfate keratan sulfate proteoglycan
LXXII Abbreviations Used in Volumes 1 and 2 LacAm LacCer LacNAc LacNAc LAD LAMP LBP Lc3 LDA Lea Lea Leb Lea, LeX LeX LeX Ley Leu Lev LNP LPS LRP LRR LUMO LYS mAb MAd MAF MAG MALDI-TOF MALDI-TOF Man ManNAc MAP MASP Man-T MBL MBP MBz MCD MDBK MDCK ME MEM MHC MK
lactosamine GalP 1+4GlcP1+ 1Cer N-acetyllactosamine N-acetyllactosamine leucocyte adhesion deficiency lysosomal associated membrane protein lipopolysaccharide-binding protein GlcNAcPl-.3Gal~1+4GlcP1+ 1Cer Lithium diisoprypropylamide GalP 1+3[Fucal+4]GlcNAcP1+3Gal~1+GlcP P-~-Gal(l-3)-[a-~-Fuc( 1-4)]-~-GlcNAc Fuca 1+2GalP 1+3[ Fucal-+4]GlcNAc~ 1+3GalP 1-4GlcP Lewis a, Lewis x determinants p-D-Gal(1-4)-[a-~-Fuc( 1-3)]-~-GlcNAc GalP 1+4[ Fucal+3]GlcNAcP 1+3GalP 1-+4GlcP Fuca 1-2GalP 1+4[Fucal+3]GlcNAcP 1+3GalP 1-4ClcP leucine levulinoyl lectin and nucleotide phosphohydrolase lipopolysaccharaide lipoprotein receptor-related protein leucine-rich repeat (amino acid/protein) lowest unoccupied molecular orbital lysine monoclonal antibody mucosal addressin Microciona prolifera aggrigation factor myelin-associated glycoprotein mass-analyzed laser desorption ionization-time of flight matrix-assisted-laser desorption ionization time of flight mass spectrometry mannose N-acetylmannosamine microtubule-associated protein MBP-associated serine protease mannosyltransferase mannose-binding lectin mannose-binding protein p-methoxybenzoyl macular corneal dystrophy Madin-Darby bovine kidney (cells) Madin-Darby canine kidney (cells) methoxyethyl methoxyethoxymethyl major histocompatibility complex myokinase
Abbreviations Used in Volumes 1 and 2 LXXIII mol.wt. MP MPEG MPR MPS M, MS MSD MTr Mu MUC NA NAc nAChR NAm NANA N-BOC NBS NCAM NCS N DP NDP NDST NEM 3NeuAcCgOse4 Neu5Ac NeuAc NGF NIH NIS NK nLc4 Nle NMK NMP NMR NOE NPG NS NTP Nu OD (2-OH)-Lea (2-OH)-Le" (2-OH)-sLea
molecular weight p-methoxyphenyl polyethylene glycol monomethyl ether mannose 6-phosphate receptor mucopolysaccharidosis apparent molecular weight mass spectrometry multiple sulfatase deficiency, Austin disease p-methoxytrityl 4-Methylumbelliferone mucin N-acetylated disaccharide unit N-acetyl nicotinic acetylcholine receptor neuraminidase N-acetylneuraminic acid N-tert-butoxycarbonyl N-bromosuccinimide neural cell adhesion molecule N-chlorosuccinimide nucleoside diphosphates nucleoside-diphosphate N-deacetylaselN-sulfotransferase N-ethylmaleimide p-~-Gal(1-3)-o-~-GalNAc( 1-4)-[a-~-NeuAc(2-3)]-P-DGal( 1-4)-D-Glc N-acetylneuraminic acid N-acetylneuraminic acid nerve growth factor National Institutes of Health N-iodosuccinimide natur a1 killer Gal~l+4GlcNAc~1+3Gal~1-t4Glc~1-+ 1Cer Norleucin nucleoside monophosphate kinase nucleoside monophosphate nuclear magnetic resonance nuclear Overhauser effect n-pentenyl glycoside N-sulfated disaccharide unit nucleoside triphosphate nucleophile optical density P-D-Gal(1-3)-[a-~-Fuc( 1-4)I-D-Glc p-D-Gal(1-4)-[a-~-Fuc(1-3)I-D-Glc a-NeuAc(2-3)-P-~-Gal(1-3)-[a-~-Fuc(1-4)]-~-Glc
LXXIV Abbreviations Used in Volumes I and 2 (2-OH)-sLe” OR ORF OSP OST OST P PA Pac PAMP PAPS PAPS PARP PBL pClP PCR PDGF PDI PEG PEGA PEP PEP PfP
pmu
PG PGM PGM Ph PhSeOTf PhSOTf Phth PI Piv PK PMB pNP-a-Man PNS PNZ POEPOP Ppase ppGaNTases PPK PRP PSA PSGL
a-NeuAc(2-3)-P-~-Gal(1-4)-[a-~-Fuc( 1-3)]-~-Glc dimethyl(methy1thio)sulfonium trifluoromethanesulfonate open reading frame 0-specific polysaccharides oligosaccharyltransferase N-oligosaccharyltransferase phosphate 2-Aminopyridine phenacyl pathogen-associated molecular pattern) 3 ‘-phosphoadenosine 5’-phosphosulfate phosphoadenosine phosphosulfate procyclic acidic repetitive protein peripheral blood leucocytes para-Chlorophenyl polymerase chain reaction platelet-derived growth factor protein disulfide isomerase poly(ethy1ene glycol) polyethylene glycol N,N-dimethylacrylamide copolymer phospho(eno1)pyruvate phosphoenolpyruvate pentafluorophenyl N,N,N’,N’-bis(tetramethylene)-O-pentafluorophenyluronium hexafluorophosphate phosphatidylglycerol phosphoglucomutase phosphoglucomutase phenyl Phenylseleniumtriflate Phenylsulfoniumtriflate phthaloyl phosphatidylinositol pivaloyl pyruvate kinase p-methoxybenzyl p-nitropheny1-a-D-mannoside peripheral nervous system p-nitrobenzyloxycarbonyl poly oxyethylene-polyoxypropylene pyrophosphatase UDP-Ga1NAc:polypeptide N-acetylgalactosaminyltransferases polyphosphate kinase proline-rich protein polysialic acid P-Selectin glycoprotein ligand
Abbreuiutions Used in Volumes I and 2 LXXV PSGL-1 PSGL- 1 PTG PYr 9.Y. RA RAr RACE rER RHAMM ROESY RT-PCR SA SAP SAT SBA SBu SDS SDS-PAGE Se SE Ser SEt SGC SGG SGGL SGLT sia sLea sLea, sLe' sLe' 6'-SLN SLRP SMC SP SPh SPH SSEA ST ST3 ST6 STF-GP SV
sw
P-selectin glycoprotein ligand-1 P-selectin glycoprotein ligand-1 peptidoglycan pyridine quantitative yield retinoic acid rheumatoid arthritis rapic amplification of cDNA ends rough endoplasmic reticulum receptor for hyaluronan-mediated motility rotating-frame nuclear Overhauser effect reverse transcription polymerase chain reaction sialyl saposin sialyltransferase soybean agglutinin Thiobutyl sodium dodecyl sulfonate sodium dodecylsulfate-polyacrylamide gel electrophoresis secretor enzyme 2-(trimethylsily1)ethyl serine Thioethyl sulfogalactosyl ceramide sulfogalactosyl glycerolipid sulfoglucuronyl glycolipid sodium-glucose transporter sialic acid a-~-NeuAc(2-3)-P-~-Gal( 1-3)-[ a-~-Fuc( 1-4)]-~-GlcNAc(sialyl Lewisa trisaccharide) sialylated Lewis a, sialylated Lewis x determinants a-~-NeuAc(2-3)-P-~-Gal( 1-4)-[a-~-Fuc( 1-3)]-~-GlcNAc(sialyl Lewis' trisaccharide) 6'4alyl-LacNAc small leucine-rich proteoglycan sialomucin complex spacer Thiophenyl saccharide-peptide hybrids stage specific embryonic antigen sulfurotransferase recombinant rat liver a-(2-3)-sialyltransferase [EC.2.4.99.6] rat liver a-(2-6)-sialyltransferase jEC.2.4.99.11 (NeuAcGalGlcNAcMan)2ManGlcNAqAsn synaptic vesicle-associated protein swainsonine
LXXVI Abbreviations Used in Volumes 1 and 2 TATU TBAF TBAI TBDMS TBDMSOTf TBDPS TBDPSCl TBTU Bu TCA Tcoc TCP TCR TDS TEMED Teoc TES TESOTf Tf20 TFA TFDMSCl TfOH TGF TGN THF Thr tlc TM TMD TMS TMSCN TMSN3 TMSOTf to1 Tos t-PA TriBz Troc Trt Ts UDP UDP UDP-Gal UDP-Gal
0-(7-azabenzotriazol-1-yl)-3-tetramethyluronium tetrafluoroborate Tetrabutylammoniumfluoride Tetabutylammoniumiodide tert-butyldimethylsilyl tert-butyldimethylsilyl trifluoromethanesulfonate (tert-butyldimethylsilyl triflate) tert-butyldiphenylsilyl tert-butyldiphenylsilylchloride 0-(benzotriazol- 1-yl)-N,N,N',N'-tetrauroniumtetrafluoroborate tert-butyl trichloroacetyl trichloroethyloxycarbonyl tetrachlorophthaloyl T cell receptor thexyldimethylsilyl tetramethylethylenediamine 2,2,2-trichloroethoxycarbonyl triethylsilyl triethylsilyl trifluoromethanesulfonate (triethylsilyl triflate) trifluoromethane sulfonic acid anhydride trifluoroacetic acid trifluoromethyldimethylsilylchloride trifluoromethanesulfonic acid (triflic acid) transforming growth factor trans Golgi network tetrahydrofuran threonine thin layer chromatography transmembrane trans-membrane domain trimethylsilyl trimethylsilyl cyanide trimethylsilyl azide trimethylsilyl trifluoromethanesulfonate (trimethylsilyl triflate) toluene p-toluenesulfonyl (tosyl) tissue plasminogen activator factor tribenzoyl
2,2,2-trichloroethoxycarbonyl triphenylmethyl (trityl) p-toluenesulfonyl (tosyl) uridine 5'-diphosphate uranyl diphosphate UDP-a-D-galactose uridine 5'-diphosphate-galactose
Abbreviations Used in Volumes I and 2 UDPGE UDP-Glc UDPGP UMD UMP UTP
uv vc
VE VL VSG
vsv
VT vWF WBP XYl XY12 Z ZP
LXXVII
uridine 5'-diphosphate-glucose epimerase [EC.5.1.3.2.] uridine 5'-diphosphate-glucose UDP-Glc pyrophosphorylase unmodified domain uranyl monophosphate uranyl triphosphate ultraviolet vitelline coat vitelline envelope vitelline layer variant surface glycoprotein vasicular stomatis virus verotoxin von Willebrand factor wheat germ agglutinin binding protein xylose p-D-xyl(1--4)-D-xyl benzyloxycarbonyl zona pellucida
Abbreviations Used in Volumes 3 and 4
A" aa ACP AGP 4 7
APC APP APS AR ARIS ASGP-R Asn Asn GT BHK bFGF BLAST BM BMP bP CAM CAT CCCP CD CDGS CDTA Cer CHO CI CMLN CMP
Angstrom amino acid acyl carrier protein arabinogalactan protein asparagine-linked glycosylation antigen-presenting cell acute phase protein adenosine phosphosulfate acrosome reaction acrosome reaction-inducing substance asialoglycoprotein receptor asparagine asparagine glucosyltransferase baby hamster kidney basic fibroblast growth factor basic alignment search tool basement membrane bis(monoacy1glycero)phosphate base pair cell adhesion molecule chloramphenicol acetyltransferase carbonyl cyanide m-chlorophenylhydrazone cation dependent carbohydrate-deficient glycoprotein syndrome diaminocyclohexane tetraacetic acid ceramide Chinese hamster ovary cation independent calf mesenteric lymph node cytosine monophosphate
LVIII
Abbreviations Uscd in Volumes 3 and 4
CNS CoA CRD CS CSPG CST CTL Da DAF DAG DBA DIG DIM DMJ DMSO DNJ Do1 Dol-P Dol-PP DP DRM DS ECM EDTA EGF eIF EM endo H EPGase ER EST EtN EtN-P F FAK FCOE FGF FSP FTIR Fuc FucT GA GAG Gal GalNAc GalT
central nervous system coenzyme A carbohydrate-recognition domain chondroitin sulfate chondroitin sulfate proteoglycan castanospermine cytotoxic T cell Dalton decay accelerating factor diacylglucosamine ((strain D x strain B) x strain A) mice detergent-insoluble glycolipid complex 1,4-dideoxy-l,4-imino-~-mannitol 1-deoxymannojirimycin dimethyl sulfoxide deoxynojirimycin dolichol dolichyl phosphate dolichyl pyrophosphate degree of polymerization detergent-resistant membrane dermatan sulfate extracellular matrix ethylene diamine tetraacetic acid epidermal growth factor eukaryotic initiation factor electron microscopy endo-p-N-acetylglucosaminidaseH endopol y galacturonase endoplasmic reticulum expressed sequence tag ethanolamine ethanolamine phosphate fusion protein focal adhesion kinase fluorophore-coupled oligosaccharide electrophoresis fibroblast growth factor fucose sulfate polysaccharide Fourier transform infra-red (spectroscopy) fucose fucosyltransferase Golgi apparatus glycosaminoglycan galactose N-acetylgalactosamine galactosyltransferase
Ahbreniutions Used in Volumes 3 and 4
GC GC-MS GDP GI GI1 Glc GlcA GlcN GlcNAc GLUT GnT GP GPI GRIFIN GRP GSL GT GTA GTB HA H Ag HAS HBP HEMPAS hCG HeP HEV HIV HL HMG HN HPLC HRGP HS HSD HSPG HUS ICAM lduA IGF Ig IgSF IL IPG
1u
LIX
glucosyl ceramide gas chromatography-mass spectrometry guanosine diphosphate gastro-intestinal glucosidase I1 glucose glucuronic acid glucosamine N-acetylglucosamine glucose transporter N-acetylglucosaminy ltransferase glycoprotein glycosyl phosphatidylinositol galectin-related interfiber protein glycine-rich protein glycosphingolipid UDP-G1c:glycoprotein glucosyltransferase human blood group A glycosyltransferase human blood group B glycosyltransferase hyaluronic acid, hyaluronate hemagglutinin hyaluronate synthesizing enzyme hexosamine biosynthetic pathway hereditary erythroblastic multinuclearity with a positive acidifiedserum-lysis test (congenital dyserythropoietic anemia type 11) human chorionic gonadotropin heparin high endothelial venules human immunodeficiency virus hepatic lectin high mobility group (proteins) hemagglutinin-neuraminidase high performance liquid chromatography hydroxyproline-rich glycoprotein heparan sulfate highly sulfated domain heparan sulfate proteoglycan hemolytic uremic syndrome intercellular adhesion molecule iduronic acid insulin-like growth factor immunoglobulin immunoglobulin superfamily of proteins interleukin inositol phosphoglycan international unit
LX
K kb kDa Kdn
Abbreviations Used in Volumes 3 and 4
kinase enzyme activity coefficient kilobase (thousand base pairs) kiloDalton 2-keto-3-deoxy-nononic acid, formally 5-desamino-5-hydroxyneuraminic acid 2-keto-3-deoxy-~-manno-octulosonic acid Kdo kinase enzyme activity coefficient, at equilibrium Keq kinase enzyme activity coefficient, for inhibition Ki kifunensine KIF kinase enzyme activity coefficient, for structural modification(s) Km keratan sulfate KS keratan sulfate proteoglycan KSPG lactosamine LacAm N-acetyllactosamine LacNAc leucocyte adhesion deficiency LAD lysosomal associated membrane protein LAMP lipopolysaccharide-binding protein LBP Lewis a, Lewis x determinants Lea, Le" leucine Leu lectin and nucleotide phosphohydrolase LNP lipopoly saccharaide LPS lipoprotein receptor-related protein LRP leucine-rich repeat (amino acid/protein) LRR lysine LYS monoclonal antibody mAb mucosal addressin MAd myelin-associated glycoprotein MAG MALDI-TOF matrix-assisted-laser desorption ionization time of flight inass spectrometry mannose Man microtubule-associated protein MAP MBP-associated serine protease MASP mannose-binding lectin MBL mannose-binding protein MBP mannosyltransferase Man-T macular corneal dystrophy MCD Madin-Darby bovine kidney (cells) MDBK Madin-Darby canine kidney (cells) MDCK major histocompatibility complex MHC molecular weight mol.wt. mannose 6-phosphate receptor MPR mucopolysaccharidosis MPS apparent molecular weight Mr multiple sulfatase deficiency, Austin disease MSD N-acetylated disaccharide unit NA N-acetyl NAc
Ahhreviutions Used in Volumes 3 and 4
nAChR NAm NANA NCAM NDP NDST NEM NGF NIH NK NMR NS ORF OST P PAMP PAPS PARP PRL PCR PDGF PDI PEP PG PGM PI pNP-a-Man PNS PRP PSA PSGL-1 PTG RA RAr RACE rER RHAMM RT-PCR SA SAP SAT SBA SDS-PAGE Se Ser SGC
nicotinic acetylcholine receptor neuraminidase N-acetylneuraminic acid neural cell adhesion molecule nucleoside-diphosphate N-deacetylaselN-sulfotransferase N-ethylmaleimide nerve growth factor National Institutes of Health natural killer nuclear magnetic resonance N-sulfated disaccharide unit open reading frame N-oligosaccharyltransferase phosphate pathogen-associated molecular pattern phosphoadenosine phosphosulfate procyclic acidic repetitive protein peripheral blood leucocytes polymerase chain reaction platelet-derived growth factor protein disulfide isomerase phosphoenolpyruvate phosphatidylglycerol phosphoglucomutase phosphatidylinositol p-nitrophenyl-a-D-mannoside peripheral nervous system proline-rich protein polysialic acid P-selectin glycoprotein ligand-1 peptidoglycan retinoic acid rheumatoid arthritis rapic amplification of cDNA ends rough endoplasmic reticulum receptor for hyaluronan-mediated motility reverse transcription polymerase chain reaction sialyl saposin sialyltransferase soybean agglutinin sodium dodecylsulfate-polyacrylamide gel electrophoresis secretor enzyme serine sulfogalactosyl ceramide
LXI
LXII
Abbreviations Used in Volumes 3 and 4
SGG SGGL SGLT sia sLea, sleX SLRP SMC ST
sv
SW TCR TGF TGN Thr tlc TM t-PA UDP UDP-Gal UMD UMP UTP UV
vc
VE VL VSG
vsv
VT vWF WBP XYl ZP
sulfogalactosyl glycerolipid sulfoglucuronyl glycolipid sodium-glucose transporter sialic acid sialylated Lewis a, sialylated Lewis x determinants small leucine-rich proteoglycan sialomucin complex sulfurotransferase synaptic vesicle-associated protein swainsonine T cell receptor transforming growth factor trans Golgi network threonine thin layer chromatography transmembrane tissue plasminogen activator factor uranyl diphosphate UDP-a-D-galactose unmodified domain uranyl monophosphate uranyl triphosphate ultraviolet vitelline coat vitelline envelope vitelline layer variant surface glycoprotein vasicular stomatis virus verotoxin von Willebrand factor wheat germ agglutinin binding protein xylose zona pellucida
Part I
Volume 1
I Chemical Synthesis of Glycosides
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
1 Introduction to Volumes 1 and 2
As no better man advances to take this matter in hand, I hereupon offer my own poor endeavours. I promise nothing complete; because any human thing supposed to be complete must for that very reason infallibly be faulty. (from Moby Dick, Herman Melville)
Our understanding of the relationship between carbohydrate structure and biological function is still far behind that of proteins and nucleic acids. Initially, carbohydrates were only recognized as structural and energy storage molecules (e.g. cellulose, chitin and glycogen). The development of novel tools to study carbohydrate function has permitted additional biological functions to be elucidated. Their roles in protein folding, cell signaling, fertilization, pathogen binding to host tissue, leukocyte trafficking and associated inflammatory responses, tumor cell metastasis, and regulation of hormone and enzyme activities are just a few selected examples. These findings led to an immense interest in the preparation of oligosaccharides and glycoconjugates as well as the understanding of their interaction with their natural receptors. Part 1 of Carbohydrates in Chemistry and Biology is designed to be a valuable collection of chemical and enzymatic methods for the synthesis of oligosaccharides. It also covers state-of-the-art knowledge of the interactions of carbohydrates with their natural receptors. Each of the chapters is written by a leading expert in the field and covers an impressive body of references. Volume 1 of the series begins with the most challenging aspect of synthetic carbohydrate chemistry, namely the stereospecific formation of glycosidic bonds. A number of chapters describe recently developed approaches and cover numerous significant advances in glycosidation techniques. Other chapters review the attachment of sugars through linkages not occurring in nature. Volume 1 concludes by summarizing one of the most exciting and rapidly evolving area in modern carbohydrate synthesis: the use of glycosyl transferases and glycosidases in the synthesis of oligosaccharides and their analogs.
4
1 Introduction to Volumes 1 and 2
Volume 2 of the series covers the interactions of carbohydrates with their natural receptors, i.e. proteins, nucleic acids, and other carbohydrates. Whereas carbohydrate-protein interactions are well documented, the investigation of carbohydratenucleic acid and carbohydrate-carbohydrate interactions is an emerging area. The Carbohydrates in Chemistry and Biology series provides a comprehensive and up-to-date compilation of information on the fields of glycochemistry (Volumes 1 and 2) and glycobiology (Volumes 3 and 4),available today. The volumes not only provide an expert overview of the entire field, but also serve as a valuable reference tool to both, established investigators and students in many disciplines. The editors are obliged to all authors for their excellent contributions. Special thanks goes to Dr. Anette Eckerle, Wiley-VCH, who managed the realization of this series. Beat Ernst, Ph.D. Professor of Molecular Pharmacy University of Base1 Pierre Sinay, Ph.D. Professor of Organic Chemistry Ecole Normale SupCrieure, Paris
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
2 Trichloroacetimidates Richard R. Schmidt and Karl-Heinz Jung
2.1 Introduction Glycoside bond formation generally requires activation of the sugar at the anomeric center. To this end, activation through anomeric oxygen-exchange reactions (the classical Koenigs-Knorr method and its modifications, sulfur based activations, etc.) or, alternatively, activation through retention of the anomeric oxygen (trichloroacetimidate method, phosphite and phosphate activation, etc.) have been proposed. The trichloroacetimidate method is well established [ 1-1 31 and although the first paper on this method was published as recently as 1980 [ 141 it has already become a classical method. This is essentially because only catalytic amounts of a promoter are required to provide very high glycosyl donor properties whereas the anomeric oxygen-exchange based methods generally require at least equimolar amounts of a promoter system, which is obviously often associated with disadvantages of various kinds. The basic principle of the trichloroacetimidate method [ 11 consists in a simple base-catalyzed addition of trichloroacetonitrile to the anomeric hydroxy group to generate the 0-glycosyl trichloroacetimidate (see Scheme 1). This reaction is generally high-yielding and, because of its reversibility, high anomeric control can often be achieved; in addition, competing reactions with non-anomeric hydroxy groups are not observed. In the second step, under mild acid/Lewis acid catalysis glycosylation of various nucleophiles (for instance, of alcohols, etc.) can be readily achieved; in general high yields and high anomeric control are obtained. The anomeric control is derived from the glycosyl donors’ anomeric configuration (inversion or retention), from anchimeric assistance, from the influence of the solvent, or from thermodynamic or kinetic effects [ 1- 131. Since 1980 (up to the end of 1998), application of the trichloroacetimidate method has been reported in more than 1100 papers and over 1200 different 0glycosyl trichloroacetimidates have been described in the literature. Obviously, the wealth of this chemistry cannot be compiled in a relatively short book chapter. A
6
*OH
2 Trichloroacetimidates
.
Base Catalysis
CI&
Reaction with CI&-CN not observed
- C - NH, .
:: t
Mild Aci? Catalysis
4 +
+OyNH
CCI,
HOR
Scheme 1.
quite comprehensive review [ 71 on the trichloroacetimidate method appeared in 1994, and this chapter is, therefore, essentially devoted to the literature since 1994. Of 686 references (1994-1998) on the use of the trichloroacetimidate method, because of space limitation, only ca 220 references are considered in this chapter. The typical examples selected illustrate the application of this method in different areas.
2.2 Methods The reported methods [ 141 for the formation and reaction of trichloroacetimidates are well established. Use of sodium hydride or cesium carbonate as a base for the reaction of 1-0-unprotected sugars with trichloroacetonitrile yields generally the thermodynamically favored 0-a-glycosyl trichloroacetimidates, whereas use of potassium carbonate yields 0-p-glycosyl trichloroacetimidates because of kinetic reaction control. Use of 1,8-diazabicyclo[5.4.01undec-7-ene (DBU) often yields a/p mixtures, mostly favoring the a-trichloroacetimidates. Recently, application of new base systems has been reported using 7-methyl-l,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD) [ 151 or phase transfer catalysis (50% potassium hydroxide, dichloromethane, tetra-n-butylammonium hydrogen sulphate) which yield exclusively atrichloroacetimidates in very good yields (96%) [ 161. Besides the well established catalysts for glycosylation reactions with trichloroacetimidates, some new catalysts have been investigated. The use of silver trifluoromethanesulfonate in the reaction with phthalimido-protected glucosamine trichloroacetimidate gave higher yields and smaller amounts of a-glucoside than the use of boron trifluoride etherate; this procedure also avoids acyl migration in the acceptor molecule; but stoichiometric amounts of the promoter are required [ 17,
2.3 Q-Glycosides
7
IS]. Use of lithium perchlorate in dichloromethane requires a large excess of promoter [ 19, 20 1, but with lithium trifluoromethanesulfonate only 0.05 equivalents are required [21]. Both catalysts give high yields, but the a/P selectivity is low and has to be improved before general application is possible. The synthesis of other 0-glycosyl imidates, i.e. O-glucopyranosyl dichlorocyanoacetimidates, 2,3,5,6-tetrafluoropyridin-4-y1 glucopyranosides, etc., is under investigation [22, 231 thus variations in reactivity and selectivity are expected.
2.3 O-Glycosides 2.3.1 Synthesis of Oligosaccharides
P-Glucosides, P-Galactosides, a-Rhamnosides, etc. 1,2-trans-glycosides (P-glucosides, 0-galactosides, P-xylosides, a-mannosides, arhamnosides, etc.) are usually prepared with glycosyl donors containing participating neighboring groups (acetyl, benzoyl, pivaloyl, etc. ) at 2-OH. Some examples for Galp(1-3)Gal [24], GalP(1-3)GalN [25], and ~-Rhaa(l-2)Glc[26] are shown in Scheme 2. Instead of the methyl glycoside of azidogalactose, the corresponding glycosyl fluoride has also been used as acceptor [27]. In general glycosylation yields are high, irres,pective of the anomeric configuration of the trichloroacetimidate, and only one anomer is obtained. Occasionally, when glycosyl acceptors with more than one free OH group are used, high regioselectivity is obtained because of the different reactivity of the OH groups. It was found that trichloroacetimidate donors are especially appropriate for deactivated (fluorine containing) donors in contrast to other glycosyl donors (thioglycosides, etc.) [28]. A highly convergent synthetic method has been developed for the synthesis of cellooctaose and other cello-oligosaccharides via di- and tetrasaccharides which can be converted either into glycosyl donors by deallylation and transformation into the trichloroacetimidates, or into acceptors by deacetylation (Scheme 3) [29].The use of p-methoxybenzyl groups (PMB) instead of acetyl groups was less successful. The combination of acetyl, pivaloyl, and benzyl groups leads to the proper balance between stability and reactivity of trichloroacetimidates as donors and the acceptor substrates. High yields were achieved by using a specially developed high-vacuum system for glycosylations under anhydrous conditions. Trichloroacetimidates have also been used for the glycosylation of more complex carbohydrate acceptors (Scheme 4). Galp( 1-3)GlcN and other disaccharides were synthesized and converted into fluorescein-labeled acceptors which are used for sialyltransferase assays [301. Glucosyl diosgenine has been used as carbohydrate acceptor for further glycosylations with various trichloroacetimidates (Scheme 4) [31, 321. All glycosides were obtained in high yields and high stereoselectivity because of participating neighboring groups at 2-OH in the synthesis of dioscin, polyphyllin D, balanitin 7, and gracillin.
8
2 Trichloroacetimidates
Baw Bzo
OBI
+
BZO
Hr&
OCH3
75%
OBZ
BzO
OBz
HO
OBZ
GalP(1-3)Gal OBZ
Ph
OAc
OBz
BZO~ O ! & & O C H ,
Aug6 et al. 1241
O YCCI, NH
AcO
.
CH TMSOTf CI ,O°C
Ph
TMSOTf CICH,CH,CI,,
-1 8%
Ga@(1-3)GalN
AcO OAc
Bock et al. [251
CCI,
N,
CCI, Ad)
OANH
BFS. OEtz A
c O AcO
H
+
0 OAll
CH,CI,, -20°Cb
AcO OAll
OAc
L-Rhaa(1-2)Glc
95% Liu et al. "261
A c O AcO R q
+
6
HO BnO
0
PhtN
I
O YCCI, NH
R = OAC, H, F
R
I cot
TMSOTf CH2C12,00C CHzCIz,OoC
*
93%
93%
A
C
~ W AcO
~
Karnerling et al. [281
,OBn
O
-
~ PhtN
I
BZ& BnO BnO
0C8H17
Scheme 2.
Aminosugar Trichloroacetimidates The use of azidosugars and N-phthalimido-protected aminosugars is well established in carbohydrate chemistry. But many new protective groups have been proposed to enable deprotection under milder conditions or to apply orthogonal protection strategies (Scheme 5). For the synthesis of P-glycosides of aminosugars, protective groups with amide structure are mainly used; these are capable of neighboring group participation. But formation of stable oxazolines reduces the reactivity of the glycosyl donors, and phthaloyl groups are therefore preferred. They can be cleaved only under strongly basic conditions, for instance, with hydrazine. As an alternative, the use of the tetrachlorophthaloyl (TCP) group has been proposed [33];this can be more readily removed with ethylenediamineor with sodium borohydride followed by acid treatment (pH 5). The glycosylation reaction gives excellent yields and in acetonitrile as solvent exclusively the P anomers are obtained. Further applications of TCP-protected trichloroacetimidates are shown below (glycosyl amino acids, C-glycosides).
2.3 0-Glycosides
9
OAll
1. Se02IAcOH
bpiv
OPiv
HBnOo
~
O A OPiv
I
I
BnO OPiv
/
disaccharide-
OPiv
\
disaccharide-
BnO
OPiv
OPiv
OPiv
tetrasaccharideimidate
OPiv
tetrasaccharideacceptor CH&,
O°C
Nishimura, Nakatsubo 1291
OPlV ACO BnO
OPlV
BnO OPlV
OPlV
OPlV
OPlV
OPlV
Scheme 3.
Similar properties have been observed for 4,5-dichlorophthaloyl (DCPhth) protected trichloroacetimidates [ 341. Deprotection can be performed with ethylenediamine. The trichloroethoxycarbonyl (Teoc, Troc-both abbreviations are in use) group has already been applied successfully to aminosugars [35].Because it can be cleaved under reductive conditions (with zinc) it can be used for base-labile compounds and more importantly for demanding orthogonal protection strategies. For glycosylation reactions with N-Teoc-protected trichloroacetimidate-activated aminosugars very high yields and stereoselectivities are reported [36]. Application was also shown for the synthesis of some complex Lipid A analogs [37-391. Glycosylation of acceptors with Teoc-protected trichloroacetimidates, both containing long chain fatty acid residues, was performed in high yields and stereoselectivities. Further applications (glycolipids, glycopeptides, bacterial polysaccharides, solid-
10
2 Trichloroucetimidates
HO HO&
"Po AcHN
BF,.OEt,
+
CCI,
CH~CI, 76%
-
1. deprotection
HO
OH
2. fluorescein isothiocyanate
O(CH,),NHCbz NHAc
Slim et al. [301
HO
CCI,
AAcOc O ~ 6 ; * o I c c , , OAc
AcO
OH
OAC CCI,
L-Rhaa(l-2)GlcP(l-O)diosgenine (dioscin)
Xyl~(l-3)Glc~(1-2)Glc~(l-O)diosgenine (balanitin 7)
L-Araa(1-2)GlcP(1-0)diosgenine (polyphyllin D)
Glc~(1-3)Glc~(l-O)diosgenine
I
Rhaa(1-2)
(gracillin)
Hui et al. [31,321
Scheme 4.
phase synthesis) of Teoc-protected trichloroacetimidates are shown below. Similar results were obtained with p-nitrobenzyloxycarbonyl (PNZ) protected trichloroacetimidates [40]. Deprotection can be performed under reductive conditions either with hydrogen and palladium-on-carbon or with sodium dithionite under neutral or slightly alkaline conditions. If protective group manipulations are incompatible with basic or reductive conditions, use of the 2,5-dimethylpyrrole group is proposed; this can be treated with hydroxylamine hydrochloride to liberate the amino group [41, 421. Glycosylations with dimethylpyrrole containing trichloroacetimidates perform well, leading selectively to P-glycosides (1,2-trans-glycosides). Recently the dimethylmaleoyl (DMM) group has been proposed as an amino-protecting group which can be cleaved under mild aqueous basic and then acidic conditions (pH 5 ) [43]. Because of the anchimeric assistance and the electron-withdrawing character of the DMM group the corresponding trichloroacetimidates give stereoselectively P-glycosides in high yields. The dithiasuccinoyl (Dts) group [44] for amino group protection is discussed below (glycopeptides).
11
2.3 0-Glycosides BnO A AcO c o ~ O y C C l , +
TCPN
OH
n BnO O OBn q CH,CN, 91% r.t.
B
NH
81%
BnO 0
BnO
CI OBn
TCP = tetrachlorophthaloyl,
Castro-Palomino, Schmidt [33]
CI
0
CI
1. TMSOTf HO DCPhthN
73%
OMP
BnO DCPhthN
NH
0
OBn
AcNH
3. AcpO, pyridine 84%
wz:
DCPhth = 4,5-dichlorophthaloyl,
b
2.ethylenediamine
Ogawa et al. [34]
0 BnO
--,
0
Teoc = trichloroeihoxycarbonyl,
-k!
R = (S)-3-(tetradecanoyloxy)tetradecanoyl = (S)-3-(dodecanoyloxy)tetradecanoyl R" = (S)-3-(benzyloxy)tetradecanoyl
-OCH,CCI,
(Troc)
% % ;A
+
PNZ'
BnO
1. TMSOTf
NH
1. BF,.OEt, CHZCI,, -3OC
&
HO BnO
2. NaOMe, 79% quant.
OBn
OBn
'yNH
.
3. Na,S,O, quant. 4. Ac,O, pyr., 93% C
H NO,
OBn
AcNH
0
CCl,
PNZ = p-nitrobenzyloxycarbonyl, - - O
Kusumoto et al. [37-391
Quian, Hindsgaul [40]
~
1. TMSOTf
CCI,
--
OCH,
~~~&o,cc13 DMMN
6
HO BnO
AcO
NH
DMM = dimethylmaleoyl,
OBn
+
1. TMSOTf C 78% H,CI,
2.NaOH 3.HCI 4. Ac,O 97%
0 $CH3 CH,
Scheme 5.
OBn
CHZCI,, -25C 78% 2. NEt,,MeOH, H,Ob 3. NH20H, 8OC 4. Ac,O, pyr.
r. t.
.
AcO
BnO OCH.
Boons et al. [41]
AEoa:o+OBn AcNH
OBn
Schmidt et al. [43]
12
2 Trichloroacetimidates CONH,
CONH,
ACO
31% OAc 0
Welzel et al. 1461
TMSOTf CH,Cldhexane, 60%
r.?
Bnd
Scheme 6.
Finally, the synthesis of moenomycin-type analogs is described (Scheme 6). Use of the trichloroacetyl protecting group for aminosugar trichloroacetimidates as previously proposed [45] gave the desired disaccharide (31% yield) [46]. Because of silyl group migration under the glycosylation conditions, the tri-TBS-protected acceptor, the diglycosylated acceptor, and the oxazolidine of the donor were also formed as by-products. The trichloroacetyl group can be converted into the acetyl group either with zinc-acetic acid or with tributyltin hydride in the presence of AIBN. Better results were obtained with a 2-(trimethylsilyl)ethanesulfonyl-protected trichloroacetimidate [47].The desired P-glycoside was obtained stereospecificallyin 60% yield and further transformations are under investigation. Trichloroacetimidate derivatives for the preparation of aminosugar a-glycosides (1,2-cis-glycosides)are rare. The use of azidosugars is well established (examples are given below), but unfortunately they are not readily available. Azidoglucose, for example, is prepared either from glucose via glucal by azidonitration [48]or from glucosamine via diazo transfer with trifluoromethanesulfonyl azide [49]. The N-l(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) group has been proposed as non-participating neighboring group for the synthesis of 1,2-cis-glycosides(Scheme 7) PI.
,OAc
Kellam et al. [501
ACO
DdeNH
~~
. .. .
0
Dde = N - l - ( 4 , 4 - d i m e t h y l - 2 , 6 ~ 1 x ~ c y c l ~ h e x ~ ~ ~ e ~ e ~ ~ ~ h y Scheme l a e n e ) e7.t 0
2.3 0-Glycosides
13
65%
CCl,
Furstner, Konetzki 1541
Scheme 8.
P-Mannosides
The synthesis of p-mannosides is crucial because p-mannoside formation is disfavored because of stereoelectronic and steric reasons. Besides the already known direct mannosylation reactions some indirect approaches which have been reported are based on the preparation of I)-glucosides and then inversion of the configuration at C-2 to yield the corresponding P-mannosides (Scheme 8). According to a previously introduced method [ 5 11, a p-glucoside, synthesized using a trichloroacetimidate as glycosyl donor, was converted into its 2-0-triflate [ 521. Intramolecular nucleophilic substitution afforded a Mano( 1-4)GlcNp( 1-4)GlcN trisaccharide occurring in the core-region of glycoproteins. In other examples 2-0-triflate intermediates were subjected to intramolecular nucleophilic substitution under inversion of the configuration by reaction with tetrabutylammonium nitrite [53] (see below, Scheme 26) or with tetrabutylammonium acetate [54]. The reaction has been significantly improved by means of ultrasound, but very good yields have been also obtained previously via a sulfonyl imidazylate [55]. 2-Deoxyglycosides
Direct glycosylation with 2-deoxyglycosyl donors is not employed because the corresponding glycosyl donors are quite labile and they lack stereocontrol by anchimerically assisting groups. A recent application of a previously developed method [56] has been reported; it uses a 2-deoxy-2-thiophenylglucosyltrichloroacetimidate which can be readily prepared from glucal (Scheme 9) [57]. After glycosylation the thiophenyl group can be removed by reduction methods. Because glucal is not readily available as starting material and the stereoselectivity of the glycosylation of carbohydrate acceptors is critically dependent on substituent X
14
2 Trichloroacetimidates
ROH, TMSOTf CH& -78% &I3
& '*OR TBDMSO
- -re$- - -b
Roush et al. [571
X = OTos, Br, H
BnO BnO
TBDMSO -&OR
PhS
a -unu- I
OCH.
1. TMSOTf
91% L 2. BuqSnH, AlBN 74%
-
BnO I
OCH.
Castro-Palomino,Schmidt I581
Scheme 9.
at the 2-position, a new method has been proposed. Glycosylation reaction with 2-O-thiobenzoyl-protected O-glucosyl trichloroacetimidates gave exclusively the 0-glucosides which could be converted into the 2-deoxy-P-glucosides by reduction with tributyltin hydride [58]. Starting from mannosyl trichloroacetimidates the 2-deoxy-a-glucosides were obtained in the same manner [ 5 8 ] . Miscellaneous Compounds
The synthesis of some rare glycofuranosyl donors has also been reported (Scheme 10). The trichloroacetimidates of D-glucofuranose [59], D-mannofuranose [59], and D-galactofuranose [59, 601 have been prepared and used for the synthesis of furanosyl glycosides. With the heptosyl trichloroacetimidate some neoglycoconjugates containing Hep( 1-4)Kdo and Hep(1-5)Kdo-4-phosphate-(2-6)GlcNAcresidues were synthesized which are part of the inner core region of bacterial lipopolysaccharides [61]. A 5-thio-~-fucosyltrichloroacetimidate was used for the synthesis of 5-S-~-Fuca( 1-2)Gal and other disaccharides which have inhibitory activity against a-L-fucosidases [621. Some 5-thiomannose-containing oligosaccharide mimetics were prepared with a 5-thiomannosyl trichloroacetimidate; their binding constants to concanavalin A were determined [63]. An example of intramolecular glycosylation has been reported with a Glca(12)Glc trichloroacetimidate [64, 651. Reaction with boron trifluoride-diethyl ether gave the cyclo-a(1-2)glucobiose (cyclokojibiose) with concomitant expulsion of the O-benzyl protecting group. 2-O-debenzylation previous to cyclization is not necessary because the oxygen is sufficiently nucleophilic and the reaction is entropically favored. A similar example has been reported with a fucose moiety [66]. Complex Oligosaccharides
Complex oligosaccharides are major parts of many biomolecules (glycolipids, glycoproteins, bacterial lipopolysaccharides, mucopolysaccharides, etc. ) and they have a variety of functions. Much interest has been focussed on Lewis antigen structures which are found in glycosphingolipids and on glycoproteins. Many syntheses have
2.3 0-Glycosides
15
AcO AAco & * I
O YCCI, NH
CCI,
LOB"
Plusquellec et al. 1591
Roy et al. 1601
Kosma et al. (611 HO O ,H
&
A d
OAc NH A d
OAc
+
Hashimoto et al. 1621
OAll
OH
BF3.OE1, CH2C12,-2OOC 86%
*
--b H : + HO
OH
,.OAc
Scheme 10.
been described for the Lewis' antigen (LeX)structure which has been found to be a tumor-associated antigen, and for the sialyl LewisX(s LeX)epitope which is involved in cell-adhesion and inflammation processes mediated by its binding properties to selectins. Several synthetic strategies with various building blocks and/or different protective group patterns have been employed, but in recent investigations two alternatives have been favored. A suitably protected azidolactose building block which is available from lactose via lactal, has been chosen (Scheme 11) because it already contains the desired Galp( 1-4)Glc:N linkage [67]. Because of the non-participating neighboring group at 2-OH, fucosylation proceeded exclusively to the a-glycoside and gave the LeXtrisaccharide repeating unit in very good yield (97%). Transformation into the trichloroacetimidate and coupling with the lactose moiety gave spacer-linked Le" which could be extended with the trisaccharide trichloroacetimidate into the spacerlinked dimeric LeXantigen. Following the alternative strategy (Scheme 12), an azidoglucose building block was chosen as starting material and glycosylated first with fucosyl trichloroacetimidate and after benzylidene ring opening with galactosyl trichloroacetimidate [68]. Both glycosylations were highly stereoselective and gave, because of suitable neighboring groups and reaction conditions, exclusively the anomers desired. The high yields were based on the use of the 'inverse procedure' (i.p.), i.e. addition of the trichloroacetimidate donor to the acceptor-catalyst mixture. The LeXtrisaccharide repeating unit was then transformed into the trichloroacetimidate donor and into the glycosyl acceptor and then coupled with trimethylsilyl triflate as catalyst in
16
2 Trichloroucetimidutes
NH
OBI
OBn
97%
Aco
OBZ
Windrnuller, Schmidt [671
Scheme 11.
~
h
T t HO
-
~
N3
~
~
~
TMSOTf, i.p. Et,O, r.t. 85%
TMSOTf, i.p. Et20, r.t. 71%
trisaccharide
hexasaccharide irnidate acceptor N3
imidate 081
dodecasaccharide imidate
n = 2 : dim.LeX n = 3 : trim.LeX n = 4 : tetram. LeX
Scheme 12.
Schmidt et al. 68
17
2.3 0-Glycosides
acetonitrile as solvent to yield stereospecifically the P-connected hexasaccharide by means of the 'nitrile effect'; this effect is particularly efficient with 2-azido sugars. The nonasaccharide and dodecasaccharide were prepared in the same way by coupling the hexasaccharide trichloroacetimidate with the tri- or hexasaccharide acceptor. Transformation into the trichloroacetimidates and reaction with the lactose acceptor yielded the octa-, undeca-, and tetradecasaccharides. After coupling with azidosphingosine and further transformations the dimeric, trimeric, and tetrameric Le" antigens were obtained. The synthesis strategy is highly convergent and allows variations in the saccharide structure and the lipid moiety. The ready a.vailability of glucosamine was reason to transfer this synthesis strategy to glucosamine building blocks which can be obtained directly from glucosamine. The N-tetrachlorophthaloyl (TCP) [69], N-trichloroethoxycarbonyl (Teoc) [70], and N-phthalimido (Pht) [711 protected glucosamine acceptors were glycosylated with the 0-galactosyl and 0-fucosyl trichloroacetimidates; both reactions proceeded with high stereoselectivity. The resulting Le" trisaccharide repeating unit was transformed to the corresponding trichloroacetimidates which were used for the glycosylation of the lactose acceptor to give, owing to neighboring group participation, the p( 1-3)-linked Le" pentasaccharide. The Lewisa (Lea) antigen is a blood group antigen; because of its structural similarity to the Re" antigen it has been synthesized in a similar manner (Scheme 13) [72]. The benzylidene-protected azidoglucose acceptor was first glycosylated with galactosyl trichloroacetimidate and, after benzylidene ring opening, with fucosyl trichloroacetimidate; both glycosylations could again be performed with high diastereoselectivity. Transformation into the trichloroacetimidate and reaction with the CCI,
CCI,
OTBS
TMSOTf Et20, r.t. 88%
N3
Ph T HO a O
T
D
BnO
TMSOTf, i.p. Et20, r.t.
AcO
*
OAc
78%
S
I
0KCC'3 NH
BF, OEt2 CH,C12, hexane
H
Schmidt et al. [;TO]
o
#
~
o
~
O
OBn B n
'
61%
FUCCU(~-4) HHO
O
aOTDS NTCP
Schmidt et al. [69]
Hio&
OTBS
I
Gal~(I-3)GlcNAc~(1-3)Gal~(1-4)Glc
N,
Toepfer, Schmidt [72]
Lea Toepfer, Schmidt [72]
Scheme 13.
18
2 Trichloroacetimidates
lactose acceptor in a non-polar solvent as catalyst with diethyl ether-boron trifluoride gave the Lea pentasaccharide. The p-linkage was stereospecifically formed under inversion of the configuration; typical conditions for this reaction type were used. The N-trichloroethoxycarbonyl (Teoc) protected glucosamine acceptor was proceeded in the same way [70]. The Lea trisaccharide was also prepared from the 3,4-di-O-unprotectedazidoglucose [72] and N-tetrachlorophthaloyl (TCP)-protected glucosamine [69] acceptors by attachment of the two sugar residues directly one after the other. The blood group antigen H and the LewisY (Ley) antigen, a stage-specific embryonal antigen (SSEA), have been synthesized using the same azidolactose building block (Scheme 14) [67]. By regioselective monofucosylation and by difucosylation of the 3,2'-di-O-unprotected azidolactose acceptor, the H-antigen trisaccharide and the Ley tetrasaccharide were synthesized in high yields. Transformation into the trichloroacetimidate and coupling with the lactose moiety furnished the spacer-
CH,Cl$hexane BF,.OEt, -10%
70%
!
o(cHd&~H, HO
OBZ
Ga@(1-4)GlcNAcp(1-3)Ga@(1-4)GlcP(1-0)Sp FuW( 1-2)
H-antigen
I
BFS.OEt2 CH,Cldhexane
H O + O - Y
OBZ
-1O°C
71%
F u c ~1(-2) F u c ~1(-3) Windmuller, Schmidt I671
Scheme 14.
2.3 0-Glycosides
19
-
trisaccharide imidate
BnO
--+ trisaccharide N3
TMSOTf. i.p. Et,O
TMSOTf, i.p. EtPO
85%
89%
acceptor BnO
TMSOTf CHSCN, -4OC 80%
I
COOCH,
FUctr(1-3)
dim. sLeX
1:
octa-
hexasaccharide imidate
Fuca(1-2) Galp(l-4)GlcNAcp(l-3) alp(l-4)Glcp(l-I)Cer
[
F U C ~-3) ('~
I
dim.LeY
BnO
Hummel, Schmidt [73]
Scheme 15.
linked H-antigen and Ley antigen, respectively. Another strategy has been applied to the synthesis of the dimeric Ley antigen (Scheme 15) [73]. According to the previously described synthesis of dimeric LeX (Scheme 12, Ref. [68].) the Le' trisaccharide has been prepared from the azidoglucose acceptor, the fucosyl and the galactosyl trichloroacetimidate, and the derived trisaccharide was used as trichloroacetimidate donor and as acceptor for the synthesis of the corresponding hexasaccharide. Coupling with the lactose moiety gave the octasaccharide which was subjected to a protective group change at the terminal galactose moiety. Regioselective protection of the 3-OH, 4-OH, and 6-OH groups and fucosylation at 2-OH gave the desired nonasaccharide. After coupling with azidosphingosine and further transformations the dimeric Ley antigen was obtained. For the synthesis of the sialyl Lewis' (sLe') antigen, the terminal galactose moiety of the octasaccharide was 6-0-benzyl-protected. Regioselective glycosylation of the 2,3,4-tri-O-unprotected acceptor with neuraminic acid phosphite as donor gave the desired 3-0-sialylated oligosaccharide which was coupled with azidosphingosine and transformed into the dimeric sLe' antigen by use of established methods. Further syntheses of the sLe' determinant follow the same strategy, but applying other glycosylation methods (Scheme 16). Fucosylation of a similar O-benzylideneprotected glucosamine acceptor performed well with a thioglycoside donor [74-761, but the galactosylation was critical because of the steric hindrance of the 4-OH group. The methylthio galactoside donor gave a 16% yield [74]; the trichloroaceti-
20
2 Trichloroacetimidates
HNAC
Ga@(1-4)GlcNAcP-X NeuAca(2-3) Fuca(1-3)
= NH2
Kretzschmar, et al. [74-761
Scheme 16.
midate was, therefore, again favored. Regioselective sialylation with the thioglycoside donor gave the sLeXdeterminant bearing an amino group or a spacer at the reducing end for attachment to peptides or for the generation of other conjugates. The synthesis was suitable for large-scale preparations 1761 and enabled the use of sialic acid derivatives with modifications at C-5 to synthesize sLeXanalogs and to investigate their binding properties to E- and P-selectins [75]. A similar strategy for the synthesis of the sLeXdeterminant was used by applying the glycal method (Scheme 17) [77]. The 3,4-di-O-unprotected glucal was regioselectively fucosylated ( a : = 7.5: 1) with fucosyl fluoride and then galactosylated with 0-galactosyl trichloroacetimidate to yield the Le" trisaccharide glycal. The 2,3,4-tri-O-unprotected galactose acceptor was then regioselectively sialylated with sialyl chloride as donor. Then application of the glycal method gave either the sLeX trisaccharide or-by extension with the tributyltin derivative of the lactal acceptor -the sLeXpentasaccharide glucal derivative which can be activated to provide a donor for the synthesis of conjugates. The synthesis is efficient and convergent, and it is worth mentioning that for the crucial galactosylation of the sterically hindered 4-OH group the trichloroacetimidate method has again been employed. The synthesis of LeXor sLeXanalogs or simplified structures, so-called mimetics, is of great interest for binding studies with selectins, thus high affinity ligands concomitantly with convenient chemical syntheses for novel anti-inflammatory drugs are evaluated. The synthesis of some only slightly modified Le" analogs containing Glc instead of GlcNAc [78], GalNAc instead of Gal [79],or 5-thiofucose instead of Fuc [801 has been similarly performed as described above using trichloroacetimidate donors. It has been found that sialylations are always crucial and expensive processes, therefore many syntheses for sLeXmimetics have been developed in which
2.3 0-Glycosides
21
OTDPS OBn
OTDPS
A
c
O
g
O
&
BZO
AgC104 SnCI, DTBP 59%
OBn
BF,~OEt, CH& -78OC 73%
Bzo
Danishefsky el al. 071
Gal~(1-4)GlcNAc~(l-3)Gal~(l-4)Glucal
Galp(1-4)GlcNAc
NeuAca(2-3) Fuca(1-3)
NeuAca(2-3) Fuca(1-3)
Scheme 17.
AcO
BnO
AcO
a
O
B
OAc
Ph q 'HO a O A l l 0 0 ~
TMSOTf CH& 87%
I
AgOTf Bu,SnCI, 90%
b
F u c ~1-4) (
n
I
AcO W o OAc & O A I I
3'-phospho-Lea-analog AcO
OBn OAc
Kiessling et al. [Sll
3'-C-carboxymethyl OBZ OBn
DMTST TMSOTf benzene, 6OC CH,CN, -1OOC 86% 67%
OBn
b
'~6
BnOoBn @OBn
I
Kiso el al.[831
NHAc
HO
Scheme 18.
Thoma el al. I841
22
2 Trichloroacetimidates
HO
OH
Wong et al. IS81
Scheme 19.
neuraminic acid is replaced by other acids (Scheme 18). A 3’-phospho-Lea analog [81] and a 3’-sulfo-Lex analog [82] were prepared from 3-0-unprotected 4,6-0benzylidene-glucose derivatives. As well established in the synthesis of LeXand sLeX (see above), for all glycosylations with galactose the trichloroacetimidates were successfully used as donors. For the synthesis of the 3’-C-carboxymethyl LeXanalog [831 the synthesis and use of the required galactose-derived trichloroacetimidate donor has been reported. A more rigidly fixed carboxylic group is contained in the sLeXmimetic with a six-membered ring fused to galactose bearing an equatorial carboxylic acid group [84]. The required branched galactose moiety has been prepared and used as trichloroacetimidate-activated donor. Further sLeXmimetics with other simplifications have been proposed (Scheme 19). The GlcNAc moiety has been replaced by cyclohexanediol which was used as acceptor and glycosylated first with the galactosyl trichloroacetimidate and then with the corresponding fucosyl donor [85]. Then a malonic acid derived group was introduced at the 2-OH of galactose. In an even more simplified structure the galactose moiety has been replaced by an aliphatic chain [86]. The acceptor which was prepared from glucal is first glycosylated with the fucosyl trichloroacetimidate and then converted into the malonic acid derivative. In a related mimetic, the malonic acid group is attached more rigidly at a benzene ring [87]. Glycosylation with the
2.3 0-Glycosides
A*
OAC
COOCH,
OBn
23
OBn
(GM, intermediate) pentasaccharide imidate
Gal~(I-3)GalNAc~(1-4)Gal~(l-4)Glc~(l-1)Cer NeuAca(2-3)
Schmidt et al. [891
Scheme 20.
fucosyl trichloroacetimidate resulted in a low yield only (35%), because of steric hindrance. A c:ompletelydifferent structure has been chosen with a terpenoid system [88]. Glycosylation of isosteriol with the mannosyl trichloroacetimidate gave a mimetic which selectively inhibits P-selectin, but not E- and L-selectins. Glycolipids other than the Lewis antigens have also been synthesized with trichloroacetimidate donors. For the synthesis of the ganglioside GM1 several strategies have been suggested [89, 901. Applying a convergent strategy (Scheme 20) [89] a 3,4-di-O-unprotected lactose acceptor was first sialylated with neuraminic acid phosphite as donor to give a GM3 intermediate, and then glycosylated with a Galp( 1-3)GalN trichloroacetimidate as donor; the latter can be easily prepared from azidoglucose and galactosyl trichloroacetimidate. Coupling with azidosphingosine by application of trichloroacetimidate activation of the pentasaccharide residue and then further transformations yielded GMI . Another strategy via a C A I intermediate performing sialylation at a later stage has also been reported [90]. The GM3 intermediate is also suitable for the synthesis of GM2 [91]Glycosylation of the GM3 intermediate with the Teoc-protected galactosamine trichloroacetimidate, which can be more readily synthesized from galactosamine than azidogalactose from galactal, and application of the established transformations, yielded GM2 which is required in a cancer immunization study with GM2-containing vaccines. A glycolipid of the neolucto series named 3-0-sulfo glucuronyl paragloboside is also of interest because of its immunological properties (Scheme 21) [92]. The syn-
24
2 Trichloroucetimidutes COOMe
k0.d-
H:p&
BzOO
OBn OSE
HO
8
BnO
gn&
OBn OSE
OBn
CCl,
OSE
94% TMSOTf
CH,CI,, r.1.
pentasaccharide imidate
N3
BF,.OEt, CHZCI,, O°C 72%
00~S-0-3GlcAP(1-3)GalP(I-4)GIcNAc~(1-3)Gal~(1-4)Glc~(l-l)Cer
3-0-sulfo glucuronyl paragloboside
Hasegawa et al. [921
Scheme 21.
thesis is based on glycosylation of a lactose acceptor with phthalimidoglucosyl trichloroacetimidate. Glycosylation with a disaccharide trichloroacetimidate, which was prepared from a glucuronic acid trichloroacetimidate and a galactose acceptor, and the usual transformations including introduction of the sulfo group yielded 3-0-sulfo glucuronyl paragloboside. A more straightforward synthesis of a related structure has been reported using a Galb( 1-4)GlcN disaccharide (lactosamine) which is contained in the neolucto structure as starting material (Scheme 22) [93]. Glycosylation of the lactose acceptor with the azidolactosyl trichloroacetimidate donor followed by sialylation and application of the standard azidosphingosine glycosylation procedure gave the neolacto ganglioside LM1. All the above mentioned examples contain P-glycosidic linkages which can be obtained by means of participating neighboring groups or-for azidosugars-by means of the nitrile effect. The synthesis of gangliosides of the globo series which contain also an a-galactose linkage is more difficult. From the azidogalactosyl trichloroacetimidate and a galactose acceptor the 2-0-benzyl-protected disaccharide was prepared (Scheme 23) [94, 951; this was converted into the trichloroacetimidate and used as donor for the glycosylation of the lactose acceptor. Using typical conditions favoring s,~-type reactions (non-participating neighboring group, strong Lewis acid, diethyl ether as solvent), the a-connected tetrasaccharide was obtained with high selectivity. Ensuing galactosylation, sialylation, and application of the
25
2.3 0-Glycosides TMSOTf CH2C12,-4OOC (SOYO) tetrasaccharide
b
I OBn
58%
OBn
OEt
pentasaccharideimidate
5
lF3.0Et2 CH2C12,O°C 43%
O h
NeuAca(2-3)Gal~(l-4)GlcNAc~(l-3)Gal~(1-4)Glc~(I-1)Cer Tietze et al. [931
LMl
Scheme 22.
Ph
Ph
Ph
TMSOTf
&:s
Ho BnO
OAll
HO
N,
OBn
BnO
OBn
OBn
1
CCI,
0
Y
NH
Y CCI,
Ph
Ph
TMSOTf, i.p EbO, -1OOC
Bds+E;ohloBn TMSOTf I
-'I
Ell0
AcOO
vNH I CCI,
TMSOTf CH,CN, -4OOC-I (a$= 3.5:l)
%En
AcO
62%
AcN
COOCH,
dAc
hexasaccharide imidate
N,
TMSOTf OBZ
Neu5Aca(2-3)Gal~(l-3)GalNac~(l-3)Gala(l-4)Gal~(1-4)Glc~(1-1)Cer Schmidt et al. 194,951
Scheme 23.
SGG
26
2 Trichloroacetirnidates
azidosphingosine glycosylation procedure yielded the sialylgalactosylgloboside (SGG), which bears the stage-specific embryonic antigen 4 (SSEA-4) structure. Another example of the globo series is the globo H (human breast cancer) antigen. It has been prepared by means of a similar strategy [96]. Although in glycopeptide synthesis, most strategies are based on the synthesis of the complete oligosaccharide moiety and then its attachment to the amino acid or peptide, glycosylations of glycosylated amino acid acceptors have also been reported (Scheme 24). Thus, glycosylation of the GalNa( I-0)Thr acceptor with
Ph
'%$
+
H
o
1. TMSOTf, CH2CI,, O°C 02%
b
2. Zn, &OH, AC20, THF 3. AqO. pyr. (80%)
TeocN
b
O YCCI, N H
core3
Fmoc[O X HC0,Bu' 3
FmocNH
Bock et al. [971 Ph
+
Ph
"q
Bock et al. 1441
1. TMSOTf, CH2C12,40°C 67% 2. Zn, 7OoC THF, AC20,HOAc)
AcNH
H
Ph
Ph
Ph
CCI,
Qiu, Koganti I981
w T s 0
'no
0
II
-15% 69%
AcN
OBn
Scheme 24.
OBn
OBn
OBn
2.3 O-Glycosides
27
N-trichloroethoxycarbonyl (Teoc)- [97] or the N-dithiasuccinyl (Dts)- [44] protected glucosamine trichloroacetimidates, respectively, afforded the core 3 building blocks. The P-glycosides were stereospecifically obtained by means of neighboring group participation. Use of the N-acetyl-protected galactosamine trichloroacetimidate gave stereoselectively the a-connected core 5 structure [98]. Thus, core 1, 2, 3 , 4, 5 , sialyl 5 , 6, and F1-a building blocks were synthesized [44, 97-99]. A sialylated glycosy1 serine building block was prepared with a disaccharide trichloroacetimidate donor and used in the solid-phase synthesis of the B-chain of human u2HS glycoprotein [loo, 1011. In the synthesis of branched complex type N-glycans the oligosaccharide part is usually prepared first; it is then attached to the amino acid, usually asparagine. For investigations of the function of glycoproteins the synthesis of libraries with variations of the antennary structures is required. A highly convergent chemoenzymatic approach has recently been reported (Scheme 25) [ 102-1041. The core trisaccharide Mar$( 1-4)GlcNP( 1-4)GlcN was synthesized via the P-glucoside and then inversion of the configuration by intramolecular nucleophilic substitution as described above (see Scheme 8:1, thus avoiding the crucial direct P-mannosylation. The trisaccharide acceptor is suitable for the attachment of various antennary structures with linear or branched trichloroacetimidate donors which were again synthesized by application of the trichloroacetimidate method. Several examples were prepared and used for investigations on further glycosylations with galactosidases and sialidases. Another versatile strategy enables the synthesis of diantennary and bisected diantennary oligosaccharides (Scheme 26) [ 531. The azidoglucose acceptor was glycosylated with the glucosyl and mannosyl trichloroacetimidates. The P-glucoside of the resulting trisaccharide was converted into the P-mannoside moiety by inversion of the configuration at C-2 via triflate formation and substitution with tetrabutylammonium nitrite. Further glycosylations with the mannosyl and the azidoglucosyl trichloroacetimidates gave the bisected diantennary pentasaccharide which was used as acceptor for extension with lactosamine trichloroacetimidate. After transformation of the nonasaccharide into the trichloroacetimidate the terminal glucosamine residue was attached at the reducing end. Thus, the synthesis is highly variable in the connection to the protein and in the antennary structures. Further syntheses applying direct 0-mannosylation have also been reported [ 105- 1071, and it was found, that the trichloroacetimidate method was superior to other glycosylation methods, especially for the synthesis of u-mannosides. The acidic mucopolysaccharides, which consist of disaccharide repeating units containing D-glucuronic or L-iduronic acid and glucosamine or galactosamine, respectively, are also members of the class of glycoproteins or proteoglycans. Because L-iduronic acid is not readily available it must be prepared. A new synthesis from Dglucose was recently described by formation of the exo-glucal, diastereoselective hydroboration, and oxidation of C-6 [ 108). Galactosamine is also a very expensive starting material and it has therefore been prepared from glucosamine by inversion of the configuration of the 4-OH group via the triflate, and used for the synthesis of dermatan sulfate fragments [ 1091. In another approach [ 1101 an L-idose acceptor was glycosylated stereoselectively (P: u = 2.5 : 1) with an azidogalactosyl trichloroacetimidate. The P-selectivity was achieved, without neighboring group participa-
28
2 Trichloroacetimidates
85% b
BnO
BnO
F
0
\
NPht
BF3 OEt2 CH2C12 84%
Ad)
Ad)
BF, OEt2 CH2CI2 -45% 76% Aco AcO
OAC
Aco Aco
GkNAc&l-6)
I MW(1-6)
I
GkNAcS(1-2)
1
Man~(1-4)GICNACP(1-4)GlcNA@N3
Unverzagt [102-1M]
I
GlcNAcP(l-P)Mana(l-3)
Scheme 25.
tion, in a non-polar solvent at low temperature by an SN2-type reaction. The resulting disaccharide was oxidized to the iduronic acid derivative and converted into the GalNp( 1 -4)IdoA trichloroacetimidate and into a corresponding acceptor which were then used for the next glycosylation step to provide a dermatan tetrasaccharide fragment; thus, because of neighboring group participation an a-glycosidic bond was formed stereospecifically. The tetra-, hexa-, and octasaccharide fragments of hyaluronic acid (Scheme 27) [ 1111 have been prepared by repeated glycosylation of a GlcNP( 1 -4)GlcA acceptor with a disaccharide trichloroacetimidate which was prepared from a trichloroacetyl
29
2.3 0-Glycosides
ACO AcO OBn
I
TMSOTf CH2C12,hexane 93%
Et20,-4OOC
68%
Bu~N'NO,
I
GlcNAc~(l-4)Man~(l-4)GlcNAc~(1-4)GlcNAc
I
Ga@(1-4)GlcNA1:b(1-2)Mana(1-3) Weiler, Schmidt 1531
Scheme 26.
(TCA)-protected glucosamine trichloroacetimidate and a glucuronic acid acceptor. All reactions gave, because of neighboring group participation, exclusively pglycosides in high yields. Some chondroitin fragments have been prepared starting from an azidoglucosyl trichloroacetimidate and a glucose acceptor (Scheme 28) [ 1 12, 1131. Despite the use of a non-participating neighboring group, the p-glycoside was obtained exclusively because in a non-polar solvent at low temperature with boron trifluoride as the catalyst an S,z-type reaction was favored. With trimethylsilyl triflate lower stereoselectivity (p :CI = 2 : 1) was observed. The disaccharide was oxidized to the glucuronic acid derivative and converted into the
30
2 Trichloroacetimidates
+
PhT-+
Ho* BzO
OMP
TCANH
OBz
O Y N H dCl,
q
89%
~
~
~
&
O
M
~
HO
TCANH
COOMe
-4 87%
Ph
TMSOTf
zqsy-;*oMe TCANH
Cm+ie
TCANH
COOMe
n=
hyaluronic acid
Blatter, Jaquinet
1,2,3
I1 111
Scheme 27.
GalNAc(1-4)GlcA trichloroacetimidate and the corresponding acceptor which were used again for glycosylation to yield a tetrasaccharide fragment. The di- and tetrasaccharide trichloroacetimidates were used for the glycosylation of a Galf3(13)Galf3(1-4)Xyl acceptor which represents a typical linkage of mucopolysaccharides to proteins. The resulting penta- and heptasaccharide trichloroacetimidates were used for the glycosylation of serine to yield chondroitin fragments with one or two repeating units; these compounds are of interest as acceptors for glycosyltransferases in enzymatic studies. Great biological interest has been shown in heparan sulfate or analogs of heparin, containing glucosamine and D-glucuronic or L-iduronic acid. A convergent synthesis has been reported (Scheme 29) [114], starting from an azidoglucose acceptor which was glycosylated with two iduronic acid trichloroacetimidates. The resulting two disaccharides were converted into the trichloroacetimidates and used consecutively for glycosylation of the suitably protected iduronic acid acceptor. Both aglycosidic bonds were formed stereospecifically with inversion of the configuration. Thus, a heparan trisulfate fragment has been prepared and used for binding studies to fibroblast growth factor-2. Syntheses of C-bridged heparan sulfate analogs [ 1151 and of heparin-like glycoconjugates which were used for thrombin binding studies [ 116, 1171, have also been reported. The synthesis of bacterial polysaccharides is of great importance for the development of antibacterial vaccines. A terminal a( 1-2)-linked rhamnose trisaccharide
2.3 0-Glycosides
31
YPh
Ph
Ph I
P' h
BF3.OEtz toluene. -5OOC
r
71%
1
5.0-70%
GalNAcP(1-4)GlcAP( n=1,2
Scheme 28.
HNFmoc
1-3)GalP(1-4)XylP(1 -0)Ser chondroitin
Ogawa et al. 1112,1131
32
2 Trichloroacetimidates
~
'
"
not reported
3
I
Lea
qn%
Lev0
OAc
w
HO
KCci3 84% TBDMSOTf NH
OAc
HO
I
OAll
toluene, -20°C
TBDMSOTf CHZCI,, -2OOC
IdoAa(l-4)GlcNa(l-4)ldoAa(i-4)GlcNa(l-4)IdoAal-OMe
6 0 , '
Sinay et al. [I141
Ao3Q
Scheme 29.
of the group B Streptococcus antigen has been synthesized with a 2-0-acetyl protected rhamnosyl trichloroacetimidate by repeated glycosylation and deprotection of, the 2-OH group [118]. A hexasaccharide fragment of the a(l-4)-linked 3-0methylmannose polysaccharide of Mycobacterium smegmatis has been convergently prepared, via a disaccharide building block [ 1191. From a 4-0-acetyl protected 3-0methylmannosyl trichloroacetimidate and a suitable acceptor a 3-0-methylmannose disaccharide was prepared which was converted into the trichloroacetimidate and used for repeated glycosylation and then deprotection of the 4'-OH group. All glycosidic bonds were stereospecifically formed owing to the participation of neighboring groups. Most bacterial polysaccharides are much more complex. Ninety 90 serotypes of Streptococcus pneumoniae are already known. The synthesis of a pyruvated tetrasaccharide repeating unit of Streptococcus pneumoniae type 27 has been described (Scheme 30) [ 1201. The disaccharide trichloroacetimidate donor was prepared from acetobromoglucose and the pyruvated glucosamine acceptor applying the KoenigsKnorr method. The a-linked disaccharide acceptor was prepared from a galactosyl trichloroacetimidate and a rhamnose acceptor. The use of a strong Lewis acid and solvent (diethylether) favoring an S,I -type reaction mechanism, gave the a-glycoside with high selectivity (a: = 9 : 1). Glycosylation of the disaccharide acceptor with the disaccharide trichloroacetimidate gave the tetrasaccharide repeating unit carrying a spacer which is suitable for the preparation of glycoconjugates. The syntheses of another tetrasaccharide repeating unit of this type [121] and four tetrasaccharide repeating units of Streptococcus pneumoniae type 6B [ 1221 have also been reported. The chemoenzymatic synthesis of a decasaccharide consisting of two repeating units of Streptococcus capsular polysaccharides type I11 group B (GBSP 111) (Scheme 31) [123] has been described. Glycosylation by the Koenigs-Knorr method of a 3,4-
a
2.3 0-Glycosides
61Yo
33
TMSOTf
6OoC
scheme 30.
di-O-unprotected glucosamine acceptor with acetobromogalactose gave regiospecifically the lactosamine disaccharide which was converted into an acceptor and into a trichloroacetimidate donor. Glycosylation of the disaccharide acceptor with acetobromoglucose, again under Koenigs-Knorr conditions, gave the trisaccharide building block which was transformed into the trichloroacetimidate donor. The disaccharide trichloroacetimidate was used for the glycosylation of a galactose acceptor; the resulting linear trisaccharide was glycosylated with a lactosyl trichloroacetimidate to give the pentasaccharide building block. Glycosylation with the trisaccharide trichloroacetimidate finally gave the desired octasaccharide. All pglycosidic bonds were formed stereospecifically, but the synthesis seems not to be straightforward because the tetrasaccharide repeating unit was not used twice as a building block. Finally, the neuraminic acid residues were enzymatically attached with a(2-3)sialyltransferase to yield the desired decasaccharide fragment. The synthesis of an octasaccharide mimic of two repeating units of the Streptococcus capsular polysaccharide type I11 group B (GBSP 111) with S-1-carboxyethyl groups replacing the siailic acids has also been reported [124]. A convergent synthesis has been reported for a hexasaccharide fragment of the Streptococcus group A cell-wall polysaccharide (Scheme 32) [ 1251. The 3 - 0 unprotected rhamnose acceptor was first glycosylated with the N-phthaloylprotected glucosamine trichloroacetimidate and then with a suitably protected
34
2 Trichloroucetimidutes
ACO
HS(CN12 tol. CHsNO2,
ACO
OSE
72%
.A;&oHg
OSE
Pht
NPhth
OAc
A
disaccharide acceptor
disaccharide imidate
J
-OAc
Ad)
Oh43
TMSOTf
Glc~(1-6)GlcNAc~(1-3)Gal~(l-4)Glc~(1 -6)GlcNAcP(1-3)GalPl-OMe
I
I
GaiP(1-4)
GalP(1-4)
1
a(2-3)sialyNransferase CMP-NedAc
Glc~(l-6)GlcNAc~(l-3)Gal~(l-4)Glc~(l-6)GlcNAc~(l-3)Gal~l-OMe
I
NeuAca(2-3)GalB(l-4)
I
NeuAca(2S)Ga@(1-4)
GBSP 111 Jennings et al. [I231
Scheme 31.
rhamnosyl trichloroacetimidate. The resulting trisaccharide was converted into the trichloroacetimidate and into an acceptor; coupling yielded the hexasaccharide with high stereoselectivity. A participating neighboring group is not necessary for the formation of the a-rhamnoside bond. An efficient synthesis has also been reported for the hexadecasaccharide fragment
35
2.3 0-Glycosides OAll
B
z
O
H
TESOTf CH2CI2, -75OC 58%
r
I '
Bz
OAc
trisaccharide imidate
Rhaa(l-2)Rhaa(1-3)-Rhaa(l-2)Rha GlcNAcp(1-3)
OBz
BZO
CH2CI2, TESOTf,-75OC 84%
TESOTf, CH2C12, r.t. 43%
GlcNAcp(1-3)
trisaccharide acceptor Pinto et al. [125]
Scheme 32.
of the Shigelln dysenteria type-1 0-specific polysaccharide (Scheme 33) [ 126, 1271. For the synthesis of the tetrasaccharide repeating unit by subsequent glycosylation of the rhamnose acceptor with azidoglucosyl chloride, methylthio galactoside, and rhamnosyl trichloroacetimidate, several glycosylation methods were applied. For the ensuing gl ycosylations with the tetrasaccharide as donor, the trichloroacetimidate method was favored. Repetitive glycosylation of the hexanoic acid acceptor gave the octa-, dodeca-, and hexadecasaccharides in high yields. Coupling with the linker to human serumalbumin (HSA) enabled the preparation of materials for vaccine studies. Further oligosaccharide fragments of the Rhizobium leguminosarum [ 128, 1291, CCI, CCI,
OANH N, SPh BnO
I
AgOTf DBMP CH&12, O°C
64%
OMBn I
I
MeOTf DBMP Et20, r.1. 58%
Brio
ClAcd
BF .OH
bez b
cH3c12,200c 8940
Rhaa(1-2)Gala(l-3)GlcNAca(l-3)Rhaa 1-O(CH,),COOMe n = 2-4
BnO
OAC
HO(CH,),COOMe
Pozsgay [126,1271
Scheme 33.
36
2 Trichloroacetimidates
BnO
OH
OBn
dBn B
n
En0 O q
68%1Sn(OTI)Z Et20/CH2CI,. r.t.
+
CCI,
Sn(OTI), Et20/CH2C12,r.t.
+
En0 B
n
O
g
0 OBn
I D-Gala(1-1)-D-rnyo-inositol
(Galactinol)
Mayer, Schmidt [ 1 1
Scheme 34.
Mycobacterium gornoae [ 1301, Mycobacterium arium [ 1311, and Vibrio cholerae [ 1321 polysaccharides have also been prepared with the help of trichloroacetimidates as glycosyl donors.
2.3.2 Inositol Glycosides The synthesis of galactinol gala( 1-l)-~-myo-inositol]which is involved in the biosynthesis of the raffinose family, has been reported (Scheme 34) [133]. The readily available L-myo-inositol derivative was glycosylated with the 0-benzyl protected galactosyl trichloroacetimidate. Because of reaction conditions favoring an S,I -type reaction mechanism, the a-glycoside was formed exclusively. In the same way D-Gala(1-1)-D-myo-inositol was obtained from the corresponding D-myoinositol derivative. The glycosylphosphatidyl inositols (GPIs) are an interesting class of compounds which serve as anchors for proteins and glycoproteins in membranes. They consist of many variants in the carbohydrate moiety and in the lipid part. The first total synthesis of a GPI anchor from yeast has been reported; it contains a ceramide residue with a phytosphingosine constituent (Scheme 35) [ 134, 1351. The synthesis has been performed in a convergent manner by glycosylation of 6-0-unprotected Dmyo-inositol acceptor with the azidoglucosyl trichloroacetimidate yielding stereospecifically the a-glycoside as a result of s,~-type reaction conditions. A similar
37
2.3 0-Glycosides
BBilOn 0
0
OAll
O-(-)-Mnt
"
G
TMSOTf
NH
o&.~~,
85% Et,O. r.t.
Pl
0
BnO
TMSOTf
00
;;oq I
uoo~OyO(-)-Mnt 0
cc13
TMSOTf
O YCCI, NH
HO
"YNH CCI,
HN
0
08 0- 6Mana(1-P)Mana(1 -P)Mana(l-4)GlcNa(1-6)lnositol-l-o,
H,Nlco':/-
I
0
Mana(l-2)
8
OH
Schmidt et al. 1134,1351
Scheme 35.
approach with a benzyl-protected azidoglucosyl trichloroacetimidate in dichloromethane gave much lower stereoselectivity [ 1361. The mannose tetrasaccharide building block was prepared by the consecutive glycosylation of the 6-0-unprotected mannose acceptor with three suitably protected O-mannosyl trichloroacetimidates. The two building blocks were coupled by glycosylation of the GlcNyx(1-1)inositol acceptor with the tetramannosyl trichloroacetimidate in very good yield. All a-
38
2 Trichloroacetimidates
mannoside bonds were stereospecifically formed. The synthesis was completed by attaching the phosphate and the ceramide phosphate moieties at the suitably deprotected positions. Other inositolglycans are of pharmaceutical interest because of their insulin-like effect. The synthesis of a 0-linked inositol glycoside using a linear strategy has been reported [ 1371. The azidoglucose acceptor was first glycosylated with mannosyl trichloroacetimidate. The resulting disaccharide was again converted into the trichloroacetimidate and used for the glycosylation of the inositol acceptor. The pseudotrisaccharide was stereoselectively (p :a = 3 : 1) obtained and then consecutively glycosylated with the three suitably protected mannosyl trichloroacetimidates. Introduction of the phosphate groups gave the desired inositolglycans; some related derivatives have also been synthesized.
2.3.3 Glycosylation of Sphingosine Derivatives and Mimics The azidosphingosine glycosylation method [ 138, 1391 is well established. It has been found that azidosphingosine is a better glycosyl acceptor than ceramide or other sphingosine derivatives. Some examples of the synthesis of glycosphingolipids with complex oligosaccharide trichloroacetimidates and azidosphingosine have already been shown (see above). Even when other glycosylation methods have been used for the synthesis of the oligosaccharide moiety, the trichloroacetimidate method has been preferentially chosen for the coupling to azidosphingosine (Scheme 36) [140]. A tetrasaccharide trichloroacetimidate of the ganglio series containing three neuraminic acid residues was used for the glycosylation of 3-0-protected azidosphingosine. Reduction of azide, attachment of the fatty acid, and deprotection
N.
TMSOTf CH,CI,. O°C 65%
I NeuAca(2-8)NeuAca(2-3)
Scheme 36.
OTBDPS
I
NeuAca(2-3)
GT'a
Hasegawa et al. [14W
2.3 0-Glycosidrs
6BZ
39
Higuchi et al. [154]
Scheme 37.
yielded the ganglioside GT1,. With the help of the azidosphingosine glycosylation method many other glycosphingolipids have been synthesized, ie. GD1, [ 1401, GD2 [141], GT3 [142], GT4 [142], GQlb [143, 1441, GQlba [145], sialylated globosides [ 1461, sulfated glucuronyl paraglobosides [ 1471, sulfated sLeX glycosphingolipids [ 1481, ganglioside lactams corresponding to G M I , GM2, GM3, and GM4 [ 1491, and some xylose analogs of glycosphingolipids [ 1501. Although the glycosylation of ceramides has been found to proceed with low yields [ 151, 1521, this method has been applied a few times, especially when special ceramide derivatives are required and their azido derivatives were not available (Scheme 37). ‘The synthesis of astrocerebroside A, which has been isolated from starfish and which is of interest because of its pharmacological activity, has been reported via glycosylation of its ceramide moiety with 0-glucosyl trichloroacetimidate [153]. A hematoside-type ganglioside called GAA-6 which has been also found in starfish, was prepared in the same way via glycosylation of the phytosphingosine derivative with the trisaccharide trichloroacetimidate [ 1541. To find potent selectin binding inhibitors, mimics not only of the carbohydrate part of glycosphingolipids, especially of LeXand sLeX(see above), but also mimics of various lipid analogs have been prepared. 2-Tetradecylhexadecanol has been proposed as lipid moiety, and was glycosylated with the sialylated lactosyl trichloroacetimidate to yield the GM3 analogous neoglycolipid [ 1551. With the same lipid moiety were also prepared the GM4 [155], LeX[156], 3’-C-carboxymethyl LeX [ 1571, and some sulfated [ 158- 1601 and phosphorylated [ 1601 neoglycolipids. Another lipid moiety containing a spacer of variable length was glycosylated with the required trichloroacetimidate to prepare sLeXneoglycolipids [ 1611. A double-chain bis-sulfone has been used for the glycosylation with the corresponding trichloroacetimidates in the synthesis of a globotriosyl neoglycolipid and some deoxy derivatives [162, 1631. Other lipid moieties have been used for various purposes (Scheme 38). The hexadecanediol derivative was glycosylated with the trisaccharide trichloroacetimidate [ 1641. The resulting glycoside was transformed into the 16-mercaptohexadecanyl glycoside and used for biosensor studies. Glycosylation of a trivalent acceptor with mannosyl trichloroacetimidate furnished a triantennary cluster mannoside; its
2 Trichloroucetimidutes
40
TMSOTf CH2C12,r.t. 71%
1 \OAc
AcO
?Oq
no
0
NH +
Y CCI,
TMSOTf CH2C12, -65°C b 74%
HO$
no
Lindhorst et al. 11651
+
m 2 w p e x a n e , r.t.
no
~
49-72%
-OH
*R
Schmidt et al. [1661
Scheme 38.
binding properties to lectins were investigated [ 1651. Long-chain diols were glycosylated with glucosyl trichloroacetimidate to synthesize bis-glycosides, furnishing new types of non-ionic surfactants of the 2/1-type (two sugar head groups, one lipophilic chain) [ 1661. Surfactants of the 2/2-type have also been prepared [ 1661. 2.3.4 Glycosylation of Amino Acids The well established method [167] for the preparation of a-glycosylated serine and threonine derivatives has also been applied to the attachment of complex oligosaccharides (Scheme 39). Glycosylation of the serine acceptor with the P-configured trisaccharide trichloroacetimidate gave the a-glycosylated product stereospecifically, thus furnishing a derivative of the Fla-antigen [ 1681. Other derivatives including the sialyl T (ST) tumor antigen [ 1691, have also been prepared. The use of N-acetyl-protected and 4,6-O-benzylidene-protectedgalactosamine trichloroacetimidates has been proposed for the synthesis of a-glycosides [170]. Because the cisbicyclic structure disfavors oxazoline formation, a-glycosides are formed exclusively even from the a-trichloroacetimidates. Thus the Thomsen-Friedenreich (TF) antigen has been prepared from the Galp( 1-3)GalNAc trichloroacetimidate and serine or threonine as acceptor [ 1711.
2.3 0-Glycosides
41
HNFmoc HO
GalP(1-4)GlcNP(l-G)GalNa(1-3)Ser
Danishefsky et al. [I681 NH
Ph HNFmoc HO . hCOOCH,COPh
BF3.0Etz,THF, -20%
Ad3
Koganti et al. [1711
68% kl,
HtjFmoc
rCm,
HO
m AGO $
R = H, CH3 R' = All, Pfp
R
TMSOTf, CH2CIz,r.t.
TeocN
HNAc
80-90% HoYNH CCI,
Saha, Schmidt 11721
k0% AcO
Ad3
TMSOTf, CHzCIz, 74%
b
I O YCCI, N H
'
O
H
% A +
DtsN
Fmoc-lle-Ser-Gly-lle-Gly- OCH,
A
Fmoc-lle-S~r-Gly-Ile-Gly
I
I
TMSOTf, CH2CI2, 78%
G Resin e +
A%$
0
I
O YCCI, N H
MeMal et a1.[1731
Fmoc-Ile-Ser-Gly-lle-Gly- OCH,
Scheme 39.
The preparation of Gala(1-3)Ser and its use for further syntheses has also been reported [98, 99, 170, 1711. The N-trichloroethoxy (Teoc)-protected glucosamine trichloroacetimidate has been employed for the synthesis of P-glycosidically linked amino acids [ 1721. Glycosylation of various protected serine and threonine esters gave P-glycosides, because of the presence of participating neighboring groups. The N-tetrachlorophthaloyl (TCP)-protected derivatives have been used similarly. After conversion into the N-acetyl derivatives, the building blocks are suitable for glycopeptide synthesis. A completely different approach for the synthesis of glycopeptides has been reported by glycosylation of the serine-containing peptides on the solid-phase [ 1731. A polyoxypropylene cross-linked polyethylene glycol resin (POEPOP, polyoxyethylene-polyoxypropylene) was chosen because it contains only ether bonds and no amide bonds, and several linkers were investigated. The use of an Ndithiasuccinyl (Dts)-protected glucosamine trichloroacetimidate as donor gave the P-glycosylated peptide whereas the use of the azidoglucosyl trichloroacetimidate
42
2 Trichloroacetimidates
gave, as expected, the a-glycosylated peptide. Galactosyl, mannosyl, and fucosyl trichloroacetimidates have been used for the synthesis of libraries of peptide acceptors with more than one serine residue. 2.3.5 Polycyclic and Macrocyclic Glycosides Glycosides of polycycles or macrocycles (anthracyclines, chalicheamicin, macrolactones, etc.) are of great interest because of their antibiotic and antitumor activity. The synthesis of calichearubicin A and B, which have the same carbohydrate moiety as calicheamicin, has recently been reported (Scheme 40) [174, 1751. The phenolic acceptor was stereospecifically glycosylated with rhamnosyl trichloroacetimidate in very good yield. The resulting a-glycoside was transformed into the trichloroacetimidate donor which was used for the glycosylation of the anthracycline acceptor with silver triflate as catalyst. The a-glycosidically linked calichearubicin A was stereoselectively ( a :P = 5 : 1) obtained. The P-glycosidically linked calichearubicin B was similarly prepared from the same trichloroacetimidate donor with boron trifluoride-diethyl ether as the catalyst and, despite the lack of a participating neighboring group, again the P-stereoselectivitywas high (P : a = 5 : 1, 35%). The syntheses of a modified carbohydrate moiety, a head-to-tail dimer of the calicheamicin oligosaccharide [ 1761, the 1,1'-disaccharide (trehalose) containing the carbohydrate moiety of everninomycin [ 1771, and the vancomycin disaccharide CH,
BF,.OEtz H " ; iTBDMGH3 ; ; . CH30
+
HO
OAc
OCH,
OCH,
CH2CI2 95%
OCH,
OCH,
TBDMSO
0%
\/ ?
H,CO
CU
OCH,
H:q Danishefsky et al. [174,1751
CH30
OH
OH
calichearubicinA
Scheme 40.
HO
a
43
2.3 0-Glycosides OTESMo
0
TMSOTf dioxaneflol., O'C 75% (a$=8:1)
OH
+
CCI,
HNWoOPMB b o T B D M s I
Nicolaou et al. [181,1821
OTBDMS
TMSOTf CH2CI , -78'C 91 % (i:a=5:1)
eleutherobin
OH
TMSOTf CH2CI2, -4O'C not reported F CCI,
C I A c O ) TBDMSO PhS
iPrC0
M &
HO
I
Me0
v
'Me
OH OH
HO
HO HO &
o
o
0
u iPrC0
iPrCO O L *k
OH OH
olivomycin A
Roush et al. [1831
Scheme 41.
[ 1781 have also been reported. The total synthesis of neocarcinostatin including the preparation of the N-methylfucosamine trichloroacetimidate and the glycosylation of the endiyne-type aglycone were recently described [ 179, 1801. The synthesis of eleutherobin, which has antitumor activity and cytotoxic properties similar to those of taxol, has also been reported (Scheme 41) [181, 1821. The carvone-derived acceptor was stereoselectively glycosylated with the arabinosyl trichloroacetimidate ( a :p = 8: 1). The resulting glycoside intermediate was treated as for sarcodictyins A and B to yield eleutherobin and eleuthosides A and B. Studies of the synthesis of olivomycin A have been reported [ 1831. Glycosylation of a glycal acceptor with a trichloroacetimidate and an acetate donor gave a trisaccharide which was transformed into the trichloroacetimidate and used for the glycosylation of the acyloin acceptor. The resulting P-glycoside is a model compound for the approach to the synthesis of olivomycin A.
44
2 Trichloroacetimidates
OH
80% (1:1)
I
BFa.OEt2 CH2C123 -80c
no
Jiang, Schmidt [184,1851
calonyctinA
Scheme 42.
The macrolactone calonyctin A, a plant growth regulator, has been synthesized (Scheme 42) [ 184, 18.51. The quinovose acceptor was glycosylated subsequently with the suitably protected quinovosyl, rhamnosyl, and quinovosyl trichloroacetimidates; all steps could be performed stereospecifically. Transformation of the tetrasaccharide into the trichloroacetimidate and glycosylation of the racemic hydroxy acid derivative gave two epimeric compounds. Separation and lactonization gave calonyctin A. A convergent strategy has been applied to the synthesis of Tricolorin A, a natural herbicide with cytotoxic activity against cultured P-388 and human breast cancer cells (Scheme 43) [186, 1871. The acceptor was prepared by glycosylation of the hydroxy acid derivative with the galactosyl and the glucosyl trichloroacetimidates. It was coupled with the disaccharide trichloroacetimidate which had been prepared before from the suitable rhamnose donor and acceptor. All steps proceeded with high stereoselectivity. The resulting tetrasaccharide was transformed into the macrolactone Tricolorin A. The synthesis of the disaccharide macrolactone lonitoside 1 has also been reported [ 1881. 2.3.6 Glycosides of Phosphoric and Carboxylic Acids
Trichloroacetimidates can be used for glycosylation of phosphoric and carboxylic acids without additional Lewis acid. Reaction of the fucosyl a-trichloroacetimidate
45
2.3 0-Glycosides TMSOTf CH,C12, r, t. 79%
AgOTf CH2C12,r. t. a4%
phT& PivO
0
A"Oo CCI,
YNH CCI,
CCI,
0
TMSOTf CH2C12,r.t. 75%
BnO
OBn
0 0
0
HO
Hd Larsson, Heathcock [186,1871
tricolorin A
Scheme 43.
with dibenzyl phosphate gave the b-fucosyl phosphate with stereospecific inversion of configuraticin (Scheme 44) [189]. Deprotection and coupling with GMP morpholidate yielded GMP fucose which was purified by preparative HPLC. Reaction of the galactosyl trichloroacetimidate with a uridine phosphonate derivative furnished a phosphonate analog of UDP-galactose [22] which might serve as glycosyl donor or as inhibitor for galactosyltransferases. Some nucleoside monophosphate sugars have also been prepared directly by reaction of 0-glycosyl trichloroacetimidates with nucleoside monophosphates [ 1901. Reaction of the glucosyl trichloroacetimidate with a ceramide- 1-phosphate derivative gave exclusively the P-glycoside, owing to the participating neighboring group [ 1911. Reaction of the benzyl-protected glucosyl P-trichloroacetimidate with a malonic acid derivative as acceptor gave, with inversion of configuration, the a-linked malonyl derivative which was designed to mimic the pyrophosphate moiety of UDP-glucose and to serve as inhibitor for glycosyltransferases [ 1921. 2.3.7 Solid-Phase Synthesis
Although solid-phase chemistry is well developed, progress has not yet been fully extended to oligosaccharide chemistry, because of the high demands on the polymer support and the lack of powerful analytical tools for monitoring reactions on solid phases. The synthesis of an a(1-2)-linked pentamannose moiety has recently been reported (Scheme 45) [193]; a Merrifield resin, a thio-linker, and a 2-0-acetyl protected mannosyl trichloroacetimidate were used. The glycosylation reaction was
46
2 Trichloroucetimidutes CCI,
0
Schmidt et al. [IS91 0 A*
OAc
AcO!&&oyCCI, OAC
0
0
II
+
NH
Schrnelzer 1221
HO AcO
OAc
Schmidt et al. [I911
CCI,
It II Wong et al. [I921
x
0
0
HO
OH
Scheme 44.
monitored by MALDI-TOF and, after oxidative cleavage from the solid phase, the oligosaccharide was obtained by preparative HPLC. The method described has been applied to other examples, i.e. the synthesis of the branched-core pentasaccharide moiety of complex type N-glycans [ 194, 1951. The same 2-0-acetyl-protected mannosyl trichloroacetimidate was used in a polymer-supported solution-phase synthesis of a tetramannose moiety with polyethylene glycol monomethyl ether (MPEG) as polymer-support [ 1961. The use of controlled-pore glass (CPG) as solid-phase support is under investigation in the synthesis of oligosaccharides [ 1971 and glyconucleotide conjugates [ 1981 with trichloroacetimidates as donors. The synthesis of polylactosamine oligosaccharides was performed with a Merrifield resin and a benzyl-type linker (Scheme 46) [199].The glycosylation was stereospecifically performed with a phthalimido-protected lactosamine trichloroacetimidate bearing a levulinoyl group as temporary protecting group which can be removed with hydrazinium acetate under mild conditions. After cleavage from the linker the octasaccharide was obtained in good yield (overall 42%).
47
2.3 Q-Glycosides r
1
bR
Rademann, Schmidt [I931
R = H, CH,
Scheme 45.
Heparan sulfate-like oligomers have been prepared by polyethylene glycol monomethyl ether (MPEG)-supported solution-phase synthesis (Scheme 47) [200]. Glycosylation of the succinyl linked acceptor (linkage not via the anomeric position) was performed with the disaccharide trichloroacetimidate up to five times, again using the levulinoyl group as temporary protecting group. The yield of the coupling step could be increased to 95% by optimizing the reaction temperature to 10 "C, and only the a-glycoside was obtained. Cleavage from the linker with lithium hydro-
,
Ph,hN
OAC
BnO
,
,
~ BnO
~
~
o
NH
A
C
C
~
NH,NH,.AcOH EtOH
,
OHn
1
TrtBF,
CH,CI,.
En0
OBn
OBn
r.t.
Ogawa et al. 11991 n = 1-3
Scheme 46.
48
2 Trichloroacetimidates
OMe
Dreef-Tromp et al. I2001
n = 2-5
Scheme 47.
peroxide and further transformations yielded the hexa-, octa-, deca-, and dodecasaccharide fragments of heparan sulfate. In a recently introduced method for the synthesis of glycopeptides the peptide was synthesized from monoglycosylated amino acid derivatives followed by glycosylation reactions on the same solid phase (Scheme 48) [201]. A polyethylene glycol N,N-dimethylacrylamide copolymer (PEGA) resin was used for the synthesis of the nonapeptide acceptor. When the resin-bound acceptor was thoroughly washed and dried before subsequent glycosylation with the benzoyl-protected trichloroacetimidate, the core 3 peptide derivative was stereospecifically obtained in good yield (62%). The synthesis of a-glycosides with trichloroacetimidates bearing no participating neighboring groups is much more difficult. The reactivity of the donor has to be well adapted. Nevertheless, glycosylation of the octapeptide acceptor with the benzoyl-protected azidogalactosyl trichloroacetimidate was stereospecific, but gave the core 5 peptide derivative in only 20% yield. Use of the corresponding less reactive 0-acetyl-protected trichloroacetimidate did not work. Further examples employed disaccharide trichloroacetimidates or performed two subsequent glycosylations to synthesize branched trisaccharides. Although the direct glycosylation of OH-unprotected serine-containing peptides did not work on this resin, the use of another resin, containing no amide bonds, gave good results (see above, Scheme 39) 11731. Further applications of polymer-supported glycosylations for the synthesis of
2.4 S-Glycosides
49
Ph ,002
I
Ac-Glu-Pro-Thr-Thr-Thr-Pro-lle-Thr-Thr7 Ac-Glu-Pro-Thr-Thr-Thr-Pro-lle-Thr-Thr-
I
‘Bu
l
‘Bu
l
‘Bu
I I
‘Bu ‘Bu
Ac H Resin
CH&
AcN H i
“I3 TMSOTf “I3
b Ac-Glu-Pro-Thr-Thr-Thr-Pro-Ile-Thr-Thr-NH2
-15OC
core 3
62%
*
TMSOTf CH2CI2, -1 5OC 20%
Ac-Pro-Thr-Thr-Thr-Pro-Ile-Thr-Thr-NH,
core 5
Bock et al. iZOl1
Scheme 48.
oligosaccharide libraries by a combinatorial approach have been reported; to this end, polymer-bound acceptors with several free hydroxy groups were glycosylated randomly with trichloroacetimidates [2021.
2.4 S-Glycosides Thio-linked analogs of oligosaccharides, i. e. Lewis’, sialyl Lewis’, etc., are of interest because of their improved stability to glycosidases. The synthesis of several examples by the trichloroacetimidate glycosylation method has been described (Scheme 49) [204, 2051. Glycosylation of the 3-thio galactose acceptor with a fucosyl trichloroacctimidate gave the a-disaccharide stereospecifically; this was converted into the 3,4-dithio glucose derivative via the triflate and then substitution with inversion of the configuration. Conversion into the disaccharide trichloroacetimidate and glycosylation of heptyl mercaptan gave the thio-linked disaccharide acceptor which was coupled with the neuraminic acid disaccharide to yield the sLeX analog containing only thio linkages and a glucose instead of a glucosamine moiety. For synthesis of the LeXanalog, the disaccharide intermediate was coupled with acetobromogalactose and transformed into the trisaccharide trichloroacetimidate. Glycosylation of the thio-linked acceptor, which was prepared from the lactosyl trichloroacetimidate and heptyl mercaptan, yielded the Le’ analogdus pentasaccharide. All p-glycosidic bonds were stereospecifically formed owing to the participation of neighboring groups. Similarly the thio-linked Lewisa [205]and ganglioside GM3 [206] epitopes have been prepared.
50
2 Trichloroucetimidutes
OH OH HO
LeXanabg
Eisele, Schmidt I2031
Scheme 49.
The benzyl-protected mannosyl a-trichloroacetimidate has been used for the glycosylation of 2-mercaptopyridine (Scheme 50) [207], but the stereoselectivity was low. The P-glycoside was separated and oxidized to the 2-pyridyl sulfone which was employed as glycosyl donor for the synthesis of C-glycosides. The corresponding azidogalactose and N-acetylgalactosamine derivatives have also been prepared [208]. An S-glycosylated amino acid derivative has been synthesized by glycosylation of a D-cysteine derivative with the L-rhamnosyl trichloroacetimidate [209]. The Sglycosyl amino acid was used for the synthesis of an 5'-glycopeptide derivative of a luteinizing hormone-releasing hormone (LH-RH) antagonist. The S-glucosylated L-
2.6 C-Glycosides
51
(pa= 5:4) Beau et al. 12071
CCI,
c
HS
50%
c
Kessler et al. [2091
Scheme 50.
cysteine has been prepared applying the same method [210]. Some glycosylations of thiophenol [21 I ] and thiobenzyl alcohol [212]derivatives with trichloroacetimidates have also been reported.
2.5 N- and P-Glycosides Trichloroacetimidates are also suitable glycosyl donors for the synthesis of Nglycosides. For instance, reaction of N-phthaloyl-protected glucosamine trichloroacetimidate with trimethylsilyl azide gave, owing to neighboring group participation, only the p-glycosyl azide (Scheme 51) [213]. In the synthesis of Nglycopeptides the glucosamine azide is a suitable building block at the reducing end. After reduction of the azide to the amino group it can be linked to aspartate via amide bond formation. Another example of the synthesis of N-glycosides is the reaction of the ribopyranosyl trichloroacetimidate with 2-(3-pyrazolyl)pyridine [2141. The resulting nucleotide was employed for investigations on non-helical supramolecular nanosystems. A P-glycoside has been prepared with the hemiacetal-type trichloroacetimidate and trimethyl phosphite [2151. The resulting diastereomeric phosphonates can be considered as P-analogs of uronic acids, and their glycosides are of interest in investigations with glycosidases.
2.6 C-Glycosides Application of trichloroacetimidates to the synthesis of aromatic C-glycosides, i. e. vitexin, isovitexin, isoembigenin, etc. which are of interest because ot their physiological properties, is well established [216, 2171. A furan derivative was recently
52
2 Trichloroucetimidutes
0
&lo phq *'
NPht
CCI,
KNH
TMSN3 TMSOTf, CH2CI,, -78'C) 700,"
lto et al. k131
NPht
Eschenrnoser et al. [2141 CCI,
BnLv OPWH,),
P(OCH& TMSOTf, CH3CN, 4'C 43%
BnO
w *
OAll
+
Bgo%oAl,
OAll
OPWH,),
1:l
Vasella et al. [2151
Scheme 51.
glycosylated with glucosyl trichloroacetimidate to yield the P-glycoside stereospecifically; this served as intermediate in the synthesis of visnagine analogs (Scheme 52) [218]. The synthesis of bis(C-glycosyl) compounds [219] and of glycosides with glucosamine [220] has also been reported. Occasionally the 0-glycoside intermediates could be isolated and subjected to Fries-type rearrangement to furnish the final C-glycosides. _OBn
+
O n% 'h
O Y N H
TMSOTt CH2C12,-300C,
&CH,
0
'
OH
g
EilO
77% OBn
iCI, BnO 0 n
Schmidt et al. [2181
TMSCN
Aa&o$cl, Ad)
OAc
CH2C12, TMSOTfr.t. qu
CN
Shiozaki et al. [222,223]
References
53
A known method [221] for the preparation of C-glycosyl cyanides from trichloroacetimidates has been applied to the reaction of the 0-acetyl-protected 0glucosyl trichloroacetimidate [222, 2231. Reaction with trimethylsilyl cyanide gave the a-glycoside, a valuable intermediate in the synthesis of carboxylic acid derivatives or other C-glycosides, in quantitative yield. The reaction of trichloroacetimidates with silylenol ethers is also well established (2211. Another interesting example, reaction of benzyl-protected galactosyl trichloroacetimidate with a silyl enol ether, which can be considered as a homoalanine carbanion equivalent [224], led to the a-glycoside; as expected from previous results [221] it was formed with high stereoselectivity. The P-glycoside could be obtained by base-catalyzed isomerization. Further transformations were performed to obtain the a- and P-C--galactosyl-L-serinederivatives.
2.7 Conclusion and Outlook 0-Glycosyl trichloroacetimidates have proven to be valuable glycosyl donors for efficient syntheses of various types of glycosides. Their ease of formation and their high glycosyl donor properties released under mild acid catalysis has led to extraordinarily wide application. Not surprisingly, 0-glycosyl trichloroacetimidates have often afforded the best results in demanding glycosylation studies. The trichloroacetimidate method will, therefore, continue to advance efficient synthesis, particularly of the complex glycoconjugates which are in demand for biological and clinical studies. Thus, the trichloroacetimidate method will also contribute strongly to the future development of glycoscience.
References 1. R. R. Schmidt, Angew. Chem. 1986, 98,213-236; Angew. Chem. Int. Ed. Engl. 1986,25, 212235. 2. R. R. Schmidt, in Stereochemistry of Organic and Bioorganic Transformation-Workshop Conferences Hoechst (W. Bartmann, K. B. Sharpless, Eds), Vol. 17, pp 169-189, VCH, Weinheim, 1987). 3. R. R. Schmidt, Pure Appl. Chem. 1986,61, 1257-1270. 4. R. R. Schmidt in Comprehensive Organic Synthesis, Vol. 6, B. M. Trost, I. Fleming, E. Winterfeld, Eds., Pergamon Press, Oxford 1991, pp. 33-64. 5. R. R. Schmidt, in Carbohydrates-Synthetic Methods and Application in Medicinal Chemistry, A. Hasegawa, H. Ogura, T. Suamin, Eds., Kodanasha Scientific, Tokyo 1992, pp. 66-88. 6. K. Toshima, K. Tatsuta, Chem. Rev. 1993, 93, 1503-1531. 7. R. R. Schmidt, W. Kinzy, Adv. Carbohydr. Chem. Biochem. 1994, 50, 21-123. 8. R. R. Schmidt in Modern Methods in Curbohydrate Synthesis, S. H. Khan, R. A. O’Neill, Eds., Hanvood Academic Publishers, Chur, Schweiz, 1996, pp. 20-54. 9. R. R. Schmidt, K.-H. Jung in Preparative Carbohydrate Chemistry, S. Hannessian, Ed. Marcel Dekker, Inc., New York, 1997, pp. 283-312.
54
2 Trichloroacetimidates
10. R. R. Schmidt in Glycosciences: Status and Perspectives, H.-J. Gabius, S. Gabius, Eds., Chapman and Hall, Weinheim, 1997, pp. 31-53. 11. R. R. Schmidt, Pure Appl. Chem. 1998, 70, 397-402, 12. R. R. Schmidt, J. C. Castro-Palomino, 0. Retz, Pure Appl. Chem., in print. 13. F. Barresi, 0. Hindsgaul in Modern Synthetic Methods, B. Ernst, C. Leumann, Eds., Verlag Helvetica Chimica Acta, Basel, 1995, pp. 283-330. 14. R. R. Schmidt and J. Michel, Angew. Chem. 1980,92, 763-764; Angew. Chem., Znt. Ed. Engl. 1980, 19, 731-732. 15. R. Hoos, J. Huixin, A. Vasella, P. Weiss, Helo. Chim. Acta 1996, 79, 1757-1784. 16. V. J. Patil, Tetrahedron Lett. 1996, 37, 1481-1484. 17. S. P. Douglas, D. M. Whitfield, J. J. Krepinsky, J. Curbohydr. Chem. 1993, 12, 131-136. 18. G. G. Cross, D. M. Whitfield, Synlett 1998, 487-488. 19. H. Waldmann, G. Boehm, U. Schmid, H. Roettele, Angew. Chem. 1994,106,2024-2025 (See also Angew. Chem., Int. Ed. Engl., 1994,33, 1994-1996). 20. U. Schmid, H. Waldmann, Liebigs Ann.lRec1. 1997, 2573-2577. 21. A. Lubineau, B. J. Drouillat, J. Curbohydr. Chem. 1997, 16, 1179-1186. 22. U. Schmelzer, Dissertation, Universitat Konstanz, 1997. 23. U. Huchel, C. Schmidt, R. R. Schmidt, Eur. J. Org. Chem. 1998, 1353-1360. 24. A. Lubineau, K. Basset-Carpentier, C. Auge, Curbohydr. Res. 1997, 300, 161-167. 25. H. Paulsen, S. Peters, T. Bielfeldt, M. Meldal, K. Bock, Curbohydr. Res. 1995, 268, 17-34. 26. M.-Z. Liu, H.-N. Fan, Z.-W. Guo, Y.-Z. Hui, J. Carbohydr. Chem. 1996,15, 1139-1145. 27. T. Tsuda, S.-I. Nishimura, Chem. Commun. (Cambridge) 1996, 2779-2780. 28. J. A. L. M. van Dorst, C. J. van Heusden, A. F. Voskamp, J. P. Kamerling, J. F. G. Vliegenthart, Carbohydr. Res. 1996,291, 63-83. 29. T. Nishimura, F. Nakatsubo, Carbohydr. Res. 1996, 294, 53-64. 30. G. Limberg, G. C. Slim, C. A. Compston, P. Stangier, M. M. Palcic, R. H. Furneaux, Liebigs Ann. 1996, 1773-1784. 31. Ch. Li, B. Yu, M. Liu, Y. Hui, Curbohydr. Res. 1998, 306, 189-195. 32. S. Deng, B. Yu, Y. Hui, Tetrahedron Lett. 1998, 39, 6511-6514. 33. J. C. Castro-Palomino, R. R. Schmidt, Tetrahedron Lett. 1995,36, 5343-5346. 34. M. Lergenmuller, Y. Ito, T. Ogawa, Tetruhedron 1998, 54, 1381-1394. 35. H. Paulsen, B. Halpap, Curbohydr. Res. 1991, 216, 195-212. 36. W. Dullenkopf, J. C. Castro-Palomino, L. Manzoni, R. R. Schmidt, Carbohydr. Res. 1996, 296, 135-147. 37. W.-C. Liu, M. Oikawa, K. Fukase, Y. Suda, H. Winarno, S. Mori, M. Hashimoto, S. Kusumoto, Bull. Chem. SOC.Jpn. 1997, 70, 1441-1450. 38. M. Oikawa, A. Wada, H. Yoshizaki, K. Fukase, S. Kusumoto, Bull. Chem. SOC.Jpn. 1997, 70, 1435-1440. 39. K. Fukase, Y. Fukase, M. Oikawa, W.-C. Liu, Y. Suda, S. Kusumoto, Tetrahedron 1998, 54, 4033-4050. 40. X. Qian, 0. Hindsgaul, Chem. Commun. 1997, 1059-1060. 41. S. G. Bowers, D. M. Coe, G.4. Boons, J. Org. Chem. 1998,63,4570-4571. 42. J. C. Castro-Palomino, Dissertation, Universitat Konstanz, 1998. 43. M. R. E. Aly, J. C. Castro-Palomino, El-Sayed I. Ibrahim, El-Sayed H. El-Ashry, and R. R. Schmidt, Eur. J. Org. Chem. 1998, 2305-2316. 44. E. Meinjohanns, M. Meldal, A. Schleyer, H. Paulsen, K. Bock, J. Chem. SOC.,Perkin Trans. 1 1996, 985-993. 45. G. Blatter, J.-M. Beau, J.-C. Jacquinet, Carbohydr. Res. 1994,260, 189-202. 46. R. Kosmol, L. Hennig, P. Welzel, M. Findeisen, D. Muller, A. Markus, Van J. Heijenoort, J. Prukt. Chem. Chem.-Ztg. 1997,339, 340-358. 47. 0. Ritzeler, L. Hennig, M. Findeisen, P. Welzel, D. Mueller, Tetrahedron 1997, 53, 16651674. 48. R. U. Lemieux, R. M. Ratcliffe, Can. J. Chem. 1979,54, 1244-1251. 49. A. Vasella, C. Witzig, J.-L. Chiara, Helv. Chim. Acta 1991, 74, 2073-2077. 50. I. Toth, G. Dekany, B. Kellam, PCT Znt. Appl., WO 9838197 A1 19980903, 42 pp., (Chem. Abstr., 1998, 129, 216855).
References
55
51. H. Kunz, W. Gunther, Angew. Chem. 1988, 100, 1118-1119; Angew. Chem. Int. Ed. Engl. 1988,27, 1086-1087. 52. J. Seifert, C. Unverzagt, Tetrahedron Lett. 1996, 37, 6527-6530. 53. S. Weiler, R. R. Schmidt, Tetrahedron Lett. 1998, 39, 2299-2302. 54. A. Furstner, I. Konetzki, Tetrahedron Lett. 1998, 39, 5721-5724. 55. S. David, A. Malleron, C. Dini, Carbohydr. Res. 1989, 188, 193-200. 56. R. Preuss, R . R. Schmidt, Synthesis 1988,694-697. 57. W. R. Roush, D. P. Sebesta, R. A. James, Tetrahedron 1997,53, 8837-8852. 58. J. C. Castro-Palomino, Schmidt, R. R., Synlett 1998, 501-503. 59. M. Gelin, V . Ferrieres, D. Plusquellic, Carbohydr. Lett. 1997, 2, 381-388. 60. A. K. Choudhury, N. Roy, Carbohydr. Res. 1998, 308, 207-211. 61. H. Sekljic, N. Wimmer, A. Hofinger, H. Brade, P. Kosma, J. Chem. Soc., Perkin Trans. 1 1997, 1973-1982. 62. M. Izumi, 0. Tsuruta, S. Harayama, H. Hashimoto, J. Org. Chem. 1997, 62, 992-998. 63. H. Yuasa, S Matsuura, H. Hashimoto, Bioorg. Med. Chem. Lett. 1998, 8, 1297-1300. 64. E. P. Dubois, A. Neszmelyi, H. Lotter, V. Pozsgay, Tetrahedron Lett. 1996, 37, 3627-3630. 65. V. Pozsgay, E. P. Dubois, H. Lotter, A. Neszmelyi, Carbohydr. Res. 1997, 303, 165-173. 66. M. Ludewig, D. Lazarevic, J. Kopf, J. Thiem, J. Chem. Soc., Perkin Trans. 1 1998, 17511752. 67. R. Windmueller, R. R. Schmidt, Tetrahedron Lett. 1994, 35, 7927-7930. 68. A. Toepfer, ‘W. Kinzy, R. R. Schmidt, Liebigs Ann. Chem. 1994, 449-464. 69. L. Lay, L. Manzoni, R. R. Schmidt, Carbohydr. Res. 1998,310, 157-171. 70. L. Manzoni, L. Lay, R. R. Schmidt, J. Carbohydr. Chem. 1998,17,739-758. 71. N. L. Pohl, I-. L. Kiessling, Tetrahedron Lett. 1997, 38, 6985-6988. 72. A. Toepfer, IR. R. Schmidt, J. Carbohydr. Chem. 1993, 12, 809-822. 73. G. Hummel, R. R. Schmidt, Tetrahedron Lett. 1997,38, 1173-1 176. 74. U. Sprengard, G. Kretzschmar, E. Bartnik, C. Huels, H. Kunz, Angew. Chem., Int. Ed. Engl. 1995, 34, 990-993. 75. G. Kuznik, W. Hoersch, G. Kretzschmar, C. Unverzagt, Bioorg. Med. Chem. Lett. 1997, 7, 577-580. 76. G. Kretzschmar, W. Stahl, Tetrahedron 1998, 54, 6341-6358. 77. S. J. Danishefsky, J. Gervay, J. M. Peterson, F. E. McDonald, K. Koseki, D. A. Griffith, T. Oriyama, St. P. Marsden, J. Am. Chem. Soc. 1995, 117, 1940-1953. 78. L. Lay, R. Windmueller, S. Reinhardt, R. R. Schmidt, Carbohydr. Res. 1997, 303, 39-49. 79. J. Bartek, R. Muller, P. Kosma, Carbohydr. Res. 1998, 308, 259-273. 80. M. Izumi, 0. Tsuruta, H. Hashimoto, S. Yazawa, Tetrahedron Lett. 1996, 37, 1809-1812. 81. D. Manning, C. R. Bertozzi, S. D. Rosen, L. L. Kiessling, Tetrahedron Lett. 1996, 37, 19531956. 82. R. Roy, W. K. C. Park, 0. P. Srivastava, C. Foxall, Bioorg. Med. Chem. Lett. 1996, 6, 13991402. 83. H. Ishida, H. Hosokawa, H. Kondo, M. Kiso, A. Hasegawa, Carbohydr. Res. 1997,303, 131133. 84. G. Thoma, F. Schwarzenbach, R. 0. Duthdler, J. Org. Chem. 1996, 61, 514-524. 85. G. Kretzschmar, A. Toepfer, C. Huels, M. Krause, Tetrahedron 1997, 53, 2485-2494. 86. A. Toepfer, G. Kretzschmar, S. Schuth, M. Sonnentag, Bioorg. Med. Chem. Lett. 1997, 13171322. 87. G. Kretzschmar, Tetrahedron 1998, 54, 3765-3780. 88. T. Ikeda, T. Kajimoto, H. Kondo, C.-H. Wong, Bioorg. Med. Chem. Lett. 1997, 7, 24852490. 89. U. Greilich, R. Brescello, K.-H. J u g , R. R. Schmidt, Liebigs Ann. 1996, 663-672. 90. T. Stauch, U. Greilich, R. R. Schmidt, Liebigs Ann. 1995, 2101-2111. 91. J. C. Castro-Palomino, G. Ritter, S. R. Fortunato, S. Reinhardt, L. J. Old, R. R. Schmidt, Angew. Chern., Int. Ed. Engl. 1997,36, 1998-2001. 92. Y. Isogai, T. Kawase, H. Ishida, M. Kiso, A. Hasegawa, J. Carbohydr. Chem. 1996,15, 10011023. 93. L. F. Tietze, C. 0. Janssen, J.-A. Gewert, Eur. J. Org. Chem. 1998, 1887-1894.
56
2 Trichloroacetimidates
94. J. M. Lassaletta, K. Carlsson, P. J. Garegg, R. R. Schmidt, J. Org. Chem. 1996, 61, 68736880. 95. J. M. Lassaletta, R. R. Schmidt, Tetrahedron Lett. 1995, 36, 4209-4212. 96. J. M. Lassaletta, R. R. Schmidt, Liebigs Ann. 1996, 1417-1423. 97. N. Mathieux, H. Paulsen, M. Meldal, K. Bock, J. Chem. Soc., Perkin Trans. I 1997, 23592368. 98. D. Qiu, Koganty, R. Rao, Tetrahedron Lett. 1997, 38, 961-964. 99. D. Qiu, Koganty, R. Rao, Tetrahedron Lett. 1997, 38, 45-48. 100. Y. Nakahara, Y. Nakahara, Y. Ito, T. Ogawa, Tetrahedron Lett. 1997, 38, 7211-7214. 101. Y. Nakahara, Y. Nakahara, Y. Ito, T. Ogawa, Curbohydr. Res. 1998,309, 287-296. 102. C. Unverzagt, Angew. Chem. 1994, 106, 1170-1173 (See also: Angew. Chem., Int. Ed. Engl., 1994,33, 1102-1 104. 103. C. Unverzagt, Angew. Chem., Int. Ed. Engl. 1996, 35, 2350-2353. 104. C. Unverzagt, Angew. Chem., h t . Ed. Engl. 1997, 36, 1989-1992. 105. H. Paulsen, M. Springer, F. Reck, E. Meinjohanns, I. Brockhausen, H. Schachter, Liebigs Ann. 1995, 53-60. 106. Z. Szurmai, L. Janossy, Curbohydr. Res. 1996,296, 279-284. 107. Y. Nakahara, S. Shibayama, Y. Nakahara, T. Ogawa, Curbohydr. Res. 1996,280, 67-84. 108. L. Rochepeau-Jobron, J.-C. Jacquinet, Curbohydr. Res. 1998, Volume Date 1997, 305, 181191. 109. L. Rochepeau-Jobron, J.-C. Jacquinet, Carbohydr. Res. 1997,303, 395-406. 110. H. Uzawa, M. Gerz, Y. Ito, T. Ogawa, Curbohydr. Lett. 1996,2, 197-204. 111. G. Blatter, J.-C. Jacquinet, Curbohydr. Res. 1996,288, 109-125. 112. K. W. Neumann, J.4. Tamura, T. Ogawa, Glycoconjugute J . 1996, 13, 933-936. 113. J.-i. Tamura, K. W. Neumann, T. Ogawa, Liebigs Ann. 1996, 1239-1257. 114. J. Kovensky, Ph. Duchaussoy, M. Petitou, P. Sinay, Tetrahedron: Asymmetry 1996, 7, 31193128. 115. A. Helmboldt, M. Petitou, J.-M. Mallet, J.-P. Herault, J.-C. Lormeau, P.-A. Driguez, J.-M. Herbert, P. Sinay, Bioorg., Med. Chem. Lett. 1997, 7 , 1507-1510. 116. R. C. Buijsman, W. H. A. Kuijpers, J. E. M. Basten, E. Kuyl-Yeheskiely, G. van der Marel, C. A. A. van Boeckel, J. H. van Boom, Chem. Eur. J. 1996,2, 1572-1577. 117. J. E. M. Basten, C. M. Dreef-Tromp, B. De Wijs, C. A. A. Van Boeckel, Bioorg. Med. Chem. Lett. 1998, 8, 1201-1206. 118. J. C. Castro Palomino, M. Hernandez Rensoli, V. Verez Bencomo, J. Curbohydr. Chem. 1996, 15, 137-146. 119. W. Liao, D. Lu, Curbohydr. Res. 1996,296, 171-182. 120. G. Schuele, T. Ziegler, Tetrahedron 1996, 52, 2925-2936. 121. G. Schuele, T. Ziegler, Liebigs Ann. 1996, 1599-1607. 122. M.-J. L. Thijssen, M. H. G. Bijkerk, J. P. Kamerling, J. F. G. Vliegenthart, Curbohydr. Res. 1998,306, 111-125. 123. W. Zou, H. J. Jennings, Bioorg. Med. Chem. Lett. 1997, 7 , 647-650. 124. W. Zou, J.-R. Brisson, Q.-L. Yang, M. van der Zwan, H. J. Jennings, Curbohydr. Res. 1996, 295, 209-228. 125. F.-I. Auzanneau, F. Forooghian, B. M. Pinto, Curbohydr. Res. 1996,291, 21-41. 126. V. Pozsgay, Angew. Chem., Int. Ed. 1998,37, 138-142. 127. V. Pozsgay, J. Org. Chem. 1998,63, 5983-5999. 128. T. Ziegler, E. Eckhardt, D. Keller, J. Curbohydr. Chem. 1997, 16, 719-738. 129. A. Banaszek, Curbohydr. Res. 1998, 306, 379-385. 130. M. K. Gurjar, S. Adhikari, Tetrahedron 1997, 53, 8629-8634. 131. I. Bajza, K. E. Kover, A. Liptak, Curbohydr. Res. 1998, 308, 247-258. 132. Y. Ogawa, P. Kovac, Glycoconjugute J. 1997, 14,433-438. 133. T. G. Mayer, R. R. Schmidt, Liebigs Ann./Recl. 1997, 859-863. 134. B. Kratzer, T. G. Mayer, R. R. Schmidt, Eur. J. Org. Chem. 1998, 291-298. 135. T. G. Mayer, B. Kratzer, R. R. Schmidt, Angew. Chem. 1994, 106, 2289-2293. 136. J. Ye, W. T. Doerrler, M. A. Lehrman, J. R. Falck, Bioorg. Med Chem. Lett. 1996, 6, 17151718.
References
57
137. W. Frick, G. Mueller, Ger. Offen., 1998, DE 19649350 A1 19980604,20 pp. (Chem. Abstracts 129 : 54536). 138. R. R. Schmidt and P. Zimmermann, Angew. Chem. 1986,98,722-723; Angew. Chem., Int. Ed. Engl. 1986,25, 725-726. 139. P. Zimmerniann, R. Bommer, T. Bar, R. R. Schmidt, J. Curbohydr. Chem. 1988, 7, 435452. 140. H. Ishida, H . Ando, H. Ito, H. Ishida, M. Kiso, A. Hasegawa, J. Curbohydr. Chem. 1997, 16, 413-428. 141. A. Hasegawa, H. Ishida, Y. Isogai, H. Ishida, M. Kiso, J. Curbohydr. Chem. 1993, 12, 12171222. 142. H. Ando, H. Ishida, M. Kiso, A. Hasegawa, Curbohydr. Res. 1997, 300, 207-217. 143. H. Ishida, H . Ishida, M. Kiso, A. Hasegawa, Curbohydr. Res. 1994, 260, ClLC6. 144. H. Ishida, H . Ishida, M. Kiso, A. Hasegawa, Tetrahedron; Asymmetry 1994, 5, 2493-2512. 145. K. Hotta, H. Ishida, M. Kiso, A. Hasegawa, J. Curbohydr. Chem. 1995, 14, 491-506. 146. H. Ishida, R. Miyawaki, M. Kiso, A. Hasegawa, J. Curbohydr. Chem. 1996, 15, 163-182. 147. Y. Isogai, H. Ishida, M. Kiso, A. Hasegawa, J. Curbohydr. Chem. 1996, 15, 1119-1137. 148. K. Yoshino. H. Ohmoto, N. Kondo, H. Tsujishita, Y. Hiramatsu, Y. Inoue, H. Kondo, H. Ishida, MI. Kiso, A. Hasegawa, J. Med. Chem. 1997, 40, 455-462. 149. M. Wilstermann, L. 0. Kononov, U. Nilsson, A. K. Ray, G. Magnusson, J. Am. Chem. Soc. 1995,117,4742-4754. 150. M. Wilstermann, G. Magnusson, J. Org. Chem. 1997,62, 7961-7971. 151. S. Fujita, M. Numata, M. Sugimoto, K. Tomita, T. Ogawa, Curbohydr. Res. 1994,263, 181-196. 152. M. Iida, A. Endo, S. Fujita, M. Numata, K. Suzuki, S. Nunomura, T. Ogawa, Glycoconjugute J. (1996), 13,203-21 1. 153. N. Chida, N. Sakata, K. Murai, T. Tobe, T. Nagase, S. Ogawa, Bull. Chem. Soc. Jpn. 1998, 71, 259-272. 154. T. Sugata, Y. Kan, Y . Nagaregawa, T. Miyamoto, R. Higuchi, J. Curbohydr. Chem. 1997,16, 917-925. 155. A. Hasegawa, N. Suzuki, H. Ishida, M. Kiso, J. Curbohydr. Chem. 1996,15,623-637. 156. Y . Wada, T. Saito, N. Matsuda, H. Ohmoto, K. Yoshino, M. Ohashi, H. Kondo, H. Ishida, M. Kiso, A. Hasegawa, J. Med. Chem. 1996,39,2055-2059. 157. H. Ishida, H Hosokawa, H. Kondo, M. Kiso, A. Hasegawa, Curbohydr. Res. 1997,303, 131133. 158. E. Tanahashi, K. Murase, M. Shibuya, Y. Igarashi, H. Ishida, A. Hasegawa, M. Kiso, J. Curbohydr. Chem. 1997, 16, 831-858. 159. T. Ikami, H. Hamajima, T. Usui, T. Mitani, H. lshida, M. Kiso, A. Hasegawa, J. Curbohydr. Chem. 1997,16, 859-875. 160. T. Ikami, N. Tsuruta, H. Inagaki, T. Kakigami, Y. Matsumoto, N. Tomiya, T. Jomori, T. Usui, Y. Suzuki, H. Tanaka, D. Miyamoto, H. Ishida, A. Hasegawa, M. Kiso, Chem. Phurm. Bull. 1998, 46, 797-806. 161. K. Adachi, Y. Yamada, H. Wada, A. Kameyama, H. Ishida, M. Kiso, J. Curbohydr. Chem. 1998, 17, 595-607. 162. 2. Zhang, G. Magnusson, J. Org. Chem. 1995,60, 7304-7315. 163. 2. Zhang, G. Magnusson, J. Org. Chem. 1996,61, 2383-2393. 164. P. I. Kitov, C:. Railton, D. R. Bundle, Curbohydr. Res. 1998, 307, 361-369. 165. S. Kotter, U. Krallmann-Wenzel, S. Ehlers, T. K. Lindhorst, J. Chem. Soc., Perkin Truns. I 1998,2193-2200. 166. R. R. Schmidt, K. Jankowski, Liebigs Ann. 1996, 867-879. 167. W. Kinzy, R R. Schmidt, Curbohydr. Res. 1987,164,265-276. 168. X.-T. Chen, ID. Sames, S. J. Danishefsky, J. Am. Chem. Soc. 1998, 120, 7760-7769. 169. D. Sames, X.-T. Chen, S. J. Danishefsky, Nature (London) 1997,389, 587-591. 170. J. E. Yule, T. C. Wong, S. S. Gandhi, D. Qiu, M. A. Riopel, Koganty, R. Rao, Tetrahedron Lett. 1995, 36, 6839-6842. 171. D. Qiu, S. S. Gandhi, Koganty, R. Rao, Tetrahedron Lett. 1996, 37, 595-598. 172. U. K. Saha, R. R. Schmidt, J. Chem. Soc., Perkin Truns. 11997, 1855-1860.
58
2 Trichloroacetimidates
173. A. Schleyer, M. Meldal, R. Manat, H. Paulsen, K. Bock, Angew. Chem., Int. Ed. Engl. 1997, 36, 1976-1978. 174. K. M. Depew, S. M. Zeman, S. H. Boyer, D. J. Denhart, N. Ikemoto, S. J. Danishefsky, D. M. Crothers, Angew. Chem., Int. Ed. Engl. 1997, Volume Date 1996,35, 2797-2800. 175. S. H. Olson, S. Danishefsky, Tetrahedron Lett. 1994, 35, 7901-7904. 176. K. C. Nicolaou, K. Ajito, H. Komatsu, B. M. Smith, P. Bertinato, L. Gomez-Paloma, Chem. Commun. (Cambridge) 1996, 1495-1496. 177. K. C. Nicolaou, F. L. van Delft, S. R. Conley, H. J. Mitchell, Z. Jin, R. M. Rodriguez, J. Am. Chem. SOC.1997,119,9057-9058. 178. K. C. Nicolaou, H. J. Mitchell, F. L. Van Delft, F. Rubsam, R. M. Rodriguez, Angew. Chem., Int. Ed. 1998, 37, 1871-1874. 179. T. Kaneko, K. Takahashi, M. Hirama, Heterocycles 1998, 47, 91-96. 180. A. G. Myers, J. Liang, M. Hammond, P. M. Harrington, Y. Wu, E. Y. Kuo, J. Am. Chem. SOC.1998,120, 5319-5320. 181. K. C. Nicolaou, F. L. van Delft, T. Ohshima, D. Vourloumis, J. Xu, S. Hosokawa, J. Pfefferkorn, S. Kim, T. Li, Tianhu, Angew. Chem., Int. Ed. Engl. (1997), 36, 2520-2524. 182. K. C. Nicolaou, T. Oshima, S. Hasokawa, F. L. van Delft, D. Vourloumis, J. Y. Xu, J. Pfefferkorn, S. Kim, J. Am. Chem. SOC.1998, 120, 8674-8680. 183. W. R. Roush, K. Briner, B. S. Kesler, M. Murphy, D. J. Gustin, J. Org. Chem. 1996, 61, 6098-6099. 184. Z.-H. Jiang, R. R. Schmidt, Liebigs Ann. Chem. 1994, 645-651. 185. Z.-H. Jiang, A. Geyer, R. R. Schmidt, Angew. Chem., Int. Ed. Engl. 1995,34, 2520-2524. 186. D. P. Larson, C. H. Heathcock, J. Org. Chem. 1997,62, 8406-8418. 187. D. P. Larson, C. H. Heathcock, J. Org. Chem. 1996, 61, 5208-5209. 188. R. T. Brown, S. P. Mayalarp, J. J. Watts, Chem. Soc., Perkin Trans. 11997, 1633-1637. 189. R. R. Schmidt, B. Wegmann, and K.-H. Jung, Liebigs Ann. Chem. 1991, 121-124. 190. R. R. Schmidt, H. Braun, K.-H. Jung, Tetrahedron Lett. 1992, 33, 1585-1588. 191. T. J. Martin, G. Dufner, B. Kratzer, R. R. Schmidt, GlycoconjugateJ . 1996, 13, 547-553. 192. R. Wang, D. H. Steensma, Y. Takaoka, J. W. Yun, T. Kajimoto, C.-H. Wong, Bioorg. Med. Chem. 1997,5,661-672. 193. J. Rademann, R. R. Schmidt, J. Org. Chem. 1997, 62, 3650-3653. 194. J. Rademann, A. Geyer, R. R. Schmidt, Angew. Chem., Znt. Ed. 1998,37, 1241-1245. 195. J. Rademann, R. R. Schmidt, Tetrahedron Lett. 1996,37, 3989-3990. 196. L. Jiang, R. C. Hartley, T.-H. Chan, Chem. Commun. (Cambridge) 1996, 2193-2194. 197. A. Heckel, E. Mross, K.-H. Jung, J. Rademann, R. R. Schmidt, Synlett 1998, 171-173. 198. M. Adinolfi, G. Barone, L. De Napoli, A. Iadonisi, G. Piccialli, Tetrahedron Lett. 1998, 39, 1953-1956. 199. H. Shimizu, Y. Ito, 0. Kanie, T. Ogawa, Bioorg. Med. Chem. Lett. 1996, 6, 2841-2846. 200. C. M. Dreef-Tromp, H. A. M. Willems, P. Westerduin, P. van Veelen, C. A. A. van Boeckel, Bioorg. Med. Chem. Lett. 1997, 7, 1175-1180. 201. H. Paulsen, A. Schleyer, N. Mathieux, M. Meldal, K. Bock, J. Chem. SOC.,Perkin Trans. 1 1997,281-293. 202. Y. Ding, J. Labbe, 0. Kanie, 0. Hindsgaul, Bioorg. Med. Chem. 1996,4,683-692. 203. Th. Eisele, R. R. Schmidt, Liebigs Ann.jRecl. 1997, 1303-1313. 204. Th. Eisele, A. Toepfer, G. Kretzschmar, R. R. Schmidt, Tetrahedron Lett. 1996, 37, 13891392. 205. Th. Eisele, R. Windmuller, R. R. Schmidt, Carbohydr. Res. 1998, 306, 81-91. 206. Th. Eisele, R. R. Schmidt, Liebigs Ann.jRecl. 1997, 865-872. 207. T. Skrydstrup, 0. Jarreton, D. Mazeas, D. Urban, J.-M. Beau, Chem. Eur. J. 1998, 4, 655671. 208. D. Urban, T. Skrydstrup, J.-M. Beau, J. Org. Chem. 1998, 63, 2507-2516. 209. K. Michael, V. Wittmann, W. Konig, J. Sandow, H. Kessler, Int. J. Pept. Protein Res. 1996, 48, 59-70. 210. L. Kaesbeck, H. Kessler, Liebigs Ann./Recl. 1997, 165-167. 211. E. Bozo, S. Boros, J. Kuszman, Carbohydr. Res. 1998, 311, 191-202. 212. H. C. Hansen, G. Magnusson, Carbohydr. Res. 1998,307, 243-251.
References
59
213. A. Dan, M. Lergenmuller, M. Amano, Y. Nakahara, T. Ogawa, Y. Ito, Chem. Eur. J. 1998, 4, 2182-2190. 214. A. Eschenmoser, C. Miculka, N. Windhab, H.-U. Hoppe, PCTZnt. Appl., 1998, WO 9825943 A1 19980618, 26 pp., (Chem. Abstracts, 129: 67987). 215. R. Hoos, J. Huixin, A. Vasella, P. Weiss, Helv. Chim. Acta 1996, 79, 1757-1784. 216. J.-A. Mahling, R. R. Schmidt, Synthesis, 1993, 325-328. 217. J.-A. Mahling, K.-H. Jung, R. R. Schmidt, Liebigs Ann. 1995, 461-466. 218. E. El Telbani, S. El Desoky. M. A. Hammad, A. R. H. A. Rahman, R. R. Schmidt, Eur. J. Org. Chem. 1998,2317-2322. 219. E. El Telbani, S. El Desoky, M. A. Hammad, A. H. Abdel Rahman, R. R. Schmidt, Carbohydr. Rex 1998, 306, 463-461. 220. J. C. Castro-Palomino, R. R. Schmidt, Liebigs Ann. 1996, 1623-1626. 221. M. Hoffmdnn, R. R. Schmidt, Liebigs Ann. Chem. 1985, 2403-2419. 222. M. Shiozaki, T. Mochizuki, T. Wakabayashi, S.4. Kurakata, T. Tatsuta, M. Nishijima, Tetrahedron Lett. 1996, 37, 7271-7274. 223. M. Shiozaki, S.4. Kurakata, T. Tatsuta, H. Maeda, M. Nishijima, Tetrahedron 1997, 53, 16041-1 6060. 224. A. Dondoni, A. Marra, Chem. Commun. (Cambridge) 1998, 1741-1742.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
3 Iterative Assembly of Glycals and Glycal Derivatives: The Synthesis of Glycosylated Natural Products and Complex Oligosaccharides Lawrence J. Williams, Robert M. Garbaccio, and Samuel J. Danishefsky
3.1 Introduction Polypeptides, oligonucleotides and oligosaccharides are three classes of oligomers that play key roles in transacting the processes of life. While the functions of proteins and DN,4 have been appreciated, at least qualitatively, for over fifty years, basic information as to the vital missions of carbohydrates, beyond that of energy storage, are still being elucidated. Thus, the roles of oligosaccharides in cellular adhesion, protein folding, cell signaling, fertilization, pathogen binding and inflammation have been recognized only recently. The detection, purification and structure determination of complex oligosaccharides have been critical in the emergence of glycobiology. The continuing maturation of this science has added to the incentives for preparing complex oligosaccharide conjugates in homogeneous form. Among these incentives is the possibility of evaluating vaccines comprised of synthetic, cell free, carbohydrate-based tumor antigens for enhancing immune response to cancers which bear such cell surface epitopes. In addition to the necessity for such homogeneous constructs, the preparation of complex oligosaccharides poses no small challenge to the science of chemical synthesis. While the successes achieved in oligonucleotide and oligopeptide synthesis arose only after long term and exacting research, the issues associated with oligosaccharide synthesis are of a much higher level of complexity. In assembling either amide bonds for polypeptide construction, or internucleotide phosphate bonds in DNA synthesis, no new stereochemical issues are encountered. By contrast, each glycosylation reaction in oligosaccharide construction involves a question of stereochemical outcome at the anomeric site (a or P-linkage, Figure 1). Moreover, every glycosylation event requires identification of a specific hydroxyl group to serve as an acceptor center (Scheme 1, A). In a typical hexose, the pinpointing of an acceptor site obliges one to identify one of the five potential hydroxyl groups to serve as the reaction locus for glycosylation. In some instances, this specificity or selectivity of action may reflect inherent reactivity margins between free
62
3 Iterative Assembly of Glycals and Glycal Derivatives
Oligopeptides
Oligonucleotides
Oligosaccharides
RO
Glycoside Bond
1-
RO RO RO
OR
Figure 1.
hydroxyl groups. Alternatively and much more commonly, oligosaccharide construction involves extensive reliance on the use of protecting group strategies. For purposes of reiteration, a specific protecting group at the reducing end of an oligosaccharide, having been brought in at the glycosyl acceptor stage, would then be converted into a donating group. In addition to the requirement for manipulating the acceptor and donor character of the terminal hexose with each elongation cycle, the need for inclusion of N-acetyl functions, as well as occasional C-methyl, 0-methyl, 0-acyl, or sulfated alcohol groups, not to speak of sialic acid units, serves to further complicate an already daunting set of challenges. It was with these considerations in mind that we began to explore the possible advantages of a new strategy, ie. glycal assembly, to facilitate oligosaccharide construction [l]. While the use of glycals as direct glycosyl donors or precursors of glycosyl donors had been practiced long before our entry to the field in 1988, it seemed that several potentially interesting and promising synthetic opportunities had not been recognized. Upon casual inspection, it appeared that the complexities associated with differential hydroxyl protection would be significantly reduced in a glycal. Thus, in a hexose glycal, only three hydroxyls need be distinguished (Scheme 1, B). Further-
3.1 Introduction
63
A
1
1
a
-
O
H _L Po
HO
--+bop :bop OP
PO
OH
+
PO
Po
OP
OP
HO
Ohgosaccharide
4
X PO
OP Po
OP
B Po
E+
PO Po
Po
Reiterate Ohgosaccharide
4
Po
Scheme 1.
more, each hyclroxyl could well differ from the others in its expected reactivity, since one is primary.,one is allylic, and the other is a more hindered secondary alcohol. It seemed, therefore, that the need for selective protection could be lessened and those protections which are needed could be simplified in the context of glycals. The most significant point of departure of ‘glycal assembly’ originated from the use of glycals as both glycosyl donors and glycosyl acceptors. This two modality
64
3 Iterative Assembly of Glycals and Glycal Derivatives
usage sets up the possibility of synthesizing polysaccharides by the assembly of glycals in an iterative fashion. In this approach, the activated, glycal-derived donor would be captured by a glycal acceptor in a glycosylation reaction (Scheme 1, B). The glycal linkage of the newly generated disaccharide, though initially introduced as a glycosyl acceptor, could now serve as a donor for the next elongation cycle. Thus, glycals would have the potential advantage that coupling maneuvers could be implemented, and reiterated, with minimal protecting group manipulation. It is well to emphasize that the success of the glycal paradigm could not have been assumed in advance of experiment. Thus, employment of a glycal-housed hydroxyl as the glycosyl acceptor means that the sensitive enol ether linkage, bearing a potential allylic leaving group at C3, must withstand the action of the promoter required for the glycosylation event. Yet, glycal linkages bearing such leaving groups are often subject to Ferrier rearrangement under conditions similar to those that would be used in glycosylation. Hence, it was necessary to find promoter conditions that were effective for glycosylation while consistent with survival of the acceptor glycal. Through the continued investigation of these ideas, we have developed a broad based approach to synthesize complex oligosaccharides, including glycosylated natural products, via glycals. In the context of specific target structures, the program has served as an incentive for exploring new issues of chemical reactivity. For the purpose of this review, the program description will be confined to examining, and demonstrating by example, the feasibility of these theorems in the context of selected total syntheses of polysaccharides and their conjugates. While there is a unified theme governing this chapter, i.e. that of fostering progression of the methodology and strategy of synthesis of complex glycans, the discussion is divided into several parts. For purposes of presentation, each of the first four sections introduces a key methodological advance in glycal technology, typically under the umbrella of the natural product that inspired them. The oligosaccharide targets discussed in the last three sections demonstrate the integrated application of these methodologies, and the iterative glycal assembly strategy, to access tumor-associated oligosaccharides used in our expanding anti-cancer vaccine program.
3.2 Ciclamycin 0 The ciclamycin complex of antibiotics possesses high inhibitory activity against experimental tumors [2] and is claimed to be of value against human neoplasia [3]. These antibiotics are largely or entirely composed of q- and 8-pyrromycinones joined at C7 by glycosidic linkages to variously permuted carbohydrate domains [4, 51. One component of this complex, which is particularly difficult to obtain, is ciclamycin 0. At the time of our investigation, the structural assignment for 1 was based solely on spectroscopic arguments. Our strategy focused on construction of an appropriately protected trisaccharide and its use as a donor to achieve glycosylation of the anthracyclinone nucleus.
3.2 Ciclamycin 0
OH 0
OH
65
P
e &eM
1 Ciclamycin 0
0
Figure 2.
Given our interest in iterative assembly, a trisaccharide glycal was envisioned to be the target. The requirement for a-linked 2-deoxy hexoses prompted the utilization of the iodoglycosylation reaction developed by Lemieux [6] and Thiem [7] (Scheme 2). Adapting this methodology to iterative glycal assembly required strict control of the roles of glycals, since substochiometric amounts of iodine could, in principle, react with either the donor glycal or the acceptor glycal resulting in coincident symmetrical coupling and possibly polymerization. It had long been known that acyl protecting groups lower the reactivity of glycosy1 donors [gal, but the idea of exploiting this effect in the coupling of two potential glycosyl donors had not been conceptualized until the seminal experiments of Fraser-Reid [Sb]. Consequently, it was hoped that the nature of such a coupling could be controlled by the resident substituents on two different glycals [9]. Glycal6 has its oxygen atoms alkylated, while those of 7 are acylated. Consistent with expectations, glycal 6 proved to be more reactive towards the iodonium electrophile, thereby generating the active glycosyl donor selectively in the presence of acceptor 7 (Scheme 3). Glycal 7 has a free hydroxyl group and, therefore, can act as the only acceptor. Iodinative coupling of 6 and 7, via trans-diaxial attack, gave rise to iodoglycoside 8 (58%). The C3 hydroxyl, allylic to the double bond of the glycal, is the most obvious position where the electronic difference between and acyloxy and an alkoxy group might be manifested. However, the clean and successful coupling of 6 and 9 to give 10 (76%) indicates that even without an acyloxy group at C3, subtle effects can be responsible for an ordered glycosylation.
2
Scheme 2.
3
4
66
3 Iterative Assembly of Glycals and Glycal Derivatives
B BnOn
O
g
+
no
O
S
/
+
O
e
b -
I+ 58%
BZos HO
6
B BnOn
O
7
6
BB n
HBZO
9
e
+
8
10
-
BZO
11
Scheme 3.
A true test for this strategy of controlling glycal-glycal coupling arrived with the total synthesis of ciclamycin 0 [lo], which embodied the additional feature of containing 2-deoxyhexoses (Scheme 4) [l 11. Coupling of benzoylated acceptor 14 to benzylated donor 13, followed by Ph3SnH reduction of the resultant iodide gave the 2-deoxy-disaccharide 15 in 57% yield. Following protecting group adjustment, silylated donor 16 was coupled to the same benzoylated acceptor 14 followed again by Ph$nH reduction, in 54% overall yield. Manipulation of the resulting trisaccharide into a suitable glycal donor was followed by a successful coupling to Epyrromycinone (19), which gave a mixture of a-linked iodides 20 in 60% overall yield. Protecting group removal and iodide reduction afforded ciclamycin 0. Up to this time, the direct coupling of an antrhacyclinone to a fully synthetic oligosaccharide en route to a naturally occurring antibiotic had not been accomplished
WI. 3.3 Allosamidin Allosamidin (21, Figure 3) was identified as a selective and powerful chitinase inhibitor in 1987 [13]. As such, it generated interest as a potential insecticide and fungicide. Allosamidin is composed of a 3,3’-epi-chitobiose P-linked to a novel aglycone sector termed ‘allosamizoline’ [ 141. This unique structure is believed to impart to it the function of a transition-state mimic for the hydrolysis of chitin [15]. The possibility that the electrophilic nature of the oxazoline linkage could allow it
3.3 Allosamidin
>
-&
67
->
OH Bno
14
13
MePo --
1. I+(symcoll)*cIo,&
2. PhaSnH, 54%
BnO 16
15
14
BnO OH 0
Mw 0
I+(symcoll)2c104-
>
19
18
20
60% M0
e
d
o
Scheme 4.
to function as a potential locus for covalent attachment to the chitin-degrading enzyme was also considered [16]. The combination of the interesting structure, valuable biological activity and scarcity in nature of allosamidin prompted our attempts at its total synthesis. In the contlext of carbohydrate synthesis, a key feature of this effort included a novel and efficient synthesis of axial glycal derivatives utilizing a Ferrier-type rearrangement [ 171 followed by a [2,3]-sigmatropic rearrangement [18, 191. The allosamidin effort also spurred development of the method of sulfonamidoglycosylation [20] for the construction of P-linked 2-aminohexoses. Following the invention and
21 Allosamidin
Figure 3.
3 Iterative Assembly of Glycals and Glycal Derivatives
68
50% overall SPh 22
OPC
23
25
24
Scheme 5.
application of methods addressed to these ends, an expeditious synthesis of allosamidin was enabled. A general objective of the efforts in this area was an efficient route to glycals bearing C3 axial alcohol derivatives in the context of a range of substituents at C4. Glycals of this type had been difficult to synthesize [21] and rapid access to such derivatives would broaden the expanding collection of glycosyl donors and acceptors that could be incorporated into the assembly program. We found that Ferriertype rearrangement of tri-0-acetyl-D-gluca122 with thiophenol yielded 23 (Scheme 5) [22]. Oxidation of the a-(pheny1thio)pseudoglycal with DMDO and exposure of the resultant sulfoxide (24) to diethylamine triggered a [2,3]-sigmatropicrearrangement which resulted in the desired allal 25 in 96% yield. This sequence was expanded to include both allal and gulal derivatives as well as a range of substituents at C4. The issue of access to 2-aminohexose moieties has been impressively addressed by the azidonitration protocol of Lemiuex [23] and relies on the character of the C2 substituent to direct the outcome of any subsequent glycosylation. In this regard, azidonitration, while far from ideal in terms of stereoselection, remains one of the most useful methods to access the 2-amino-a-linkage. We, however, sought a new, direct method for introducing equivalent functionality through which stereoelectronic factors, rather than local steric factors, would determine the outcome of subsequent glycosylation. To this end, a key reaction, sulfonamidoglycosylation, was developed [22] (Scheme 6).
I(syn-Colli)zCi04
RRO o
> -HzNSO2P or h BrZNSOzPh
e
NH41
1
Scheme 6.
LTMP, AgOTf > -
RO
Ph02SNH 27 X = Br, I
26
28
R:q
29
30
3.4 KS-502 and Rebeccamycin
Bny% BnO
69
OSugar Ph02SNH 31
PhOzSNH
32 52%
34
64%
23%
Scheme 7.
This method, which involves trans diaxial addition of an N- halobenezesulfonamide to a glycal, leads to the 1-aminohexose 27. Alternatively, the action of IDCP (iodonium syn-collidine perchlorate) followed by benzenesulfonamide provides the same type of 2-halo-1-aminohexose. Treatment of 27 with base, likely proceeds through the 1,2-sulfonyloaziridine 28, which then reacts quickly and selectively with acceptors to form a P-linkage at the anomeric carbon. The scope of this general method was shown to include glycal- and nonglycal-containing carbohydrates as potential acceptors. Donors could include di- or higher saccharides terminating in a glycal linkage (Scheme 7). An additional benefit of this method was the finding that the intermediate halosulfonamides were often stable crystalline solids. The efficiency of coupling was later enhanced by the combination of stannylalkoxides in the presence of AgBF4 for the actual glycosylation reaction [24]. Furthermore, an iodosulfonamide can be readily converted to a corresponding ethyl thioglycoside, which in turn functions as a useful and alternative glycosyl donor. The adaptability of the sulfonamidoglycosylation to the iterative mode (glycals as donors and acceptors) made it particularly attractive for the synthesis of allosamidin (Scheme 8) [25]. The route to allosamidin utilized the sulfonamidoglycosylation for both glycosylation reactions, as well as the [2, 31 sigmatropic rearrangement methodology for both hexoses. Bromosulfonamidation of 36 (63%) followed by direct rollover with KHMDS in the presence of glycal 38 gave the P-linked disaccharide 39 in 81% yield. In turn, 39 then served as a donor in a second azaglycosylation with the aglycon derivative 41 to afford, after several steps, allosamidin (21). The efficiency of this synthesis served to reflect the power of iterative glycal assembly.
3.4 KS-502 and Rebeccamycin Glycosylated natural products, and other carbohydrate conjugates, present a challenging problem to synthetic chemists who must modify traditional carbohydrate
3 Iterative Assembly of Glycals and Glycal Derivatives
70
BnO
B n O KHMDS, 30%
-
-w
21 Allosarnidin
Scheme 8.
domain methodology to tolerate greater diversity present in the non-carbohydrate sector. In principle, incompatibility of synthetic methods to reach one domain can often limit options along a proposed route. KS-502 (43) and rebeccamycin (44) posed precisely this challenge and became the targets of our synthetic efforts in 1992-1 993. KS-502 (43) was isolated in 1989 [26] and identified as a specific inhibitor of calmodulin-dependent cyclic-nucleotide phosphodiesterase (CaM-PDE). The important roles of CaM and CaM-PDE in cultured cells and living systems underscored the utility of KS-502, perhaps as a valuable biological tool. Its structure comprises an interesting aryl furanoside for which new methodology was devised. Rebeccamycin (44)was shown to have antitumor activity against P388 leukemia, L1210 leukemia and B16 melanoma implanted in mice [27]. It is believed to impart this effect through the single strand cleavage of DNA. Water-soluble analogues Ho*
n-C7H 15
OH 42 KS 501 R = H 43 KS 502 R = C02H
Figure 4.
3.4 KS-502 and Rebeccamycin
71
have shown increased potency and are currently advancing through clinical trials [28]. The rebeccamycin structure consists of a symmetrical indolocarbazole chromophore P-N-linked to a 4-0-methylglucose residue. Synthetic efforts had concentrated on the aglycon portion of rebeccamycin, and an effective general methodology for the glycosylation step was sought to facilitate the total synthesis of this family of antitumor agents. While a unifying solution was pursued for these two glycoconjugate problems, total synthesis was not the sole inspiration for such efforts. Though sulfonamidoglycosylation provided routes from glycals to 2-aminoglycosides [ 14, 161, there was a more general need for a reliable and far-reaching route that would serve to convert glycals to common glycosides of glucose, galactose and mannose. The trichloroacetimidate donor methodology advanced by Schmidt [29], probably the single most powerful glycosylation method available, can often provide selective access to either a- or P-glycosides. We alternatively focussed on the application of glycal epoxides to tackle these and other problems. Such epoxides can participate in iterative strategies and can provide, in some circumstances, strikingly direct routes to polysaccharides. The existing record of a-epoxides as stereoselective glycosylating reagents was, at best, unimpressive when we launched our study. Access to such 1,2-anhydrosugar derivatives was awkward and their applications to glycosylations were beset by serious problems in stereoselectivity [30]. We first sought a direct and reliable route to 1,2-anhydrosugars. Furthermore, we hoped to gain advantages at the level of stereoselectivity by careful selection of resident functions. In particular, it was felt that resident groups bearing neighboring group participating functions, would tend to undercut clean formation of P-glycoside from a-epoxides. A major advance toward this goal came with the recognition that a variety of glycals react smoothly with 3,3-dimethyldioxirane (DMDO) [311. Glucal derivative 45 reacted with DMDO to give near quantitative yields of 46 and with >20: 1 aselectivity (Scheme 9). Galactal derivative 47 yielded the same result. In the allal series, glycal 49 underwent selective epoxidation instead from the P-face, while the gulal derivative 51 yielded a 1 : I mixture of epoxides. With these results in hand, the glycosyl donating abilities of these epoxides was explored (Scheme 10). Considerable experimentation indicated that ZnClz promoted selective p-glycosylation with alcohol acceptors, (e.g. 54, 55) whereas basic conditions promoted effective P-glycosylation only with moderately acidic acceptors (e.g. 56), such a s phenols. In some instances stannyl derived acceptors, conveniently generated in situ, afforded superior results. Subsequent to these investigations, it was found that oxirane 46 is actually among the poorest of donors [32], and that glycosylation yields could be improved by constraining the C3-C4 or C4-C6 oxygens into a cyclic motif. An interesting and useful variation on this procedure arises from the AgBF4 promoted reaction of an epoxide with a stannyl ether, which gives a-linked product. For example, treatment of 46 with 57 in the presence of AgBF4, followed by acetylation, cleanly affords 58 [33]. This method is presently limited to primary alcohol acceptors. These constraints underscore the need for a comprehensive solution to the challenging problem of stereospecifically generating a-glucosides.
3 Iterative Assembly of Glycals and Glycal Derivatives
72
0%
> -
>95%
45
0 % > -
TBSO
>95%
47
a:D
> 20:l
0% > -
>95%
TBSO
TBSO
49
Ph 0% > I
TBSO 51
>95%
50 > 2O:l P:a
?G
TBSO
52 1:l 8:a
Scheme 9.
R O *RO
OH
0-sugar
53
88%
42%
55
56
""d-""' /
Scheme 10.
67% (KzC03,18-C-6)
3.4 KS-502 and Rebeccamycin
73
Roao
1 .DMSO AqO
RO
pyridine F
RO 59
RO
61
60
Scheme 11.
The glycal epoxide method of glycosylation results in the placement of a free hydroxyl group at the 2-position of the hexose following coupling. This feature of the method enabled two alternative and useful advances (Scheme 11). First, the unique C2-hydroxyl group could be removed by the free-radical reduction of the derived pentafluorophenyl thionocarbonate (Barton deoxygenation). This sequence provides a synthesis for 2-deoxy-~-glycosides(60) [32, 341. Alternatively, the C-2 hydroxyl group could be inverted via an oxidation (AclO/DMSO)/reduction (NaBH4) protocol furnishing P-mannosides (61) [33, 351. Furthermore, the efficient and reliable route to a-glycal epoxides, facilitates numerous other methodologies leading to established donor functions (Scheme 12). Of particular importance is the fluoro-sugar type (cf. 66). Following benzylation of the uniquely free C2 hydroxyl group, derivatives such as 67 are produced. This method provides access to glycosyl donors ready for the powerful Mukaiyama promoter system [36]. We have often applied this protocol to allow for the stereoselective introduction of a-glycosides, as in 68.
62
63
BnNb
TBAF
<:
ZnCl2.71%
/
6
Bno BnO
NHB
OH 64
I
B\%c V
65
Scheme 12.
HX
X
Bno& BnO
AR'OH S & OR
66R=H 67 R = Bn
74
3 Iterative Assembly of Glycals and Glycal Derivatives " 5 ; H
nC5H11
184-6
H % H
q$(c!F?: En0
81% 69
"OH
15
,q
Rn6,H
OH
71
42 KS 501 R = H 43 KS 502 R = COfl
3 --L
48% 72
73
Rebeccamycin
Scheme 13.
The finding that the acceptor for a glycal epoxide can itself be a glycal established the basis for an iterative strategy for the synthesis of repeating p-glycosides. This concept has been a central component of our approach to oligosaccharide synthesis and has been adapted to solid support strategies as well [37]. The glycosidic bonds present in KS-502 and rebeccamycin provided especially attractive contexts for evaluation of the glycal epoxide method (Scheme 13). Treatment of the furanose glycal with DMDO gave the furanose epoxide 69 stereoselectively [38].Reaction of this epoxide with the phenol 70 afforded an 81% yield of the desired aryl-glycoside 71. Subsequent steps completed the synthesis of KS501 (42) and KS-502 (43). These furanoid epoxides were also used in the synthesis of nucleosides [39]. The extension of this procedure to the glycosylation of indoles was pursued in the context of rebeccamycin [40]. In this example, deprotonation of the differentiated indolocarbazole 72 followed by exposure of the resultant anion to glycal-epoxide 73 gave a 48% yield of the glycosylated product 74. Subsequent 2,2'-bis indole bond formation and deprotection yielded the glycoconjugate rebeccamycin (44).
3.5 Extension to Thioethyl Donors Galactal derived epoxy donors, such as 48, are particularly effective in enabling pgalactosylation. By contrast, glucal derived epoxides, lacking a comparable inter-
3.5 Extension to Thioethyl Donors
75
Lewis Acid
OH RO 75
77
RO 76
Scheme 14.
locking pattern between C3 and C4, are considerably more labile to typical Lewis acid promoters. This heightened instability brings with it significant limitations to the scope of their effectiveness as p-glycosyl donors. When the acceptor reactivity of the hydroxyl group in ROH is diminished, the yields of the glycosylation are compromised. In addition, the glycal epoxide method had not provided an efficient route to mannosides. For these reasons, we sought to extend the glycal epoxide method and turned to the use of thioethyl donors (75) for glycosylation and mannosylation (Scheme 14). Thioethyl and thioaryl glycosyl donors have previously been shown, in impressive studies by Garegg [41], to provide access to PI-4 glucosyl linkages in good yield. Donor activation is accomplished by either heavy metal salts or, directly and more efficiently, by other thiophilic reagents. The stereoselectivity of the glycosylation reaction is determined by the nature of the protecting group on C2. Indeed, it was found that glycals can be smoothly converted to thioethyl donors via glycal epoxides [42] (Scheme 15). The thioethyl glycosides thus produced, could be converted into the mannosides (75 + 85, Scheme 16). This overall inversion of configuration at C2 proceeded via an oxidation-reduction sequence to give, exclusively, the mannose derivative. The
EtSH cat. TFAA
B nO
71%
>95% 70
BnO& B nO
>95% w
BnOO 46
Scheme 15.
EtSH
-*cat. TFAA 78%
s OH . 81
EtSH cat. TFAA
03
BnO 6 BnO
U
-0TIPS
82
OH
80
79
E
45
BnO
-
U
84
76
3 Iterative Assembly of Glycals and GlycaI Derivatives 1. protect
R RO
asEt
O
OH
2. MeOTf DTBP 4A MS
75 1. ACZO DMSO pyridine 76 % 2. NaBH4
77
R
RO
O
MeOTf b -
F&SEt
85 R' = Ac, Bz, PIV
R O = L
S
* rR RO
DTBP 4A MS
a7
RORO -
Scheme 16.
resultant C2 hydroxyl groups can be protected with a variety of groups of known participatory capacity. The pivalate (Piv) derivatives were generally found to be the neighboring groups of choice for installing the desired linkage in that they provide very good margins of p-stereoselection without incurring problems of orthoester formation in competition with glycosylation (Scheme 17) [42].
3.5 Lewisy The antigenic blood group determinant LewisY (Ley) [43] is over-expressed in glycolipid and mucin glycoprotein form on many human tumor cells including those found in colon, lung, breast, and ovarian cancers. With this in mind, we set out to fashion vaccines based on the lipid and protein forms of Ley (Figure 5) [44]. To achieve these goals, we settled on the following target systems: (i) the natural product Ley-ceramide 94, (ii) a conjugate form for vaccination purposes 95, and a (iii) mucin mimic en route to a polypeptide-based vaccine (vide infra). The Ley tetrasaccharide determinant consists of a bis-fucosylated N-acetyl lactosamine. We reasoned that the iodosulfonamidation methodology could be used to install the amine function and would favor the subsequent incorporation of additional glycal units. It also seemed likely that both the Ley-ceramide target and the Ley-conjugate could be prepared from a common, but late, glycal epoxide intermediate. Concise construction of the Ley tetrasaccharide 98 commenced with lactal (96, Scheme 18) [44, 451. Protection of the primary alcohols, followed by engagement of the syn-vicinal diols as a cyclic carbonate, provided the lactal acceptor 97. Stereoselective introduction of the fucose moieties was achieved by applying a modifica-
77
3.5 LewisY MeOTf OTIPS B BnO n O
a
Brio*
DTBP
s E OPlV
t
OTIPS
OSugar
BnO
b *
OPlV
88
89
91
80%
BnO
MeOTf TIPSO n o % ,., BnO
4AMS DTBP ~
~
Bno
TIPSO BnO
93
92
0-Sugar
78%
91%
76%
Scheme 17.
tion of the original Mukaiyama conditions, using either 99 or 100. By starting with the glycal, the protecting group manipulations were minimal (2 steps). It remained however to convert the glycal function to the required lactosamine and to effect glycosylation to the pentasaccharide. In the event, 98 could be cleanly converted, by application of the iodosulfona-
OH
Figure 5.
94; R = Ceramide 95 R = Ally1
78
3 Iterative Assembly of Glycals and Glycal Derivatives
I
V
IDCP, 94% Oh
OBn
&
OB"
p
PhS02HN
102
75%
Scheme 18.
midation protocol, to 101 bearing the anomeric sulfonamide in excellent yield (94%). The latter underwent rearrangement to 102, using the 3-stannyl ether of 6Tips galactal, in 75% yield. This product contained the necessary lactosamine core linked in the desired p-configuration to the terminal galactal unit and constituted a protected Ley-pentasaccharide. After conversion of 102 to peracetate 103, the stage was set for divergent pathways to the ceramide (94) as well as to the ally1 glycoside (95). The later was the form we selected for conjugation to protein carriers, bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). These were obtained from 103 upon treatment with DMDO followed by addition of the corresponding acceptors to the glycal epoxide. By application of iodosulfonamidation and the glycal epoxide methodologies, the targets were readily obtained. These syntheses paved the way for preclinical, and eventually clinical, investigations based on the Ley-pentasaccharide [46]. In these first generation vaccines, clinical evaluation of the immune response revealed that the antibodies displayed an apparent selective binding to Leyglycolipids, while relatively low antibody reactivity was noted with Ley-mucin pro-
3.5 Lewis”
79
teins. Implicitly, these immunological results posed the challenge of designing and synthesizing a closer mimic of a Ley-bearing m u c h glycoprotein. Before any effort to construct a mucin mimic could be launched, we were faced with the issue of glycoprotein-targeted vaccine design. We set out, therefore, to construct the simplest, and yet most realistic, molecules that would serve to direct the immune response against mucins. However, unlike Ley-ceramide, which is a discrete molecule, glyco-patterns in mucins may vary along the backbone and from one protein to the next. Hence, the uncertainties regarding glycoprotein structure render it quite difficult to design, with high confidence, molecules that will stimulate an immune response which targets mucin bearing cancer cells. The complexities of mucin structure notwithstanding, we proposed to synthesize several Ley glycopeptide-based vaccine candidates [45b, c]. To achieve this, we elected to incorporate the key common features of a mucin [43d, 471, including a primary structure characterized by sequentially arranged, and glycosylated, serine or threonine residues. The first carbohydrate of the glycodomain is a-0-linked GalNAc, and a number of intervening Gal and GlcNAc spacer units may insulate the core from the terminal region, which may bear, for instance, a blood group determinant like Ley. The glycopeptide domains of three of our mucin vaccines are shown in Figure 6 (104-106). Clustered pentasaccharide 104 satisfies the minimum requirements of the Ley tetrasaccharide attached to GalNAc-a-0-Ser. The GalNAc-P-0-Ser derivative 105, containing unnatural p-linkages, was also prepared. We wondered if such a ‘non-self’ structure might prove more immunogenic than 104 and still elicit antiLey antibodies. The hexasaccharide cluster, 106, has the natural GalNAc-a-0-Ser core as well as a spacer Gal between the Ley tetrasaccharide and the first carbohydrate and as such might be considered an even more realistic mimic of mucin structure.
B
R
Figure 6.
R
80
3 Iterative Assembly of Glycals and Glycal Derivatives
Having settled on a design, we returned to our original synthesis of Ley and identified the pentasaccharide 103 as a subgoal. If this pentasaccharide could be converted to the appropriate glycosyl amino acid bearing constructs, derivatives 104-106 should be accessible through standard solution phase peptide coupling. While iodosulfonamidation of glycals is often high yielding, and the adduct ready for subsequent glycosylation, removal of the 2-benzenesulfonamido group requires Birch (metal/ammonia) reduction conditions. This protocol was developed to accommodate the glycal moiety in the context of a complex oligosaccharide and achieves removal of benzyl groups without recourse to hydrogenation. However, dissolving metal reduction is not compatible with the base- and acid-sensitive glycoamino acid function. Thus, the iodosulfonamidation method could not be appropriated for the synthesis of Ley pentasaccharide bearing glycopeptides. To gain access to 104 and 105, pentasaccharide 103 would have to be transformed to a suitable donor substrate. The latter would be combined with the amino acid core for eventual peptide coupling (Scheme 19). In practice pentasaccharide 103 was subjected to Lemeiux azidonitration [48].This transformation was followed by reduction of the nitrate ester, and conversion of the resultant anomeric alcohol to the Schmidt trichloroacetimidate [49]. Donor 107, thus prepared, was ready for coupling with a serine acceptor. Compound 107 and FmocSerOBn (108), under promotion by TMSOTf, afforded the desired O-linked glycoamino acid, albeit in a ratio of 2.7: 1, a(109):b(ll0). After considerable experimentation, it was found that by altering the solvent composition, the relative ratio could be reversed to 1 : 1.7, a(109) :P(ll0). Thus both building blocks necessary for the construction of 104 and 105 were obtained from a common donor. Further transformations afforded the acids 111 and 112, respectively. These products were ready now for peptide coupling. In several instances, as illustrated above, glycosylation of elaborated 2-azido galactosyl donors seemed to be dependent upon the nature of the donor and the identity of the serine or threonine acceptor. In this context an acceptor such as 113 was preferred (Scheme 20). Assuming pentasaccharide 103 could be converted into a suitable donor, the glycoamino acid required for assembling hexasaccharide cluster 106 could be obtained by direct glycosylation with acceptor 113 [50]. To prepare 119, pentasaccharide glycal 103 was subjected to the action of DMDO, generating the glycal-epoxide donor. However, under normal promotion conditions acceptor 113 failed to couple with the donor epoxide. In order to enhance the reactivity of the donor, the epoxide was converted to the thioethyl donor 114, which, after protecting the newly generated 2-hydroxyl group as the benzoate (116), was coupled with 113 under MeOTf promotion. The glycosylation did proceed, but in low yield and was accompanied by N-methylation of the acetamide functionality, affording both the desired 118, as well as, the undesired methyl imidate. Thus, methyl triflate promotion of thioethyl donors, found to be fully compatible with glycal acceptors, becomes complicated in the presence of acetamides and this difficulty appears to be general. Though the imidate, formed as byproduct in the methyl triflate reaction, could be hydrolyzed back to the acetamide, other more direct methods were pursued. In this regard, it was found that in the presence of NIS/TfOH [51], the desired coupling was achieved in minutes and in excellent yield. Following further optimization, the preferred route to 118 is as follows. Pen-
3.5 LewisY
81
1. CAN, Nab, (66%) b
103
OCNHCCI,
2. PhSH, iP?!NEt (92%)
3. CbCCN. I(,CO,
(89%) 107
> 75%, 1:1 .7 109:110
75%#,2.7~1
WFmcc 108
0 109
AcHN
NiFmoc
OAc
,&&
OAc d 0 r A c
0 OAc
112
ACOoAc
Scheme 19.
tasaccharide glycal 103 was subjected to the action of DMDO and subsequently converted to the pentenyl donor 115 [52]. The 2-hydroxy glycoside was then protected as the benzoate. In the event, NIS/TfOH-promoted coupling of donor 117 with acceptor 113 cleanly afforded the desired serine-O-linked hexasaccharide 118. After manipulating the protecting groups, the glycoamino acid 119 was obtained. Sequential peptide coupling of 111, 112, and 119 provided the glycopeptide domains 104--106 and eventually several vaccine candidates. It was gratifying in this regard to find that preclinical immunological evaluation demonstrated that we had indeed constructed a vaccine which provokes an antibody response targeting
82
3 Iterative Assembly of Glycals and Glycal Derivatives
oac Ho
oms
-%J--.
1. DMDO
103
2. HSEI or 4-penlen-1-01
AcHN
'NHF~~:
113
116 w117
f
3
114: R ,
= SEI. & = H
115: R ,
= 0-4-Penteny1, R,= H
BzCl
(45% from 103)
116 RI = SEt. R2 = Bz
1
117 R1 = 0-4-Penteny1, R, = Bz
Scheme 20.
Ley-mucin bearing cells. Clinical evaluation of an anti-cancer vaccine based on 106 is currently well underway.
3.7 Globo H The hexasaccharide designated globo H (120, Figure 7) has been identified on human prostate, breast and small cell lung carcinomas, as well as in a restricted number of normal epithelial tissues [53]. Globo H was originally isolated in ceramide form from the human breast cancer cell-line MCF-7 1541. The enhanced expression of antigens assumed to be globo H on both primary and metastatic prostate cancer specimens drew us to the possibility of initiating a globo H-based vaccine strategy in prostate and other cancers 1551. At the planning stage of the synthesis 1561 it seemed reasonable that, similar to the Ley synthesis, late stage divergence from a hexasaccharide glycal epoxide could pro-
83
3.7 Globo H
H type 111 trisaccharide
Globotriose
120: R = Ceramide 121: R = Ally1
Figure 7.
vide both the ceramide form and the ally1 glycoside (121) for immuno-conjugation to KLH. As i1.s name indicates, globo H contains globotriose and an H type I11 module. The chemical union of two trisaccharides, a globo trisaccharide acceptor with an H type donor, would constitute a highly convergent approach this complex target. The galactosamine unit constituted added complexity, since iodosulfonamidation had not been attempted on a galactal unit. Additionally, a final 3 3 coupling using a galactal donor derived from an iodosulfonamide could serve to provide a dramatic demonstration of the method. The galactosyl units, including the galactosamine, might be derived from a common galactal building block. Similarly, the glucose unit of the lactosyl ceramide would come from glucal, while the terminal fucose could be installed at any convenient stage in the synthesis. Compound 122 serves as an excellent acceptor, due in part to its allylic nature, and does not suffer from competing glycosylation at the axial 4-position (see 123, Scheme 21) [56].In keeping with our earlier precedents, conversion of 122 to cyclic carbonate 124 followed by treatment of 124 with DMDO generates the expected glycal epoxide 125. This intermediate was coupled with 122 and 127 to give disaccharide building blocks 126 and 128 respectively. Disaccharide 125 was carried forward without protecting group manipulations and was fucosylated under Mukaiyama conditions (125 + 130, Scheme 22). This step required selective functionalization of the unreactive C2'-hydroxyl over the,
+
(123) (not observed)
Scheme 21.
126
128
84 0
3 Iterative Assembly of Glycals and Glycal Derivatives 0
OTPS
OH 0T1p
-
AgC104, SnCl2
\ k k & O e OH 125
130: R = H
48%
131: R = Protecting Group
BnO BnO
129
Scheme 22.
presumably less reactive, axial C6hydroxyl. In the event, under optimized conditions, the desired product was obtained in 48% yield. The undesired regioisomer was a minor byproduct. Still, this sequence provided the most direct route to the target trisaccharide glycal (a total of four steps from the common precursor, 6-Tips galactal). Protection of the lone hydroxyl was achieved in a straightforward manner (130 -+ 131). As will be shown, an even more serious unanticipated turn of events arose in attempting to use this glycal as a donor. The acceptor for the projected 3 + 3 coupling was prepared from fluoro-donor 134 and lactal 132 (Scheme 23). Compound 127 was readily converted to lactal acceptor 132, while 134 was prepared from selectively protected galactal 133. It seemed prudent to convert 133 to the more reactive 0-derivative 134 by direct opening of the epoxide with TBAF, followed by protection of the resultant hydroxyl. Mukaiyama coupling of 132 with 134 proved troublesome. While the yield was high (72%), the major impediment was selectivity ( a :p, 3: 1). The desired
I33
I27
OH OBn
o&
BnO-
Bno Bm 134
135
132
AgC104, SnCI2 132
+ I34 M 135
(88%, l O : l , a:p)
136: R = PMB 137: R = H
Scheme 23.
3.7 Globo H
85
product was obtained in only about 50% yield. Furthermore, as the reaction proceeded the reactive p-derivative 134 was slowly converted into the unreactive aderivative 135. Eventually, application, with slight modification, of the Suzuki [ 571 promotion procedure, using the superiluorophile CplZr(OTf)2, provided the desired product in 10: 1 ratio in an overall yield of >80% [ 5 8 ] . In fact, this promoter allowed for smooth conversion of either 134 or 135 to the desired product. This finding simplified the sequence considerably, in that treatment of 133 with DMDO could be followed by HF:pyridine effecting clean and rapid conversion to a-derivative 135. After removing the protecting group from 136, the acceptor trisaccharide (137) was ready for the key glycosylation reaction. As depicted in Scheme 24, compound 131 underwent smooth conversion to iodosulfonamide 138 in good yield. However, in practice, 138 failed to couple with trisaccharide 137. Undaunted we pursued the complementary route. Thus iodosulfonamide 138 was converted to the thioethyl donor 139. The 3 3 coupling of 139 and 137 was effected using methyl triflate in excellent yield, particularly considering the sophistication of the j’oined partners. A small quantity of an isomeric hexasaccharide was obtained. At the stage of hexasaccharide (140) it was difficult to rigorously assign the structure of the product, and it was primarily precedence upon which we base the structural assignment. Only after eventual conversion of oligosaccharide 140 to the deprotectetl ceramide, 141, did we realize that in the 3 + 3 coupling gave the undesired a-glycoside. This unpredicted turn of events is actually rather general [591. It was found that even monsaccharides consisting of 2-sulfonamide thioethyl galactosides resulted in glycosylation with poor selectivity. In the case at hand, modifying the protecting group on the C4-hydroxyl (see ‘R’ in 139) failed to reverse selectivity. Eventually, we would conduct the key glycosylation without any protecting group on the C4-position. However, C4 deprotection of 139 to give 143 (see Schemes 24 and 25) proved to be no simple matter, regardless of the identity of the protecting group. After considerable experimentation, it appeared as if the iodosulfonamidation and the ‘roll over’ reaction would have to be performed on the unprotected glycal 130.
+
131
IDCP L (82%. PhSqNH,) W
O
B
BnO OBn
13’,
Hexasaccharlde
139 b
60%
140
... Scheme 24.
UH
n 139
86
3 Iterative Assembly of Glycals and Glycal Derivatives 0 OTIpS 130
IDCP, PhSO N 2
50%
2
Oxo&-
p
LHMDS, HSEt L
PhS0,NH
79x
oy*o& p
om SEt !‘hSO,NH
143
I43
137, MeOTf L
70 - 85%
L
L
I20 and121
Scheme 25.
While this approach did eventually succeed, the path was not traversed without further obstacles (Scheme 25). Treatment of 130 with IDCP gave the desired iodosulfonamide. However, unlike similar derivatives, the immediate product was not stable to purification or storage and isomerized to an unproductive stereoisomeric donor. Usually, instead of attempting to purify the intermediate, 142 was taken on to the ‘roll over’ step providing the desired donor 143. Remarkably, methyl triflate promoted coupling of 143 with 137 gave the desired product in good yield. This compound was accompanied by a small amount of an undesired isomer. That the major product was indeed hexasaccharide 144 was demonstrated ultimately by its conversion to the natural product. In the end, 144 was converted to a peracetate glycal, which was converted to globo H-ceramide 120 and the ally1 glycoside 121. Compound 121 was taken on to vaccination trials in the form of a KLH conjugate and continues to be studied in various clinical settings with encouraging early results. While hard won, the lessons garnered in the globo H venture paid large dividends, in that a number of related carbohydrates were accessible using ‘unprotected 4-hydroxy’ donors similar to 143. Thus, asialoGM1 [59], GM1 [60], and the demanding fucosyl GM1 [61] have each been prepared for study. In retrospect, globo H was synthesized by implementing most of the glycal methods available, including glycal donors, acceptors, epoxides, and fluorides, as well as by extension of the iodosulfonamidation protocols.
3.8 KH-1 KH-1 (145, Figure 8) is one of the most formidable carbohydrate-based tumor antigens characterized to date [ 621. This nonasaccharide has been identified on the cell surfaces of all human adenocarcinoma cells thus far studied. Furthermore, its pres-
3.8 KH-1
P
AcHN
LeY
OH
AcHN
LeX
OH
87
OH
Lactose
145: R = Ceramide 146: R = Ally1
Figure 8.
ence has never been detected in normal colonic extracts. In keeping with our program goals we targeted both natural ceramide (145) and ally1 glycoside (146) forms for total synthesis [63]. Structurally. KH-1 can be viewed as a lactosyl ceramide bearing the Lewis' (Le') trisaccharide upon which is mounted the Ley tetrasaccharide [62]. In the synthetic planning stage, however, we set, as the cardinal strategic objective, maximum relief from blocking group manipulations. In an effort to implement a highly convergent route, we hoped to introduce all three fucosyl units in a single operation. The subgoal would then consist of assembling, in an iterative fashion, glycal disaccharides to form the hexasaccharide core. Importantly, the three oxygen centers to be fucosylated would be formulated such that a single deprotection maneuver would serve to unveil them concurrently. Pursuit of the target began with glycal building blocks 147-149, and from these three glycals, and fucosyl donor 150, the entire molecule would be constructed (Scheme 26). TES ether protection, for example resident in 151, was chosen as the mask for sites of eventual fucosylation. Thus, treatment of 147 with DMDO cleanly afforded the a-epoxide, to which the acceptors 148 and 149 could be added, in the usual way, in a parallel sequence giving glycals 151 and 152. Reiteration of glycal assembly was attempted after independently applying either the acetate (153) or an additional TES ether (154) to the newly generated hydroxyl of 151. Compound 152 was destined to house the lactose bearing the ceramide unit and was therefore converted to trio1 155. While 155 has several potential glycosylation sites, in principle, the C3' hydroxyl is the most reactive position. A suitable donor could be fashioned by subjecting 153, the core of the Le' sector, to IDCP iodosulfonamidation (see 156). Addition of ethanethiolate effected the roll over reaction and the donor was ready for coupling with 155. In the event, treatment of 157 with 155 under promotion of MeOTf did, in fact, give the desired tetrasaccharide 158, albeit often in low yield (35-55%). In addition, the reaction mixture appeared to contain significant quantities of at least one regioisomer.
88
3 Iterative Assembly of Glycals and Glycal Derivatives
\KO&
?go& m, TESO
OH En0
\,
151
KzC03
80%
I,,":
c
c
LHMDS. HSEt OYKo%
-*
TESO TESO PhS0,HN I60
PhS02HN 156
I59 R=SnBu,
oy&q Eso
Aco
SEl PhSO,HN
91%
1
I 159, AQEF,
V
I55 R = H
81%
/
157
155, MeOTf
35 - 55%
Scheme 26.
We wondered if the donor, one of our most activated, might be so reactive as to be insensitive to the subtle differences in the other hydroxyl units of the acceptor. To test this hypothesis, we attempted the union of 156 with the tin ether of the triol, 159. Direct 'roll over' proved to be quite slow, but very sensitive to the differences in hydroxyl reactivity in the acceptor. This chemistry constituted a very pleasing and attractive solution to the assembly of 158. The next plateau to be reached was seen to be that of the full hexasaccharide core (Scheme 26). In an effort to maintain the tempo of the synthesis, we proceeded on to another assembly iteration with minimal protecting group-based transformations. The use of a thioethyl donor was explored; however, in the end the direct roll over route proved more reproducible. To this end, iodosulfonamidation of 154 afforded 160. While presumably even more demanding than the earlier transformation, we opted to attempt the selective coupling of the tin ether of pentaol 161 with 160 (Scheme 27). This acceptor was readily available from the tetrasaccharide by simple removal of the cyclic carbonate (158 +.161). The reaction between 160 and 162 was performed, as before, in the form of the tin ether acceptor. Fortunately, the required
3.8 KH-1
89
161 R = H
162 R = SnBu,
163 R=H.R,=TES 164 R = A c . R ( = H
\\,
Scheme 27.
coupling could be executed in good yield (>60% of 163) and without detectable formation of side product arising from glycosylation at alternative acceptor sites. The culminating stage of the synthesis was confronted after protecting the four remaining hydroxyls and removing the silyl ether protection (163 + 164). The critical step, threefold fucosylation, was indeed accomplished through the reaction of 150 and 164. An exemplary yield (60%) of the nonasaccharide 165 was obtained. In the final steps, direct epoxide opening with ally1 alcohol proved optimal for preparing 146. Our alternative procedure was used to install the ceramide. Thus, thiolation of the epoxide generated from the reaction of 165 with DMDO. This was followed by protection of the resultant free hydroxyl to give donor 166 ready for coupling with a suitable acceptor. Following this sequence, the glycal was successfully taken on to the natural product (145). Conjugation of 146 to KLH provided a vaccine and pre-clinical immunological testing is under way. Application of iterative glycal methodologies requires glycal compatible promotion systems, and these promoters are now available. The ability to attenuate donor reactivity with the acceptor is frequently desirable, especially in the search for optimized routes, and was used to effect in the case at hand. By applying the full complement of glycal technology the target was obtained in a highly convergent and efficient way. In summary, the synthesis of KH-1 serves as a cogent demonstration
90
3 Iterative Assembly of Glycals and Glycal Derivatives
of glycal assembly and the minimized protecting group manipulations that this strategy can provide.
3.9 Concluding Remarks As shown above the logic of glycal assembly has been field tested in a variety of different contexts. While our focus here has been on the total syntheses themselves, clearly, much has been learned about the chemistry of glycals and their amenability to iterative coupling with suitable acceptors and donors. At this stage, with the required compounds in hand our attention is directed toward vaccine performance, with respect to eliciting active immunity against various cancers.
Acknowledgments
This work was supported by the National Institutes of Health (Grant Numbers: CA28824, A116943, HL25848). Postdoctoral Fellowship Support is gratefully acknowledged by L.J.W. (Grant Number: NIH: F32CA79120) and R.M.G. (NIH F32CA85894). We gratefully acknowledge Dr. George Sukenick of the SloanKettering Institute’s NMR Core Facility (Grant Number: CA-08748) for NMR and Mass Spectral Analyses. References 1. S. J. Danishefsky, M. T. Bilodeau, Angew. Chem. Int. Ed. 1996, 35, 1380-1419. 2. V. Q. G. De Lima, C. A. Albert, 0. G. De Lima, An. Acad. Bras. Cienc. 1964,36, 317. 3. J. J. Afora, C. F. Santana, 0. G. De Lima, Ann. CVIIth Int. Congr. of Hematology and Hemotherapy, Paris, 1978, p. 165. 4. (a) 0. G. Da Lima, F. D. Monache, I. L. D’Albuquerque, G. B. Marini-Bettolo, Tetrahedron Lett. 1969,471; (b) L. W. Bieber, A. A. Da Silva Filho, J. F. De Mello; W. Von Der Saal, 0. G. De Lima, Rev. Znst. Antibiot. (Recife) 1982-1983, 21, 27. 5. T. Oki, Anthracycline Antibiotics [Pap,. Symp. On Anthracylines, Aug 24-25,1981, New York] Khakeme, E. Ed.; Academic Press: New York, 1982; p. 75. 6. (a) R. U. Lemiew, S. Levine, Can. J. Chem. 1964,42, 1473; (b) R. U. Lemieux, A. R. Morgan, ibid 1965, 43, 2190. 7. (a) J. Thiem, H. Karl, J. Schwentner, Synthesis 1978, 696; (b) J. Thiem, H. Karl, Tetrahedron Lett. 1978, 4999; (c) J. Thiem, P. J. Ossowowski, Carbohydr. Chem. 1984, 3, 287; (d) J. Thiem, A. Prahst, I. Lundt, Justus Liebigs Ann. Chim. 1986, 1044; (e) J. Thiem, W. Klaffke, J. Org. Chem. 1989, 54, 2006; (f) J. Thiem, Trends in Synthetic Carbohydrate Chemistry, D. Horton, L. D. Hawkins, G. L. McGarvey (Eds.), ACS Symposium Series #396, American Chemical Society, Washington, D. C., 1989, Ch. 8. 8. (a) H. Paulsen, Angew. Chem. Int. Ed. Engl. 1982,21, 155; (b) D. R. Mootoo, P. Kondradsson, U. Udodong, B. Fraser-Reid, J. Am. Chem. SOC.1988,110, 5583. 9. (a) R. W. Friesen, S. J. Danishefsky, J. Am. Chem. SOC.1989,111, 6656; (b) R. W. Friesen, S. J. Danishefsky, Tetrahedron 1990, 46, 103. 10. K. Suzuki, R. Friesen, G. Sulikowski and S.J. Danishefsky, J. Am. Chem. SOC. 1990,112,8895.
References
91
11. J. Gervay and S.J. Danishefsky, J. Org. Chem. 1991, 56, 5448. 12. For an even more concise synthesis of ciclamycin 0, see: S. Raghavan, D. Kahne, J. Am. Chem. Sac. 1993, 11'5,1580. 13. (a) D. Koga: A. Isogai, S. Sakuda, S. Matsumoto, A. Suzuki, S. Kimura, A. Ide, Agric. Biol. Chem. 1987, 51, 471; (b) S. Sakuda, A. Isogai, S. Matsumoto, A. Suzuki, J. Antibiot. 1987, 40, 296. 14. B. M. Trost, D. L. Van Vranken, J. Am. Chem. SOC.1990, 112, 1261. 15. M. L. Sinnot, Chem. Rev. 1990, 90, 1171. 16. (a) T. Iversen, D. R. Bundle, J. Chem. SOC.Chem. Commun. 1981, 1240; (b) L. J. Mathias, Synthesis 1979, 561; (c) L. A. Clizbe, L. E. Overman, Org. Synth. 1978, 58, 4. 17. R. J. Ferrier, N. Prasadi, J. Chem. Soc. C 1969, 570. 18. (a) P. Bickart, F. W. Carson, J. Jacobus, E. G. Miller, K. Mislow, J. Am. Chem. Soc. 1968, 90, 4869; (b) R. Tang, K. Mislow, J. Am. Chem. Soc. 1970, 92, 2100. 19. (a) D. A. Evans, G. C. Andrews, C. L. Sims, J. Am. Chem. SOC.1971, 93, 4956; (b) D. A. Evans, C. A. Bryan, C. L. Sims, J. Am. Chem. SOC.1972, 94, 2891; (c) D. A. Evans, G. C. Andrews, Arc. Chem. Res. 1974, 7, 147. 20. D. A. Griffith, S. J. Danishefsky, J. Am. Chem. Soc. 1990, 112, 5811. 21. S. J. Danishefsky, Chemtracts Org. Chern., 1989,2, 273. 22. M. D. Wittman, R. L. Halcomb, S. J. Danishefsky, J. Org. Chem. 1990, 55, 1979. 23. R. U. Lemieux, T. L. Nagabhushan, Can. J. Chem. 1968,46,401. 24. S. J. Danishefsky, K. Koseki, D. A. Griffith, J. Gervay, J. M. Peterson, F. E. McDonald, and T. Oriyama, J. Am. Chem. SOC.1992, 114, 8329. 25. D. A. Griffith and S. J. Danishefsky, J. Am. Chem. Soc. 1991, 113, 5863. 26. Isolation: (a) S. Nakanishi, K. Ando, I. Kawamoto, W. Kase, J. Antibiot. 1989, 42, 1049; Structure determinaton: (b) T. Yasuzawa, Y. Saitoh, H. Sano, J. Antibiot. 1990, 43, 336. 27. J. A. Bush, B. H. Long, J. J. Catino, W. T. Bradner, K. Tomita, J. Antibiot. 1987, 40, 668. 28. (a) T. Kaneko, H. Wong., J. Utzig, T. W. Doyle, J. Antibiot. 1990, 43, 125; (b) A dibromo analogue of rebeccamycin has been prepared K. Sing Lam, D. R. Schroeder, J. M. Veitch, J. A. Matson, S. Forenza, J. Antibiot. 1991, 44, 934. 29. R. R. Schmtdt, Angew. Chem., Znt. Ed. Enyl. 1986, 25, 212. 30. (a) W. J. Hickinbottom, J. Chem. SOC.1928, 3140; (b) E. Hardegger, J. de Pascual, Helv. Chim. Acta 1948, 31, 281; (c) L. L. Klein, W. W. McWhorter, Jr. S. S. KO, K.-P. Pfaff, Y. Kishi, J. Am. Chern. SOC. 1982, 104, 7362; (d) V. Bellosta, S. Czernecki, J. Chem. Soc. Chem. Commun. 1989, 199. 31. S. J. Danishefsky, R. L. Halcomb, J. Am. Chem. Soc. 1989, 111, 6661. 32. (a) C. M. Timmers, G. A. van der Marel, J. H. van Boom, Red. Trau. Chim. Pay-Bus 1993, 112, 609; (b) Ibid. Chem. Eur. J. 1995, I , 159. 33. K. K.-C. Liu, S. J. Danishefsky, J. Org. Chem. 1994,59, 1892. 34. (a) A direct method for the formation of 2-deoxy-P-glycosides from glycals employing Ph3P.HBr has been developed: V. Bolitt, C. Mioskowski, S.-G. Lee, J. R. Falck, J. Org. Chem. 1990, 55, 5812; (b) D. H. R. Barton, J. C. Jaszberepyi, Tetrahedron Lett. 1989, 30, 2619. 35. H. B. Boren, G. Ekborg, K. Eklind, P. J. Garegg, A. Pilotti, C.-G. Swahn, Acta. Chem. Scand. 1973,27, 2639. 36. T. Mukaiyama, Y. Murai, S. Shoda, Chem. Lett. 1981, 431. 37. (a) J.T. Randolph and S.J. Danishefsky, Angew. Chem. Int. Ed. 1994, 33, 1470; (b) K. F. McClure, J. T. Randolph, R. Ruggeri, S. J. Danishefsky, Science 1993, 260, 1307; (c) S. J. Danishefsky, J. Y. Roberge, Pure Appl. Chem. 1995,67,-1647;(d) J. Y. Roberge, X. Beebe, S. J. Danishefsky, Science 1995,269, 202. 38. R. G. Dushin, S. J. Danishefsky, J. Am. Chem. Soc. 1991, 114, 655. 39. K. Chow, S. J. Danishefsky, J. Org. Chem. 1990, 55, 4211. 40. M. Gallant, J. T. Link, S. J. Danishefsky, J. Org. Chem. 1993, 58, 343. 41. P. Fugedi, P. J. Garegg, H. Lonn, T. Norberg, Glyccoonj. J. 1987, 4, 97. 42. P. Seeberger, M. Eckhardt, C. Gutteridge and S.J. Danishefsky, J. Am. Chem. SOC.1997, 119, 10064. 43. (a) K. 0. Lloyd, E. A. Kabat, E. J. Layug, F. Gruezo, Biochem. 1966, 5, 1489; (b) T. Kaizu, S. B. Levery, E. Nudelman, R. E. Stenkamp, S. Hakomori, J. Biol. Chem. 1986, 261, 11254; (c) S. B. Levery, E. Nudelman, N. H. Anderson, S. Hakomori, Carbohydr. Res. 1986, 151, 311;
92
3 Iterative Assembly of Glycals and Glycal Derivatives
(d) B. W. T. Yin, C. L. Finstad, K. Kitamura, M. J. Federici, M. Welshinger, V. Kudryashov, W. J. Hoskins, S. Welt, K. 0. Lloyd, Int. J. Cancer 1996, 65, 406. 44. V. Behar, S. J. Danishfefsky, Angew. Chem. Int. Ed. Engl. 1994, 33, 1468. 45. (a) S. J. Danishefsky, V. Behar, J. T. Randolph, J. Am. Chem. SOC.1995, 117, 5701; (b) P. W. Glunz, S. Hintermann, J. B. Schwarz, S. D. Kuduk, X.-T. Chen, L. J. Williams, D. Sames, S. J. Danishefsky, V. Kudryashov, K. 0. Lloyd, J. Am. Chem. SOC.1999, 121, 10636; (c) Glunz, P. et al. Unpublished Results. 46. (a) V. Kudryashov, H. M. Kim, G. Ragupathi, S. J. Danishefsky, P. 0. Livingston, K. 0. Lloyd, Cancer Imunol. Immunother. 1998, 45, 281; Clinical data: (b) P. Sabbatini, V. Kudryashov, S. Danishefsky, P.O. Livingston, G. Ragupathi, W. Bornmann, M. Spassova, A. Zatorski, D. Spriggs, C. Aghajanian, S. Soignet, M. Peyton, C. O’Flaherty, J. Curtin, K.O. Lloyd, International Journal of Cancer, In Press. 47. (a) I. Brockhausen, in Glycoproteins, Montreuil, J., Schachter, H., Vliegenthart, J.F.G. Eds.; Elsevier: Netherlands 1995; pp. 201-259; (b) K. 0. Lloyd, J. Burchell, V. Kudryashov, B. W. T. Yin, J. T. Taylor-Papadmitrou, J. Biol. Chem. 1996,271, 33325; (c) S. Muller, S. Goletz, N. Packer, A. Gooley, A. M. Lawson, F. G. Hanisch, J. Biol. Chem. 1997, 272, 24780; (e) P. M. Rudd, R. A. Dwek, Crit. Rev. Biochem. Mol. Bio. 1997, 32, I ; (f) I. Carlstedt, J. R. Davies, Biochem. SOC.Trans. 1997,25, 214; (g) P. van den Steen, P. M. Rudd, R. A. Dwek, G. Opdenakker, Crit. Rev. Biochem. Mol. Bio. 1998, 33, 151. 48. R. Lemieux, R. M. Ratcliffe, Can. J. Chem. 1979, 57, 1244. 49. R. R. Schmidt, Angew. Chem. Int. Ed. 1986, 25, 212; R. R. Schmidt, W. Kinzy, Adv. Carbohydr. Chem. Biochem. 1994, 50, 84. 50. (a) X.-T. Chen, D. Sames, S. J. Danishefsky, J. Am. Chem. SOC.1999, 121, 10636; (b) J. B. Schwarz, S. D. Kuduk, X.-T. Chen, D. Sames, P. W. Glunz, S. J. Danishefsky, J. Am. Chem. SOC.1999,121, 2662. 51. (a) G. H. Veeneman, S. H. van Leeuwen, J. H. van Boom, Tetrahedron Lett. 1990, 31, 1331; (b) P. Konradsson, U. E. Udodong, B. Fraser-Reid, Tetrahedron Lett. 1990, 31, 4313. 52. D. Gordon, S. J. Danishefsky, Curb. Rex 1990,206, 361. 53. (a) P. 0. Livingston, Cancer Biol. 1995, 6, 357; (b) S. Zhang, C. Cordon-Cardo, H. S. Zhang, V. E. Reuter, S. Adluri, W. B. Hamilton, K. 0. Lloyd, P. 0. Livingston, Int. J. Cancer 1997, 73, 42; (c) S. Zhang, H. S. Zhang, V. E. Reuter, S. F. Slovin, H. I. Scher, P. Livingston, Clin. Cancer Res. 1998, 4 , 295. 54. (a) R. Kannagi, S. B. Levery, F. Ishijamik, S. Hakomori, L. H. Schevinsky, B. B. Knowles, D. Solter, J. Biol. Chem. 1983, 258, 8934; (b) E. G. Bremer, S. B. Levery, S. Sonnino, R. Ghidoni, S. Canevari, R. Kannagi, S. Hakomori, J. Biol. Chem. 1984,259, 14773. 55. (a) G. Ragupathi, S. F. Slovin, S. Adluri, D. Sames, I. J. Kim, H. M. Kim, M. Spassova, W. G. Bornmann, K. 0. Lloyd, H. I. Scher, P. 0. Livingston, S. J. Danishefsky, Angew. Chem. Int. Ed. 1999, 38, 563; (b) S. F. Slovin, G. Ragupathi, S. Adluri, G. Ungers, K. Terry, S. Kim, M. Spassova, W. G. Bornmann, M. Fazzari, L. Dantis, K. Olkiewicz, K. 0. Lloyd, P. 0. Livingston, s. J. Danishefsky, H. I. Scher, Proc. Natl. Acad. Sci. USA 1999,96, 5710; (c) Z.-G. Wang, L. J. Williams, X.-F. Zhang, A. Zatorski, V. Kudryashov, G. Ragupathi, M. Spassova, W. Bornmann, S. F. Slovin, H. I. Scher, P. 0. Livingston, K. 0. Lloyd, S. J. Danishefsky, Proc. Nat. Acad. Sci., 2000, 97, 2719. 56. T. K. Park, I. J. Kim, S. Hu, M. T. Bilodeau, J. T. Randolph, 0. Kwon, S. J. Danishefsky, J. Amer. Chem. SOC.1996, 118, 11488. 57. (a) K. Suzuki, H. Maeta, T. Suzuki Tetrahedron Lett. 1989, 30, 6879; (b) T. Matsumoto, H. Maeta, K. Suzuki, G. Tsuchihashi, G. Tetrahedron Lett. 1988, 29, 3567; (c) K. Suzuki, H. Maeta, T. Matsumoto, G. Tsuchihashi Tetrahedron Lett. 1988,29, 3571. 58. J. R. Allen, J. G. Allen, X.-F. Zhang, L. J. Williams, A. Zatorski, S. J. Danishefsky Eur. J. Chem. 2000, 1366. 59. 0. Kwon, S. J. Danishefsky, J. Am. Chem. SOC.1998,120, 1588. 60. S. K. Bhattacharya, S. J. Danishefsky, J. Org. Chem. 2000, 65, 144. 61. J. Allen, S. J. Danishefsky, J. Am. Chem. SOC.1999, 121, 10875. 62. E. Nudelman, S. B. Levery, T. Kaizu, S . 4 . Hakomori, J. Biol. Chem. 1986,261, 11247. 63. (a) P. P. Deshpande, S. J. Danishefsky, Nature 1997, 387, 164; (b) P. P. Deshpande, H. M. Kim, A. Zatorski, T.-K. Park, G. Rapgupathi, P. 0. Livingston, D. Live, S. J. Danishefsky, J. Am. Chem. SOC.1998,120, 1600.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
4 Thioglycosides Stefan Oscarson
4.1 Introduction Thioglycosides are not very common in nature, only a few simple alkyl and aryl thioglycosides have been found as constituents of antibiotics from Streptomyces species [ 1-31. Although not really thioglycosides, glucosinolates with an anomeric sulfur are also well-known natural compounds [4, 51. In spite of their low natural abundance, thioglycosides and their chemistry have for a long time been a field of interest for many researchers, the first thioglycoside was synthesized in 1909 [6], and their basic chemistry was early reviewed [4, 71. But it is only recently that their excellent glycosyl donor qualities have been recognized. In the beginning of the seventies the first successful synthesis of a disaccharide was performed [S], but it was not until the middle of the eighties that enough efficient promoters were discovered to make glycosylation with thioglycoside donors into a general and accepted method. Since then and in a very short time-span, thioglycosides have become one of the most used type of donors, especially in glycosylations with oligosaccharide donors (block synthesis). There are already some reviews on the subject [9-121, and in the most recent complete list of published glycosylations during one year (1994) [ 131, thioglycosides were used as donors in about 20% of all reported glycosylations and in 35% of all couplings using oligosaccharide donors, competing well with the long established halide donors and the trichloroacetimidate method. The strength of thioglycosides as glycosyl donors is their easy synthesis (Section 4.2) combined with their stability, withstanding most reaction conditions used in e.g. protecting group manipulations and glycosylations, and at the same time their effective acth ation using chemoselective thiophilic promoters (Sections 4.3.2 and 4.4.1) and also easy transformation into other types of glycosyl donors (Section 4.3.1). Hence, they are perfectly fitted to be used in block synthesis of oligosaccharides, where stable oligosaccharide donors are a prerequisite. Their stability also makes them very apt in synthetic schemes where a number of chemical modifications are performed just prior to the glycosylations, as, e.g., in various types of
94
4 Thioglycosides
internal glycosylations (Section 4.4.2). In this article the synthesis of thioglycosides and their activation and application as glycosyl donors are discussed.
4.2 Synthesis of Thioglycosides Obviously one important feature of attractive glycosyl donors are their easy availability. The synthesis of the most used types of thioglycoside donors, mainly simple alkyl and aryl glycosides, is straight-forward and can be performed on a large scale using cheap chemicals. Often the product is crystalline and can be purified without chromatography. Numerous ways to prepare thioglycosides have been described [4, 10, 111, but for the easy production of thioglycosides mainly two synthetic methods are commonly employed, namely the reaction of a peracetylated glycose with a thiol in the presence of a Lewis acid and the reaction of an acylated glycosyl halide with a thiolate.
4.2.1 From Anomeric Acetates As depicted in Scheme 1 an anomeric acetate is usually easily substituted with a thiol under Lewis acid promotion to give thioglycosides. A standard procedure is to react a peracetylated aldose dissolved in CH2C12 with a slight excess of a thiol using a hard Lewis acid, e.g. BF3-etherate [ 141 or FeC13 [ 151, as promoter to give high yields of the 1,2-truns-product (neighbouring group participation). @-Acetateprecursors usually react faster [16], which is an advantage since this diminish the formation of 1,2-cis-impurities through Lewis acid catalysed anomerisation of the pthioglycoside [ 171. a/p-Mixtures are often obtained with less reactive mercaptans [18-201 or glycosyl acetates [21, 221. Variations of this method include the utilization of trialkylstannyl [23] or silyl derivatives [24] of the mercaptan. Advantages are less odor and sometimes higher yields owing to less formation of (1,Zcis- and dithioacetal) side products, disadvantages are the accessibility and cost of the reagents. Also, thioacetic acid can be used as nucleophile in this approach, to give anomeric thioacetates, which, in a two step-procedure, can be selectively S-deacylated and alkylated to give thioglycosides, a sequence often used in the synthesis of thiooligosaccharides [251.
A AcOc
O
Scheme 1.
GOAC OAc
BF3-Et20 EtSH CHC13 83%
AcO
SEt OAc
4.2 Synthesis of Thioglycosides ,OAc
95
,OAc
5 Et,O/H,O ACO*~~,, AcO AcO Br
84%
OAc
Scheme 2.
4.2.2 From Glycosyl Halides
This is a classical way to thioglycosides [6], in which an acylated glycosyl halide is reacted with a thiolate in a polar solvent to yield the 1,2-truns-thioglycoside (Scheme 2). Both alkyl-and aryl mercaptans have been used and good yields are usually obtained, although, especially with the more basic alkyl thiolates, a reacetylation step is often necessary prior to work-up and isolation of the product. If controlled conditions and an aprotic polar solvent are used, a S~2-typedisplacement reaction can be performed to give the inverted anomeric configuration in the product, e.g. a-glycosides from p-glycosyl halide precursors, in spite of the presence of participating 2-@protecting groups (Scheme 3) [26, 271. A modification is the use of phase-transfer conditions, allowing the use of free thiols and non-polar solvents in the reaction [28-301. Also with glycosyl halides as donors, alkylstannyl derivatives of the thiol have been employed as acceptors [23]. The lower reactivity of these reagents necessitates the use of elevated temperature or a promoter in these reactions, which often result in alp-product mixtures. Syntheses of thioglycosides are sometimes possible using Koenigs-Knorr glycosylation conditions (Scheme 4) [3l], although mercury(I1) salts have been rather frequently used to promote glycosylation with thioglycoside donors (Section 4.3.2). Indirect formation of thioglycosides can also be performed from glycosyl halides. Reaction with thiourea yields a pseudothiouronium salt, which (as the thioacetate above) can be hydrolyzed under mild, selective conditions to give the anomeric thiol, which then can be alkylated in an efficient, non-smelling procedure to produce
Scheme 3.
Scheme 4.
96
4 Thioglycosides
czA,& " 3 S
Aito+
acetone A c o ~ r 80%
AcO OAc
+
H20
Aco&sH AcO
YNH2 90% NH2
OAc
Scheme 5.
I IV
OH
Scheme 6.
thioglycosides (Scheme 5) [321, however hampered by the believed carcinogenity of thiourea (and alkyl halides).
4.2.3 Protecting Group Manipulations in Thioglycosides All common protecting groups such as esters (acetates, benzoates, pivaloates, leviulonates), ethers (benzyl, allyl, trityl, silyl), acetals (isopropylidene, benzylidene, cyclohexane-l,2-diacetal (CDA)) and orthoesters can effectively be introduced, manipulated and removed. Problems encountered are the removal of benzyl groups using catalytic hydrogenolysis (when Birch reduction conditions is an alternative), since the sulfur generally contaminates the catalyst, and sometimes participating of the anomeric sulfur to the 2-position and a consecutive 1 -+ 2-migration of the thio group. In an attempt to introduce a 2,3-orthoester into a rhamnose thioglycoside, using a trimethylorthoester in CH3CN the methyl 2-thio-2-deoxy glycoside was found to be the main product (Scheme 6) [33]. This migration could be avoided by changing the solvent to DMF or by removing the methanol formed in the reaction [34]. Similar 1,Zmigrations of thioglycosides have been utilized to give 2-thio-2-deoxy glycosides or glycosyl fluorides, which can be processed to 2-deoxy-glycosides (Scheme 7) [35, 361. Most functional group conversions abundantly used on carbohydrate derivatives are also compatible with thioglycosides, e.g. SN~-displacementsof sulfonates [37], azo transfer [ 381, and deoxygenation by tributyltin hydride reduction of thiocarbonyl carbamates [39]. During oxidations care has to be taken so not to obtain the sulfoxide derivative (if that is not desired). DMSO and also PDC-mediated oxidations can usually be performed without problems [40]. The thioglycosides are also stable towards various metal reagents such as Grignard and alkyl lithium reagents, which allows carbon elongation of carbohydrates and is a handy way to higher carbon sugar donors [41-431.
4.3 Glycosylutions with Thioglycoside Donors
97
1) i.BupSnO
MeOH
ii. BnBr, CsF
DMF, 66%
SEt
OH
SEt
2)CIC(S)OPh DMAP, CH$N
80% BnO BnO
OMe OBn
OMe OBn
Scheme 7.
4.3 Glycosylations with Thioglycoside Donors The stability of thioglycosides was early recognized, as was also their very mild conversion into glycosyl halides. Hence, thioglycosides were initially used as convenient precursors for various glycosyl halides, which then could be used as donors in glycosylation reactions. Later, it was found that thioglycosides could be activated directly by the use of chemoselective thiophilic promoters, and this discovery opened up a new field of applicable donors and glycosylation reactions. 4.3.1 A Two-step Procedure: Transformation of Thioglycosides into Other Types of Glycosyl Donors Glycosyl bromides and chlorides are effectively produced using bromine [44, 451 (also I-Br [46]) or chlorine [47] (also I-Cl [46]), nowadays often with CHzClz as solvent. Under these mild conditions even unstable glycosyl halides can be produced, as e.g. furanosyl [48, 491 and alkylated oligosaccharide bromides [50], and various acid-labile protecting groups are allowed as well. Glycosyl fluorides have been produced using numerous reagents [51], e.g. dimethyl(methy1thio)sulfonium tetrafluoroborate (DMTSB) [52], NBS-DAST [ 531, HF/pyridine [53]and Selectfluor [54]. Glycals can be obtained from phenyl thioglycosides by treatment with lithium naphtalenide under conditions compatible with acid4abile groups [ 551. Trichloroacetimidates may be obtained by the hydrolysis of the thioglycoside followed by the subsequent transformation into the imidate using trichloroacetonitrile and a base [56). Hydrolysis have been performed using various promoters (e.g. AgNO:3 [57], DMTSB [58],NOBF4 [59], and DMTST (dimethyl(methylthi0)sulfonium triflate) [43])in the presence of water. Sulfoxides are formed by oxidation using MCPBA or dimethyldioxirane [60].
98
4 Thioglycosides
/
J
-cl S
L
-0Ac o
7
W
N
O HN&I,
Scheme 8.
Anomeric esters have been synthesized through the action of silver [61] or mercuric [62]carboxylates, NOBF4/Ac20 [59] or NIS/RCOOH [63] on a thioglycoside. Since it is possible to activate a thioglycoside in the presence of a pentenyl glycoside [12], it should be possible to transform a thioglycoside into a pentenyl glycoside, but to my knowledge this sequence has not been explored. This convertibility is still an attractive feature of thioglycosides and the two-step glycosylation procedure is continuously being used in glycosylations and is a good complement to the direct one-step activation of thioglycosides. It is an important asset, since in oligosaccharide synthesis it is difficult to foresee which donor that will function best in which coupling, and, furthermore, since it allows thioglycosides as the sole source of donors, which simplifies the synthesis and diminishes the number of precursors that has to be made. Of the possible donors it is mainly the halides that have been employed. As mentioned, this was the original way to use thioglycosides in glycosylation reactions and already in the early publications on the conversion to halides, alkyl 0-glycosides was prepared by activation of the formed halide by a halide promoter, Ag2C03 [49] or Hg(CN)2 [64], in the presence of an alcohol (Scheme 9) both in situ and after purification of the halide. Both procedures have been used also with glycosyl acceptors. The in situ procedure [65-671 is advantageous if the isolation of the glycosyl halide is a problem. However, with the mild conditions used, this is normally not a problem and the halide is most often crudely purified before the glycosylation reaction, which, at least in our experience, generally gives a cleaner coupling reaction with less side-products. The in situ method, of course, also disqualifies thioglycoside acceptors in orthogonal couplings to build thioglycoside blocks (Section 4.4.1).
1) clp, CHpCIp
Ac N
SEt
benzene 69%
Scheme 9.
~
2) EtOH,Hg(CN)P
OEt
4.3 Glycosylutions with Thioglycoside Donors
99
BnO
SMe
BnO
OBn
OBn
AgOTf 63%
BnO
OBn
Scheme 10.
These transformations are possible to perform on complex oligosaccharides and a number of large, biologically important structures have been synthesized with this approach both using bromides 1501 (Scheme 10) and fluorides [68]. Treatment of perbenzylated thioglycosides with iodine in the presence of an acceptor gave good yields of 0-glycosides, but there is no discussion of glycosyl iodides as intermediates in the reactions [69]. No additional promoter was necessary, and acetylated thioglycosides were not affected.
4.3.2 Direct .4ctivation of Thioglycoside Donors Heavy Metal Salt Promotors The first efforts to use thioglycosides directly as donors were performed mainly with mercury(I1) salts as promoters. It was early recognized that mercury had a high affinity for the sulfur functionality [4], and effective hydrolysis of 1-thiofuranosides using mercuric chloride as catalyst instead of a mineral acid was reported [70]. An early attempt to methanolyze ethyl tetra-0-acetyl-1 -thio-P-D-mannopyranoside using HgC12 was nct successful [71], but when Ferrier et al. tried alcoholysis on unprotected phenyl 1-thio-D-glucopyranosides using Hg(0Ac)z or HgClz/HgO as promoters good yields of the corresponding alkyl glycosides with inverted anomeric configuration was obtained [8, 721. Using the perbenzylated phenyl 1-thio-p-Dglucopyranoside as donor and HgS04 as promoter the first disaccharide was produced (Scheme 11) 181. Other similar promoters were then tried, i.a. HgC12 1731, Hg(0Bz)z [74] and PhHgOTf [75],but especially with less reactive donors, e.g. peracylated, the yields were low and it was difficult to find a general high-yielding glycosylation method.
100
4 Thioglycosides
HgS04 THF, reflux, 3h 54%
Scheme 11.
However, if a heterocyclic aryl thioglycoside was used as donor a more efficient activation with metal salts was accomplished. HgClz-promoted methanolysis of phenyl thioglycosides had been found to be faster than for the corresponding ethyl thioglycoside (compare Section 4.4.1), and with the heteroatom in the aryl system an even faster metal-ion promoted reaction took place, indicating that the heteroatom facilitated the activation of the thioglycoside (remote activation concept [76]). Mainly 2-pyridyl thioglycosides have been employed (Scheme 12), but also e.g. 2-pyrimidinyl [771 and benzothiazole thioglycosides have been used, and also different metal salt promoters, e.g. Hg(N03)Z [78], CuOTf2 [79], Pb(C104)2 [80, 811, or AgOTf [80, 82, 831. More recently, Mereyela et al. in a number of papers introduced methyl iodide as a promoter for 2-pyridyl thioglycosides [ 84-87]. Halonium, sulfonium and carbonium type promoters Owing to the many advantages of thioglycoside donors and the promising but so far limited success with heavy metal salt promoters a quest for more generally effective promoters was launched. Starting with NBS in 1983 [88] and especially with methyl triflate (MeOTf) in 1984/5 [89-911 and DMTST in 1986 [92] a seemingly never ending spectrum of new promoters has been reported (Table l), most examples variation of a common theme, soft electrophile reagents (halonium or sulfonium) with non-nucleophilic counter ions (triflate or perchlorate), but also electrochemical and radical activation have been described (Section 4.3.2). In spite of the array of different promoters and thioglycoside donors developed and reported, only a few have so far been proven generally useful in more extensive oligosaccharide synthesis. Among donors the methyl, ethyl and phenyl thioglycosides are by far the most utilized, and MeOTf, IDCP(T), DMTST and NIS alone or
OBn Ac0-q
Me0
scheme 12.
N
+ HO& Me0
Pb(C'04)2 THF,8h 59%, C@J 3:l
MeO
4.3 Glycosylations with Thioglycoside Donors
101
Table 1. Thiophilic promotors. Promoter
References
MeOTf DMTST/DMTSB NBS N BS/TfOH NBS/BQNOTf NBS/LiC104 NIS, NIS/TfOH or TMSOTf NIS/AgOTf or I3t3SiOTf IDCP IDCT Methylsulfenium triflate/bromide (MeSOTf/Br) 12
IBr Iz/AgOTf PhSOTf PhSeOTf NOBF4 N-PhSePhthOTf S02C12/TfOH PhIO-Tf20
in combination with triflic acid or silver triflate cover more or less completely the spectrum of more frequently used promoters. Numerous highly complex structures have been synthesized using these combinations verifying the impressive utility of the method. A few examples, exhibiting the synthesis of structures from N-linked glycans [91], the Salmonella LPS-core [ 1lo], and gangliosides [ 1111, are shown in Schemes 13-15. The methodology are compatible with almost all types of donors, and thioglycoside donors of e.g. furanoses, heptoses, Kdo, neuraminic acid, deoxy sugars, and uronic acids have been prepared and used successfully in glycosylation reactions. These promoters also complement each other nicely regarding reactivity, from the least reactive IDCP and MeOTf through the intermediate DMTST up to the most reactive NIS/TfOH. This promoter reactivity difference in combination with the different reactivity of donors and acceptors allows many practical orthogonal couplings between thioglycosides (Section 4.4.1). Notwithstanding the efficiency of these promoters and thioglycosides as donors, problems can, of course, be encountered in various applications. The problems are connected both to the type of thioglycoside used as well as to the promoter employed. Regarding the promoter, MeOTf can, if the acceptor is unreactive, give methylation instead of glycosylation of the acceptor hydroxyl group [ 1121, although this alkylation is normally surprisingly slow in the absence of base. The use of DMTST together with N-acetamido sugars might lead to thiomethylation of the amide group [113]. NIS, due to the
102
4 Thioglycosides
MeOTf
1
W OH L n OBn
Scheme 13.
nucleophilicity of the counter ion (as compared to triflate in most other promoters), can, once more in the case of unreactive acceptors, give rise to N-succinimide glycosides of the donor as the major product (Scheme 16) [39, 114-1 161. The obvious considerations that soft nucleophilic centres, like e.g. double bonds, should not be allowed in the donor/acceptor, since these would compete with the sulfur in its reaction with the electrophilic promoter, is not as adamant as perhaps expected. Accordingly, glycosidation with thioglycoside donors and allyl and pentenyl groups both in the donor and the acceptor have been performed using controlled conditions (Scheme 17) [ 117-1 191. Problems have been encountered when the acceptor hydroxyl group is adjacent to an allyl protecting group, which have resulted in internal addition to an activated double bond being the major reaction (Scheme 18) [97, 1131. Common problems in oligosaccharide synthesis not directly related to the method of direct activation of thioglycoside donors, such as for example transacylations or formation of orthoesters, when using 2-0-participating groups, or glycal or 1,6-anhydro derivative formation of the donor can, of course, also be encountered [90, 1lo]. As is the case with most glycosylation reactions, i.a. because of their complexity, no truly thorough mechanistic investigation has been performed on the direct activation of thioglycosides. An obvious general mechanism (Scheme I9), very similar to the one suggested for other types of glycosylation reactions, in which the soft
4.3 Glycosylutions with Thioglycoside Donors
OSP
BnO
BnO
BnO
OAc SM.3
BzO BzO
AcO
EtNBr, 90%
HO
OAc
OH
0
Scheme 14.
103
104
4 Thioglycosides
BzO
BzO?'$ko&sph BzO
:
r
s
E
e
OBn OBn
OB+oBn
'f
1) NlSRfOH 86%
NPhth
OBn
2) NaOMe
OBn
OBn
OBn
SMe
OAc
') DMTST 2) Ac20, pyridine, DMAP 3) CAN 55%
OBz
AcO
OBn
NlSKfOH 60%
OBn
OBn
OBn OBn
Scheme 15.
SE = CH2CH2SiMe3
OBn
4.3 Glycosylutions with Thioglycoside Donors
105
BnO
BnO O(CH2)&02Me NHAc
HO
Scheme 16.
DMTST
EQO OSE
92% dp 2.3/1 or NIS/AgOTf (75%)
N3
OBn
Scheme 17.
DMTST
OBn
SEt
SEt
Scheme 18.
X-
A
+HX
Scheme 19.
electrophile E+ (e.g. I+ (NIS, NBS, IDCT(P), Mef (MeOTf) or MeS+ (DMTST(B), MeSOTf(Br)) attaches to the sulfur functionality, whereafter the anomeric carbonsulfur bond is broken to generate an oxycarbenium ion (with some counterion X-, e.g. TfO- or C104), which then is attacked by the acceptor nucleophile, sometimes preceded by the formation of a cyclic acyloxonium ion if 2-0-participating groups are used, to give the 0-glycoside, is more or less commonly accepted, but the intrinsic details, like the exact nature of the intermediates or rate-determining step, are not known, and are also probably very different for different combinations of acceptor/donor/promoter and reaction conditions. No kinetic studies have been performed. Some studies of the anomerisation of alkyl p-D-thioglycosides with IDCP have been carried out showing this to be an intermolecular process and bulky
106
4 Thioglycosides
SEt
DMTST Et20 76% a
OMe
OMe
OBz
63% p
Scheme 20.
alkyl groups to be inert (however possible to use as donors) [17, 1201, and Crich and Sun have identified a stable anomeric triflate as intermediate when activating thioglycosides with PhSOTf at -78 “C [loll, but mostly mechanistic suggestion are based on product structure and distribution in combination with the understanding about mechanisms in similar reactions. Since the major outlines of the mechanism is the same, with common intermediates, as for most other types of glycosylations, much of the knowledge about the stereochemical outcome of glycosylation reactions with other type of donors is valid also for thioglycoside donors. Consequently, the use of 2-0-participating groups will lead to 1,2-trans-glycosides, if there is not a mismatch between donor and acceptor [ 1211. Furthermore, with non-participating groups, the use of diethyl ether as solvent will improve the alp-ratio [ 1221, whereas the use of acetonitrile will give predominantly P-glycosides, especially with primary acceptors [ 1231. Boons et al. advocate the use of toluene/dioxane as an even better a-directing solvent mixture [124].The stereochemical outcome is also dependent on the promoter used and on the protecting group pattern (even apart from the 2-0-protecting group). To generalize, IDCP and DMTST are good a-directing promoters whereas MeOTf and NIS are not (Scheme 20 [ 1251) [ 124, 1261. Bulky groups at 0-6 of glucopyranoside donors [ 1271 and electron-donating ester groups at 0-4 of galactopyranosides [ 1281 have been shown to increase the a//p-ratio in the disaccharide products due to steric and participating effects, respectively. As so often in glycosylation reactions these findings are only guidelines, and an optimisation is normally necessary for each specific reaction. Single-Electron Activation
Thioglycosides can also be activated by a single-electron transfer mechanism (Scheme 21). In these reactions a radical cation is generated by the transfer of a single electron from the sulfur to a suitable electron acceptor. Subsequent heterolytic cleavage of the anomeric linkage yields a sulfur radical, which dimerize to a disulfide, and an oxocarbenium ion, which, as in “normal” glycosylations, can undergo a nucleophilic attack by an alcohol to generate an 0-glycoside. The single electron transfer can be accomplished either photochemically [ 1291, electrochemically [130, 1311, or chemically [132]. Aryl thioglycosides in CH3CN/MeOH (9: 1) were irradiated in the presence of
107
4.3 Glycosylutions with Thioglycoside Donors single-
+I12 RSSR Scheme 21.
1,4-dicyanonaphtalene to give methyl glycosides [ 1291. However, the choice of protecting groups was a problem. Benzyl protected thioglycosides gave mainly other products, whereas acetylated derivatives were not activated. Hence, only permethylated compounds were compatible with the reaction conditions to give the desired glycosides. Electrochemical glycosylation was possible using both phenyl (also p-methoxyphenyl [131]) and ethyl thioglycosides [130, 1331. Acetylated donors were found to be less efficient whereas both benzylated and benzoylated donors gave good yields of disaccharides (Scheme 22). Acylated donors gave, as expected, exclusively the 1,2-truns-products, and also the benzylated donors gave predominantly the pproducts, probably due to the use of CH3CN as solvent in the reactions. Chemically, single electron transfer glycosylations have been effected by the use of the commercially available electron transfer reagent tris(4-bromo-pheny1)ammoniyl hexachloroantimonate (BAHA) as promoter. Once more, both ethyland phenyl thioglycosides, benzylated and benzoylated, could be utilized as donors to afford disaccharides in high yields (Scheme 23) [ 1321. The efficient synthesis of a trisaccharide have been reported [134],but, in spite of the promising results no other oligosaccharide syntheses have been published using this methodology. Glycosylations performed with an electron transfer quencher (1,2,4,5-tetramethoxybenzene) present showed that the reactions are completely quenched in CH2C12 but only partly in CH3CN (28% yield), giving evidence for the single-electron transfer mechanism, but also suggesting alternative mechanistic possibilities in CH3CN [135].
BBnOn
O
a SPh + OBn
Brio%
HO
anodic oxidation BBnO n -e__.___) CH3CN
BnO OMe 73%, a/p 1.3
OBz Bzo*SPh BzO
OBz
+
anodic oxidation -eCH&N
&fo&$,
~
BnoOMe
Scheme 22.
45%
O BnO
G
o
6
BnO BnoOMe
B5:oGo&$, BzO
Brio
BnoOMe
108
4 Thioglycosides
Q BnO& BnO
SPh +
OBn
::o
4
*+
SbCIi
a
Br B r * BBnO n BnO CH3CN OMe 86%, a l p 1 :2
OBz BzO Bzo$&SEt
OBz
O BnO
G 0 BnO
G BnoOMe
OBz
+
:to+
CH&N BAHA BnoOMe
81%
Bi!o&oG BzO
Brio BnoOMe
Scheme 23.
Other Types of Donors With an Anomeric Sulfur
Although not proper thioglycosides, various other derivatives with an anomeric sulfur have been synthesized and used as glycosyl donors. Kotchetkov et al. introduced, as a complement to their cyanoorthoester 1,2-truns glycosylation method, (3-glucopyranosyl thiocyanates as 1,2-cis-glycosyl donors with tritylated acceptors and trityl perchlorate as promoter [136]. With a nonparticipating group at 0-2 the glycosylation is completely stereoselective to give the inverted anomeric configuration in the product (Scheme 24). Also polycondensation using this approach has been reported [137]. Anomeric carbodithioates, both 0-and N-linked have been investigated. The 0ethyl derivative, i.e. the ethyl S-glycosyl xhantate, of 2-azido-galactopyranoseswas found to be an efficient donor activated by either Cu(0Tf)z or DMTST [138, 1391, but especially the same type of derivative of N-acetyl-neuraminic acid has proven to be an excellent alternative as a sialic acid donor, which has found general applications activated by thiophilic promoters as DMTST or MeSOTf (Scheme 25) [ 1401421. The analogous anomeric N-piperidine derivatives could be activated not only by thiophilic promoters (MeOTf and DMTST) but also by AgOTf and various Lewis acids, e.g. SnC14 and FeC13, to allow orthogonal couplings with thioglycosides and the building of thioglycoside donor blocks (Scheme 26) [ 1431.
AcoG,,, + Tf)o&oAc
AcO
OBn
= +
OAc
CHzC12 77%
OAc AcO
Scheme 24.
4.4 Applications of Thioglycosides OAc OAc COOMe - - ...-
-
Acol*%
OEt
'dS\
AcHN AcO
MST CH3CN CH&
~
109
AcHN AcO
Scheme 25.
AAcOc
O
a
s
OAc
K
Ng CH& 79%
'OBn
Scheme 26.
Also anomeric phosphorothioates have been explored. Perbenzylated S-glycosyl phosphorodiamidimidothioates promoted by 2,6-lutidinium p-toluenesulfonate gave good yields of disaccharides and 1,2-cis-selectivity(Scheme 27) [ 1441. If BQNI was used as an additive in the glycosylation even higher 1,2-cis-selectivitywas obtained. S-glycosyl phosphorodithioates as glycosyl donors are discussed in a number of articles, both 2-deoxy derivatives activated by AgF [ 1451 or iodonium promoters [ 1461 to give alp-mixtures of disaccharides, and 2-0-pivaloyl derivatives activated by MeOTf to give 1,2-trans products (Scheme 27) [147]. Although stable under some reaction conditions, none of the donors above exhibit the superior stability of thioglycosides and are therefore not an alternative to
BnO toIuene 85%
'OBn
BnO
BnO& BnO bpiv
Scheme 27.
-
MeOTf
CH2CIz 55%
-
PivO
110
4 Thioglycosides
thioglycosides, e.g. in block synthesis, but rather an addition to the spectrum of less stable glycosyl donors, which might find use in applicable cases.
4.4 Applications of Thioglycosides The stability of thioglycosides in combination with their easy activation makes them highly suitable as donors in most glycosylation strategies and technologies, e.g. in block synthesis as well as in techniques where the donor is manipulated before glycosylation and in solid phase synthesis. 4.4.1 Block Syntheses, Orthogonal Glycosylations The concept of orthogonal glycosidations will be discussed in more detail in other chapters in this book, but since the possibility of oligosaccharide block syntheses is one of the major advantages of thioglycosides, a few aspects of orthogonal glycosidations involving thioglycosides will complementarily be discussed also here. Thioglycosides as Acceptors
Most other donors can effectively be used in couplings to thioglycoside acceptors to give thiooligosaccharide blocks, e.g. halide donors using halide-assisted conditions or heavy-metal salt promoters, trichloroacetimidate donors with hard Lewis acid promoters, selenoglycosides with AgOTf activation and sulfoxide donors with TMSOTf or triflic anhydride as promoter. The efficiency in the couplings are dependent on the reactivity of the acceptor and donor and complications can arise especially with activated thioglycoside acceptors, when transglycosylation of the thiol is an often observed side reaction (Scheme 28) [89, 148, 1491. Taking the knowledge of the protecting group influence on the reactivity into account, it is normally possible to design an efficient way to thiooligosaccharide blocks, however, the protection group manipulations might be tedious and surprises might be encountered (Scheme 29) [ 1171.
Hg(CN)z or HgBr2 or . . O M B r OAc
Scheme 28.
+
A c O W S E t toluene
OAc 6O-8OYo
4.4 Applications of Thioglycosides
I
tES,&&ThTBDMSO P
11 1
bBZ
c AgOTf --
BnO
SEt
Br
OBz
Scheme 29.
Thioglycosides as Both Donors and Acceptors
The protecting group pattern is known to have a large influence on the reactivity of the donor (and of the acceptor). This has been recognized for a long time, but only been possible to utilize effectively in synthetic schemes with the event of stable donors, which has allowed the formation of donors with almost any type of protecting groups and pattern. This reactivity difference can be utilized to permit orthogonal glycosylations between thioglycoside donors (reactive, alkyl protecting groups) and acceptors (inreactive, acyl protecting groups) exploiting less reactive promoters, which only react with activated thioglycosides (Scheme 30) [97]. The idea of using the reactivity difference more systematically was developed by Fraser-Reid et al. in orthogonal couplings between armed and disarmed pentenyl glycosides [ 1501. This concept has been further developed by quantitative determination of the reactivity of donors by the use of competitive glycosylations [ 1511. Recently, fifty thioglycosides with different protecting group patterns have been synthesized and their reactivity has been determined. Through this approach it has been possible lo assign a reactivity constant to each thioglycoside, which allows the planning of synthetic schemes involving orthogonal glycosylations between thioglycosides and one-pot synthesis of oligosaccharides and also a possible computation of the selection process [ 1521. However, with thioglycosides there is an additional possibility to change the reactivity, namely through changing the alkyl (aryl) group of the anomeric thiol. This anomeric activation/deactivation prospect might be compared to the reactivity dif-
B"O& BnO
SEt
OBn
Scheme 30.
&-
+BB'po
IDCP
OBz
SEt (CICH2)2/Et20 84%
BnO SEt OBz
1 12
4 Thioglycosides OBn
SEt + H O
BnO OBn
OBn
72%
Scheme 31.
ference between halide sugars (iodo > bromo > chloro > fluoro) and the inactivation of pentenyl glycosides by bromination of the double bond, but the spectra allowed with thioglycosides are broader. Both steric effects and electronic effects can be utilized to influence the reactivity. Hence, thioglycosides of sterically crowded mercaptans, as e.g. 1,l-dicyclohexylidenemethyl mercaptan, show lower affinity for thiophilic promoters and can be used as acceptors in condensations with ethyl thioglycosides as acceptors [ 201. The higher stability of phenyl thioglycosides as compared to ethyl thioglycosides with IDCP as promoter has been shown [ 1191, and this reactivity difference have been further proved by glycosylations with p-tolyl thioglycoside donors and thioethyl acceptors using NIS/TfOH as promoter (Scheme 31) [153]. By introducing various substituents into the aromatic ring of phenyl thioglycosides a variety of reactivity can be obtained. On the extreme of the unreactivity side is the p-nitrophenyl thioglycoside, where activation does not have to be feared even with efficient promoters. On the contrary, sometimes deactivation of the acceptor hydroxyl group might be a problem and for continued use as donor the p-nitrophenyl group has to be activated as the acetamido analogue. This sequence has been investigated [ 154, 1551, but problems often arise and the scope of the methods appears to be limited, however effective with sialyl derivatives [ 1561. p-Halogenphenyl thioglycosides might be interesting compounds of intermediate reactivity. With methyl triflate or DMTST as promoters, acylated derivatives are not activated, whereas the use of NIS/triflic acid smoothly allows the use of these derivatives as glycosyl donors [ 1571. 4.4.2 Intramolecular Glycosidations To obtain desired regio- and stereoselectivity in glycosidations, new glycosylation methodology are continuously developed. Recently two intramolecular variants, internal acceptor delivery and intramolecular glycosidations of spacer prearranged glycosides, were described. In both of these approaches a number of reactions are performed on the acceptor/donor pair before the glycosidation reaction, and therefore thioglycosides as donors have been an obvious choice. In the internal delivery approach the acceptor hydroxyl group is linked to the 2-OH of a thioglycoside donor via a labile acetal linkage. Activation of the thioglycoside also breaks the acetal bond and the acceptor is transferred stereospecifically to the anomeric site of the activated donor to give high yields of
References
1 13
1,Zcis-glycosides, also the otherwise difficult obtainable P-mannopyranosides and P-fructofuranosides [ 158-1621. In the intramolecular glycosidation using prearranged glycosides the donor and acceptor are linked through a spacer via other hydroxyl groups than the ones that will be part of the new glycosidic linkage. By using various attachment sites and various spacers, regio- and stereoselective glycosylations can be performed [ 1631651. 4.4.3 Solid Phase Synthesis Since thioglycosides are possible to synthesize with almost any type of protecting group pattern and since they are shelf-stable, they should be very suitable as donors in a commercial approach to solid-phase synthesis of oligosaccharides. That they are excellent donors also in a solid-phase approach is shown, i.e., by the publications from Nicolaou et al. in which up to dodecasaccharides are synthesized on polystyrene beads using thiophenyl and thiomethyl glycoside donors and DMTST as promoter [1166, 1671. References 1. R. R. Herr, J , Am. Chem. Soc., 1967,89,2444-2447. 2. H. Hoeksema, B. Bannister, R. D. Birkenmeyer, F. Kagan, B. J. Magerlein, F. A. MacKellar, W. Schroecler, G. Slomp, R. R. Herr, J. Am. Chem. Soc., 1964,86, 4223-4224. 3. H. Hoeksema, J. Am. Chem. Soc., 1964,86, 4224-4225. 4. D. Horton, D. H. Hutson, Adv. Carbohydr. Chem., 1963, 18, 123-199. 5. V. Gil, A. J. MacLeod, Phytochern., 1980,19, 2071. 6. E. Fischer, K. Delbriick, Ber., 1909, 42, 1476-1482. 7. A. L. Raymond, Adv. Carbohydr. Chem., 1945, 1, 129-145. 8. R. J. Ferrier, R. W. Hay, N. Vethaviyasar, Carhohydr. Res., 1973, 27, 55-61. 9. P. Fugedi, P. J. Garegg, H. Lonn, T. Norberg, Glycoconj. J., 1987, 4, 97-108. 10. T. Norberg, in S. H. Khan, R. A. O’Neill (Eds.): Modern methods in Carbohydrate Synthesis, Harwood Academic Publishers 1995, p. 82-106. 11. P. J. Gareg,g, Adti. Carbohydr. Chem. Biochem., 1997, 52, 179-205. 12. G. H. Veeneman, in G.-J. Boons (Ed.): Carbohydrate Chemistry, Blackie Academic & Professional 1998, p. 98-174. 13. F. Barresi, 0. Hindsgaul, J. Carbohydr. Chem., 1995, 14, 1043-1087. 14. R. J. Ferrier, R. H. Furneaux, Carbohydr. Res., 1976, 52, 63-68. 15. F. Dasgupta, P. J. Garegg, Acta Chem. Scand., 1989,43,471-475. 16. R. U. Lemieux, Can. J. Chem., 1951,29, 1079-1091. 17. B. Lindberg, B. Erbing, Acta Chem. Scand., 1976, B30, 611-612. 18. W. V. Dahlhoff, Liebigs Ann. Chem., 1990, 1025-1027. 19. S. A. Galema, J. B. F. N. Engberts, H. A. v. Doren, Carbohydr. Res., 1997, 303, 423-434. 20. G. J. Boons, R. Guertsen, D. Holmes, Tetrahedron Lett., 1995, 31, 6325-6328. 21. P. J. Garegg, L. Olsson, S. Oscarson, J. Org. Chem., 1995, 60, 2200-2204. 22. T. Nakano, Y. Ito, T. Ogawa, Tetrahedron Lett., 1990, 31, 1597-1600. 23. T. Ogawa, M. Matsui, Carbohydr. Res., 1977, 54, C17-C21. 24. V. Pozsgay, H. Jennings, Tetrahedron Lett., 1987,28, 1375-1376. 25. H. Driguez, Topics Curr. Chem., 1997, 187, 85-1 16. 26. M. Appani, M. Blanc-Muesser, J. Defaye, H. Driguez, Can. J. Chem., 1981, 59, 314-320.
114 27. 28. 29. 30.
4 Thioglycosides
M. Blanc-Muesser, J. Defaye, H. Driguez, Carbohydr. Res., 1978, 67, 305-328. J. Bogusiak, W. Szeja, Pol. J. Chem., 1985, 59, 293-298. F. D. Tropper, F. 0. andersson, C. Grand-Maftre, R. Roy, Synthesis, 1991, 734-736. S. J. Meunier, F. 0. andersson, M. Letellier, R. Roy, Tetrahedron: Assymetry, 1994, 5, 23032312. 31. M. Gerz, H. Matter, H. Kessler, Angew. Chem. Znt. Ed. Engl., 1993, 32, 269-271. 32. D. Horton, Methods Carbohydr. Chem., 1963,2,433-437. 33. F. I. Auzanneau, D. R. Bundle., Carbohydr. Res., 1991, 212, 13-24. 34. V. Pozsgay, Carbohydr. Res., 1992,235, 295-302. 35. K. C. Nicolaou, T. Ladduwahetty, J. L. Randall, A. Chuchulowski, . I Am. Chem. Soc., 1986, 108. 2466-2467. 36. H. M. Zuurmond, P. A. M. v. d. Klein, G. A. v. d. Marel, J. H. v. Boom, Tetrahedron Lett., 1992,33, 2063-2066. 37. M. Nilsson, T. Norberg, J. Chem. Soc. Perkin Trans. I , 1998, 1699-1702. 38. T. Buskas, P. J. Garegg, P. Konradsson, J.-L. Maloisel, Tetrahedron: Asymmetry, 1994, 5, 2187-2194. 39. S. Oscarson, U. Tedebark, D. Turek, Carbohydr. Res., 1997, 159, 159-164. 40. P. J. Garegg, L. Olsson, S. Oscarson, J. Org. Chem., 1995, 60, 2200-2204. 41. G. J. P. H. Boons, G. A. v. d. Marel, J. H. v. Boom, Tetrahedron Lett., 1989, 30, 229-232. 42. G. J. P. H. Boons, Doctoral thesis, Leiden University 1991. 43. C. Bernlind, S. Oscarson, J. Org. Chem., 1998,63, 7780-7788. 44. W. A. Bonner, J. Am. Chem. Soc., 1948, 70,770-772. 45. F. Weygand, H. Ziemann, H. J. Bestmann, Chem. Ber., 1958,91,2534-2537. 46. R. Kartha, R. Field, Tetrahedron Lett., 1997, 38, 8233-8236. 47. M. L. Wolfrom, W. Groebke, J. Org. Chem., 1963,28, 2986-2988. 48. M. L. Wolfrom, P. McWain, R. Pagnucco, A. Thompson, J. Org. Chem., 1964,29, 454-457. 49. F. Weygand, H. Ziemann, Liebigs Ann. Chem., 1962, 657, 179-198. 50. K. Ekelof, S. Oscarson, J. Org. Chem., 1996, 61, 7711-7718. 51. P. J. Card, J. Carbohydr. Chem., 1985, 4 , 451-487. 52. L. Blomberg, T. Norberg, J. Carbohydr. Chem., 1992, 11, 751-760. 53. K. C. Nicolau, R. E. Dolle, D. P. Papahatjis, J. L. Randall, J. Am. Chem. Soc., 1984, 106, 4189-4192. 54. M. D. Burkart, Z. Zhang, S.-C. Hung, C.-H. Wong, J. Am. Chem. Soc., 1997, 119, 1174311746. 55. A. Fernandez-Mayoralas, A. Marra, M. Trumtel, A. Veyriirres, P. Sinay, Carbohydr. Res., 1989,188,81-95. 56. R. R. Schmidt, W. Kinzy, Ado. Carbohydr. Chem. Biochem., 1994,50, 21-117. 57. P. J. Garegg, H. Hultberg, C. Lindberg, Carbohydr. Res., 1980, 83, 157-162. 58. S. Nilsson, H. Lonn, T. Norberg, Glycoconjugate J., 1991, 8, 9-16. 59. V. Pozsgay, H. Jennings, J. Carbohydr. Chem., 1990, 9, 333-343. 60. D. Kahne, S. Walker, Y. Chang, D. v. Engen, J. Am. Chem. Soc., 1989,111,6881-6882. 61. C . Pederson, H. G. Fletcher, J. Am. Chem. Soc., 1960, 82, 3215-3217. 62. R. J. Ferrier, S. R. Haines, Carbohydr. Res., 1984, 127, 157-161. 63. G. H. Veeneman, S. H. v. Leeuwen, J. H. v. Boom, Tetrahedron Lett., 1990,31, 1331-1334. 64. M. L. Wolfrom, H. G. Garg, D. Horton, J. Org. Chem., 1963,28, 2989-2992. 65. J. 0. Kihlberg, D. A. Leigh, D. R. Bundle, J. Org. Chem., 1990, 55, 2860-2863. 66. S. Sato, M. Mori, Y.lto, T.Ogawa, Carbohydr. Res., 1986, 155, C6-C10. 67. F. 0.Andersson, P. Fugedi, P. J. Garegg, M. Nashed, Tetrahedron Lett., 1986,27, 3919-3922. 68. K. C. Nicolau, H. Ueno, in S. Hanessian (Ed.): Preparative Carbohydrate Chemistry, Marcel Dekker, Inc. 1997, p. 314-338. 69. R. Kartha, M. Aloui, R. Field, Tetrahedron Lett., 1996, 37, 5175-5178. 70. M. L. Wolfrom, K. Anno, J. Am. Chem. SOC.,1953, 75, 1038-1039. 71. J. Fried, D. E. Waltz, J. Am. Chem. SOC.,1949, 71, 140-143. 72. L. Hough, M. I. Taha, J. Chem. Soc., 1956,2042-2048. 73. T. Y. R. Tsai, H. Jin, K. Wiesner, Can. J. Chem., 1984,62, 1403-1405. 74. J. W. v. Cleve, Carbohydr. Res., 1979, 70, 161-164.
References
1 15
75. P. J. Garegg, C. Henrichson, T. Norberg, Curbohydr. Res., 1983, 116, 162-165. 76. S. Hanessian, in S. Hanessian (Ed.): Preparative Carbohydrate Chemistry, Marcel Dekker, Inc. 1997, p. 381-387. 77. Q. Chen, F. Kong, Carbohydr. Rex, 1995,272, 149-157. 78. S. Hanessian, C. Bacquet, N. Lehong, Carbohydr. Rex, 1980, 80, C17-C22. 79. T. Mukaiyama, T. Nakatsuki, S. Shoda, Chem. Lett., 1979, 487-490. 80. R. B. Woodward, e. al., J. Am.Chem. Soc., 1981, 103, 3215-3217. 81. P. G. M. U'uts, S. S. Bigelow, J. Org. Chem., 1983, 48, 3489-3493. 82. K. Tsuboyama, K. Takeda, K. Torii, M. Ebihara, J. Shimizu, A. Suzuki, N. Sato, H. Furuhata, Chem. Pharm. Bull., 1990, 38, 636-638. 83. Z. Zhiyuan, G. Magnusson, Carbohydr. res., 1994,262, 79-101. 84. G. V. Reddy, V. R. Kulkarni, H. B. Mereyala, Tetrahedron Lett., 1989, 30, 4283-4286. 85. H. B. Mereyala, G. V. Reddy, Tetrahedron, 1991, 47, 6435-6448. 86. H. B. Mereyala, V. R. Kulkarni, D. Ravi, G. V. M. Sharma, B. V. Rao, G. V. Reddy, Tetrahedron, 1992, 48, 545-562. 87. H. B. Mereyela, V. R. Gurijala, Curbohydr. Rex, 1993,242, 277-280. 88. K. C. Nicolaou, S. P. Seitz, D. P. Papahatjis, J. Am. Chem. Soc., 1983, 105, 2430-2434. 89. H. Lonn, Chem. Commun. Stockholm Univ., 1984, No2, 1-30. 90. H. Lonn, Carbohydr. Res., 1985, 139, 105-113. 91. H. Lonn, C'arbohydr. Rex, 1985, 139, 115-121. 92. P. Fugedi, P. J. Garegg, Curbohydr. Res., 1986, 149, C9-Cl2. 93. M. Sasaki, K. Tachibana, H. Nakanishi, Tetrahedron Lett., 1991, 32, 6873. 94. K. Fukase, A. Hasuoka, S. Kusumoto, Tetrahedron Lett., 1993, 34, 2187-2190. 95. K. Fukase, A. Hasuoka, Kinoshita, S. Kusumoto, Tetrahedron, 1995, 51, 4923-4932. 96. P. Konradsson, U. E. Udodong, B. Fraser-Reid, Tetrahedron Lett., 1990, 31, 4313-4316. 97. G. H. Veeneman, J. H. v. Boom, Tetruhedron Lett., 1990,31, 275-278. 98. G. H. Veeneman, S. H. v. Leeuwen, Z. H, J. H. v. Boom, J. Curbohydr. Chem., 1990,9, 783796. 99. F. Dasgupta, P. J. Garegg, Curbohydr. Rex, 1988, 177, C13-Cl7. 100. F. Dasgupta, P. J. Garegg, Curbohydr. Rex, 1990,202, 225-238. 101. D. Crich, S. Sun, J. Am. Chem. SOC.,1998,120,435-436. 102. D. Crich, S. Sun, Tetrahedron, 1998, 54, 8321-8348. 103. Y. Ito, T. Ogawa, Tetrahedron Lett., 1988, 29, 1061-1064. 104. Y. Ito, T. Ogawa, M. Numata, M. Sugimoto, Carbohydr. Rex, 1994,202, 165-175. 105. V. Pozsgay, H. J. Jennings, J. Org. Chem., 1987, 52, 4635-4637. 106. H. Shimizu, Y. Ito, T. Ogawa, SynLett, 1994, 535-536. 107. H. Shimizu, SynLett., 1998, 84-86. 108. H. Lonn, Glycoconjugute J., 1987, 4, 117-118. 109. K. Fukase, A. Hasuoka, I. Kinoshita, S. Kusumoto, Tetrahedron Lett., 1992, 33, 7165-7168. 110. S. Oscarson, H. Ritztn, Carbohydr. Rex, 1994,254, 81-90. 111. A. Hasegawa, M. Kiso, in S. Hanessian (Ed.): Preparative Carbohydrate Chemistry, Marcel Dekker, Inc. 1997, p. 357-379. 112. P. J. Garegg, S. Oscarson, M. Szonyi, Carbohydr. Res., 1990,205, 125-132. 113. A.-K. Tiden, Doctoral thesis, Stockholm University 1991. 114. L.-X. Wang, N. Sakiri, H. Kezukura, J. Chem. SOC.Perkin I , 1990, 1677-1682. 115. C. Krog-Jensen, S. Oscarson, J. Org. Chem., 1996, 61, 1234-1238. 116. M. Anderson, P. Konradsson, S. Oscarson, L. Oberg, J. Carbohydr. Chem., in press. 117. M. Alpe, S. Oscarson, unpublished results. 118. S. K. Das, R. Ghosh, N. Roy, J. Carbohydr. Chem., 1993, 12, 693-701. 119. H. M. Zuurmond, S. C. v. d. Laan, G. A. v. Marel, J. H. v. Boom, Carbohydr. Res., 1991, 215, clLc3. 120. G.-J. Boons, T. Stauch, Synlett, 1996, 906-908. 121. N. M. Spijker, C. A. A. van Boeckel, Angew. Chem. Znt. Ed. Engl., 1994, 30, 180-182. 122. H. Lonn, J. Curbohydr. Chem., 1987, 6, 301--306. 123. I. Braccini, C. Derouet, J. Esnault, C. H. d. Penhoat, J. M. Mallet, V. Michon, P. Sinay, Curbohydr. Rex, 1993,246, 23-41.
1 16
4 Thioglycosides
124. A. Demchenko, T. Stauch, G.-J. Boons, Synlett, 1997, 818-820. 125. P. Svahnberg, Licenciate Thesis, Stockholm University 1999. 126. P. J. Garegg, S. Oscarson, H. Ritztn, M. Szonyi, Carbohydr. Rex, 1992, 228, 121-128. 127. S. Houdier, P. J. A. Vottero, Carbohydr. Res., 1992,232, 349-352. 128. A. V. Demchenko, E. Rousson, G.-J. Boons, Tetrahedron Lett., 1999, 40, 6523-6526. 129. G. W. Griffin, N. C. Bandara, M. A. Clarke, W . 3 . Tsang, P. J. Garegg, S. Oscarson, B. A. Silwanis, Heterocycles, 1990,30, 939-947. 130. C. Amatore, A. Jutand, J.-M. Mallet, G. Meyer, P. Sinay, J. Chem. SOC.Chem. Commun., 1990, 718-719. 131. G. Balavoine, A. Greg, J.-C. Fischer, A. Lubineau, Tetrahedron Lett., 1990,31, 5761-5764. 132. A. Marra, J.-M. Amatore, C. Amatore, P. Sinay, SynLett., 1990, 572-574. 133. J.-M. Mallet, G. Meyer, F. Yvelin, A. Jutand, C. Amatore, P. Sinay, Carbohydr. Rex, 1993, 244,237-246, 134. Y.-M. Zhang, J.-M. Mallet, P. Sinay, Curbohydr. Res., 1992, 236, 73-88. 135. S. Mehta, B. M. Pinto, Carbohydr. Rex, 1998, 310, 43-51. 136. N. K. Kochetkov, E. Klimov, N. N. Malysheva, Tetrahedron Lett., 1989,30, 5459-5462. 137. N. K. Kochetkov, N. N. Malysheva, E. Klimov, A. V. Demchenko, Tetrahedron Lett., 1992, 33, 381-384. 138. A. Marra, L. K. S. Shun, F. Gauffeny, P. Sinay, Synlett, 1990, 445-448. 139. A. Marra, F. Gauffeny, P. Sinay, Tetrahedron, 1991, 47, 5149-5160. 140. A. Marra, P. Sinay, Carbohydr. Res., 1990, 195, 303-308. 141. H. Lonn, K. Stenvall, Tetrahedron Lett., 1992, 33, 115-116. 142. U. Ellervik, G. Magnusson, J. Orq Chem., 1998, 63, 9314-9322, 9223. 143. P. Fugedi, P. J. Garegg, S. Oscarson, G. Rosen, B. A. Silwanis, Carbohydr. Res., 1991, 211, 157-1 62. 144. S. Hashimoto, T. Honda, S. Ikegami, Tetrahedron Lett., 1990,31, 4769-4772. 145. H. Bielawska, M. Michalska, J. Curbohydr. Chem., 1991, 10, 107-1 12. 146. L. Laupichler, H. Sajus, J. Thiem, Synthesis, 1992, 1133-1136. 147. 0.J. Plante, P. H. Seeberger, J. Org. Chem., 1998,63, 9150-9151. 148. D. A. Leigh, J. P. Smart, A. M. Truscello, Carbohydr. Res., 1995, 276, 417-424. 149. F. Belot, J.-C. Jaquinet, Carbohydr. Res. 290, 1996, 290, 79-86. 150. D. R. Mootoo, P. Konradsson, U.Udodong, B. Fraser-Reid, J. Am. Chem. Soc., 1988, 110, 5583-5584. 151. N. L. Douglas, S. V. Ley, U. Lucking, S. L. Warriner, J. Chem. SOC.Perkin Trans. I , 1998, 51-65. 152. Z. Zhang, I. R. Ollman, X.-S. Ye, R. Wischnat, T. Baasov, C.-H. Wong, J. Am. Chem. SOC., 1999.121. 734-753. 153. A. K. Choudhury, I. Mukherjee, B. Mukhopadhyay, N. Roy, J. Carbohydr. Chem., 1999,18, 361-367. 154. S. Cao, F. Hernandez-Mateo, R. Roy, J. Carbohydr. Chem., 1998, 17, 609-631. 155. L. A. J. M. Sliedregt, K. Zegelaarjaarsveld, G. A. van den Marel, J. H. van Boom, Synlett, 1993, 335-337. 156. R. Roy, F. 0. Andersson, M. Letellier, Tetrahedron Lett., 1992, 33, 6053-6056. 157. M. Lahmann, S. Oscarson, unpublished results. 158. F. Baressi, 0. Hindsgaul, J. Am. Chem. SOC.,1991, 113, 9376-9377. 159. M. Bols, J. Chem. SOC.Chem. Commun., 1992,913-914. 160. F. Baressi, 0. Hindsgaul, Can. J. Chem., 1994, 72, 1447-1465. 161. Y. Ito, T. Ogawa, Ang. Chem. Int. Ed. Engl., 1994,33, 1765-1767. 162. C. Krog-Jensen, S. Oscarson, J. Org. Chem., 1998, 63, 1780-1784. 163. T. Ziegler, R. Lau, Tetrahedron Lett., 1995, 36, 1417-1420. 164. S. Valverde, A. M. Gomez, J. C. Lopez, B. Herradon, J. Chem. SOC.Chem. Commun., 1995, 2005-2006. 165. M. Miiller, U. Huchel, A. Geyer, R. R. Schmidt, J. Org. Chem., 1999, 64, 6190-6201. 166. K. C. Nicolaou, N. Winssinger, J. Pastor, F. DeRoose, J. Am. Chem. Soc., 1997,119,449-450. 167. K. C. Nicolaou, N. Watanabe, J. Li, J. Pastor, N. Winssinger, Angew. Chem. Int. Ed. Engl., 1998,37, 1559-1561.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
5 Glycosylation Methods: Use of Phosphites Zhiyuan Zhang and Chi-Huey Wong
5.1 Introduction
In the chemical synthesis of oligosaccharides, the overall procedure can be divided into five steps: 1) design and preparation of the building blocks (glycosyl donors and acceptors) with selected protecting groups; 2) manipulation of the protecting groups to free the desired hydroxyl group; 3) separation of products after each step; 4) stereo-controlled glycosylation; and 5 ) deprotection and product purification. Of those steps, 1-3 are the most time-consuming. Considerable efforts have thus been made to facilitate the synthetic process. For example, many methods have been developed to reduce the number of steps in the synthesis of building blocks. Recently developed orthogonal protection-deprotection strategies [ 1, 21 provide the efficiency and flexibility for the preparation of highly diversified carbohydrate libraries [ 1, 31. To minimize protecting group manipulation, the armed-disarmed strategy [4], the orthogonal glycosylation strategy [ 5 ] , and the one-pot multiglycosylation strategy [6-81 have been developed. In terms of simplifying the purification procedure, solid-phase synthesis [91 and one-pot solution-phase synthesis [7d, 81 have shown great promise. Glycosylation is the central reaction of carbohydrate chemistry. Because low yield, low regioselectivity, and low stereoselectivity are frequently encountered, it is this step which requires special attention. Though chemists might be able to synthesize any oligosaccharide [lo],there is no universally employed glycosylation [ lOc, 111. Therefore, development of new and efficient methods of glycosylation is still quite challeng,ing.
118
5 Glycosylation Methods: Use of Phosphites
The use of glycosyl phosphite as a glycosyl donor was initiated in 1992. The Wong group [12] and the Schmidt group [13] independently reported that trimethylsilyl triflate (TMSOTf) could activate glycosyl phosphites in glycosylation reactions. An advantage of this method is that it provides a very high yield and aselectivity in the synthesis of sialic acid derivatives [12, 131. This method has been further developed by these two groups and others. Different promoters have been investigated and some unusual stereoselectivities have been reported. The purpose of this chapter is to review recent developments in phosphite glycosylation and its applications.
5.2 Preparation of Glycosyl Phosphites Glycosyl phosphites are prepared from their corresponding anomeric hydroxyl compounds, either by treatment with dialkyl N,N-diethylphosphoramidites (DDP) and 1,2,4-triazole [ 141 or with dialkyl phosphorochloridites in the presence of Hunig's base (Figure 1) [ 13, 151. Most phosphitylation reagents are commercially available, and both methods give glycosyl phosphites in high yield. Examples of glycosyl phosphites that can be prepared by these methods are dibenzyl phosphites, diethyl phosphites, and dimethyl phosphites. Bicyclic glycosyl phosphites such as 4 and 5 can also be prepared. In the synthesis of an N-acetyllactosamine (LacNAc) derivative it has been shown that 4 gives better yields than the corresponding trichloroacetimidate [ 14fl. When the sugar moiety is highly electron-donating, such as the 2,6-di-deoxy derivative, its diethyl phosphite derivative could not be prepared.
Et2N-P-(OBn),
PCH
A . d
I
COOMe
1,2,4-triazole AcO
1
CI-P-(0Et)2 EtNiPr2
4
3 97%
AcO
Figure 1.
COOMe
5.3 Glycosylotion using Glycosyl Phosphites
1 19
The corresponding pinacol phosphite 5 was, however, stable enough to be isolated in 67% yield [16]. Several phosphitylation reagents are now available for the preparation of glycosyl phosphites with desirable reactivity. Factors known to affect the anomeric ratio in the preparation of glycosyl phosphites include the anomeric composition of the starting hydroxyl compound. For example, a mixture of galactosyl phosphite with an a : ratio of 90 : 10 was obtained with an a : ratio of 90: 10. A different from 2,3,4,6-tetra-O-benzylgalactopyranose anomeric mixture ( a :p = 61 :39) of the phosphite product was, however, generated from an anomeric mixture of the galactopyranose ( a :p = 56:44) [17].2,3,4,6-Tetra0-acetylgalactose ( a :p = 3 : 1) and glucose ( a :p = 2: 1) gave the phosphite p isomer as a predominant product ( a :p = 1 :4 and 1 :2 respectively), whereas the 2-acetamido-2-deoxy sugar gave mainly the a-phosphite ( a :p > 7: 1) [18]. Surprisingly, only phosphite p isomers (2 and 3) were observed when sialic acid (NeuSAc) derivatives were phosphitylated using either phosphitylation method [ 12, 131. Solvents also affect the product composition. Phosphitylation of 2,3,4,6-tetra-Oacetylgalactose in CH2C12 gave the dibenzyl phosphite with an a : p ratio of 1 :2; this increased to 1 : 6 when THF was used as solvent [ 181. Effects of Lewis acid [ 191 and mild base [20] on the anomeric ratio of the phosphite have also been reported. In general the anomeric ratio of the phosphite does not have a large effect on the glycosylation step in terms of reactivity and stereoselectivity [ 171. It might, however, be a factor in the synthesis of phosphate derivatives from phosphites (see Section 5.4).
5.3 Glycosylation using Glycosyl Phosphites 5.3.1 Mechanism The first catalyst used to activate the glycosyl phosphite was TMSOTf [ 12, 131. The mechanism of the reaction has been investigated [12]. As shown in Figure 2, path a applies when TMSOTf is mixed with donor first or added into the mixture of donor and acceptor. In this method, the formation of a substantial amount of phosphonate orthoester is a considerable problem. As illustrated in path b, however, a premixed solution of TMSOTf and acceptor spontaneously generates triflic acid (TfOH). TfOH then activates the glycosyl phosphite added subsequently to give the desired product, and reduces the formation of the orthoester. The most facile method is through path c where TfOH directly catalyzes the glycosylation, and also reduces the nucleophilicity of the hydrogen phosphonate. TfOH is actually involved in each of the pathways, and is potentially the real catalyst even when TMSOTf is used [21]. In general, TfOH-catalyzed glycosylation gives the highest yield. Notably, addition of glycosyl phosphite to premixed TMSOTf and acceptor gave a better yield than addition of TMSOTf to the mixture of donor and acceptor [22]. In practice, however, it is more convenient to use TMSOTf than TfOH as catalyst. Interestingly, BF3.Et20-promoted [ 171 and TMSOTf-promoted [ 12, 13, 211 gly-
120
5 Glycosylation Methods: Use of Phosphites
a. TMSOTf reacts with donorfirst
-
TMSO-P(OBn)2
TMSOTf
HOTf
b. TMSOTfreacts with acceptorfirst
TMSOTf
TMSOR HO-P(OBn),
-
R
HP(OBn),
c. When TfOH was used
Figure 2.
cosylations using perbenzylated phosphites at low temperature (-78 "C), as reported by Hashimoto et al., gave similar stereoselectivity and yields. Both activation gave high 1,2-trans-P-selectivityeven without a participating group at the C-2 position of the glycosyl donor [ 171. BF3.EtzO-promoted glycosylations, furthermore, gave even better P-selectivity. The reason for this, and the nature of the intermediate involved in the BF3 .Et20-promoted glycosylation, remain unclear [ 171. More recently, the Hashimoto group developed a new activation method using 2,6-di-tert-butylpyridinium iodide (DTBPI) and tetrabutylammonium iodide (BmNI), and achieved high a-selectivity. The intermediate was found to be the aglycosyl iodide, which is first anomerized to the P isomer then glycosylated through a S~2-likemechanism (Figure 3). In general, a-selectivity is predominant in glycosylations using the phosphite method. Watanabe et al. [22] have investigated other Lewis acids as promoters of glycosylation using glycosyl phosphites; they found that ZnCl2, ZnCl2-AgC104, 12, and other Lewis acids can efficiently activate perbenzylated phosphites at room temperature. The stereoselectivities are generally lower than when reaction is performed out at low temperature. The effect of promoters on the glycosylation of perbenzylated glucosyl phosphite 6 with a primary alcohol was studied (Figure 4).ZnClz-
5.3 Glycosylation using Glycosyl Phosphites
121
60-95% cc/B > 90110
8 donors
Figure 3.
promoted glucosylation gave predominantly the P product, 7. In contrast, 12promoted reactions mainly gave the a product, whereas almost no selectivity was observed with the ZnClz-AgC104-promoted glycosylation [ 17b]. It is unclear why the addition of AgC104 would increase the efficiency and reduce the selectivity of the reaction. The origin of selectivity remains to be investigated [17b]. Metal perchlorate salts (e.g. LiC104), which act as weak Lewis acids, activate the glycosyl phosphite and yield the glycosyl perchlorate ion pair which leads to the product [23] This method provides a useful alternative when acid-labile protecting groups, or acid-sensitive glycosyl linkages are involved. The low yield and low stereoselectivity are potential drawbacks of this method. 5.3.2 Low Temperature-Dependent Stereoselectivity The stereoselectivity of a glycosylation is controlled by many factors, including the substituent at the C-2 position, the leaving group on the anomeric center, temperature, solvents, and the structural features both of glycosyl donor and acceptor. In the construction of a glycosyl P-linkage, a participating group at the C-2 position of the glycosyl donor is normally required. In the absence of this participating group the thermodynamically favorable a-glycosyl bond is generally formed because of the anomeric effect [24]. Thus, the stereo-controlled synthesis of the P-linkage is considered to be difficult. A solution to this problem is to use acetonitrile as solvent to provide the nitrile effect [25] to aid the formation of the P-linkage. In addition, some newly developed glycosylatioii methods using glycosyl donors with a non-participating group at the C-2 position at low temperature gave very high p-selectivities [17, 21, 22b, 26, 271. These methods include the use of sulfoxides activated with Tf20 [27, 281, glycosyl bromides activated with AgOTf [27], thioglycosides activated with RSCl-AgOTf
Lewis acid
Ph(CH&OH
B
%B
Lewis acid
6
+
BB& O(CH.&Ph
OP(O5h 7
Figure 4.
Yield
1,
92% 81/19
ZnCI,
88% 20180
ZnCI2-AgC1O4 92% 52/48
122
5 Glycosylation Methods: Use of Phosphites
I
Temp.
-78OC
-5OOC
8
3:97
8:92
11:89
1:99
2:98
3:97
-3oOc
8
BF3-Et20
Ratio
ROH
9
(b.) Watanabe eta/.
6 I
LNlS/TfOtJ CH2C12
-\
B"4 BnO 7
O(CH&Ph
3°C
-21 "C -45°C -68"C
Figure 5.
[27, 291 and glycosyl phosphites activated with TMSOTf [17, 211 or BF3.Et20 [17, 261. Low temperature seems to promote P-selectivity; the thermodynamically stable a-glycosyl triflate is formed and further reacted in a S~2-likemechanism with the added glycosyl acceptor to give the P-glycoside [27b, c]. Figure 5 shows the temperature-dependent selectivity reported by Hashimoto et al. [17] and Watanabe et al. [22b] Further study showed that P-selectivity was almost independent of solvent [ 171, although slightly dependent on the anomeric composition of the donor [17, 211. This is exemplified by the synthesis of Pgalactosyl-, P-glucosyl-, and P-glucuronyl linkages [ 14e, 16, 17, 21, 26, 301. Fucosyl phosphites behave differently under these conditions, however, and a-selectivity is predominant [21, 30, 311. The stereoselectivity issues of glycosyl phosphites will be discussed later.
5.3.3 Glycosylation of Sialyl Phosphites Sialylated oligosaccharides are often expressed on the surfaces of cells, including cancer cells, and are very attractive targets for investigation. Sialylation [ 321, especially yield and stereoselectivity, is, however, still a major problem in oligosaccharide synthesis. Synthetic difficulties stem from the tertiary-hindered anomeric center and the lack of an electron-demanding group adjacent to the anomeric center of sialylic acid (NeuAc) [ 121. Sialylation using sialyl phosphites is currently one of the most successful synthetic methods, and many sialylated compounds have been prepared by this strategy. Compounds 10-11 and 13 (see Figure 6) were prepared from dibenzyl phosphite 2 [12], and compounds 10, 12 and 13-14 were synthesized from diethyl phosphite 3 [13]. In the synthesis of sialyl Lewisa (sLea), a regioselec-
5.3 Glycosylution using Glycosyl Phosphites A
123
OOMe
AcO 10 80% (a:p6:1) from 2 70% (cx:p4:1) from 3
OMe
Ho
PcHN 11 48% only cx from 2
OpC
Ph
13 44% (a:p6:1)from 2
12 38% cx from 3
Figure 6.
tive coupling of the sialyl moiety to the 3-OH on the Gal unit of partially protected Lea gave the desired compound 14 in low yield (18%). Also isolated was 35% of the lactonized product 15 [33]. This lactone side-product 15 was isolated as the only product (37%) when the same acceptor and a different glycosyl donor and promoter were used [34].
5.3.4 Glycosylation of C-2-Acylated Glycosyl Phosphites In general, the 1,2-tuans P-glycosyl linkage is formed by use of a C-2 participating protecting group. Catalytic amounts of TMSOTf (0.5 equiv.) can promote the glycosylation of peracetylated glycosyl phosphites. Donors utilized include fucosyl phosphites, galactosyl phosphites, glucosyl phosphites, glucuronyl phosphites, and glucosaminyl phosphites. Reaction of these donors with different glycosyl acceptors yield the desired P-product with yields of 40-70% [21, 351. T h s method has also been used for the synthesis of some P-C-glycosides and P-S-glycosides [21, 301. A few side-reactions are, however, encountered when using this method; examples include the formation of glycosyl phosphonate orthoesters [21, 30, 361, which are less reactive intermediates, the ester transformation problem [21, 361, and in some instances the formation of silylated acceptor byproducts [21]. Side-reactions often depend on the type of catalyst used in the reaction. Perbenzoylated glucosyl phos-
124
5 Glycosylution Methods: Use of Phosphites
Oh
BzO 16 R' =Bn, R2,R3 = PhCH, %YO, a only 17 R' =R3 = Bn, R2= H, 73%, a:p1:1 18 R' =R2=H, R3= Bn 88%, p only
BzO 19
loo%, aonly
20 93%, ponly
Figure 7.
phite in the presence of BF3.Et20 (1 equiv.), for example, gave 24% of the phosphonate orthoesters whereas use of 1 equiv. TMSOTf at -50°C gave the desired 1,2 trans-p product in excellent yield, with no traces of the aforementioned byproducts [36]. Other promoters such as ZnC12, ZnC12-AgOTf, and metal perchlorate salts have not been used for the activation of acylated glycosyl phosphites. Stereo-controlled synthesis of fructofuranosides is regarded as a difficult problem, even with a participating group next to the anomeric center [36]. The study of TMSOTf-catalyzed glycosylation using fructofuranosyl phosphite showed that a- or P-selectivity was highly dependent on the properties of the acceptor (Figure 7) [36]. 5.3.5 Glycosylation with C-2-0-Benzylated Glycosyl Phosphites
The glycosylation reaction using glycosyl phosphites without a participating protecting group at C-2 is more complicated. The stereoselectivity seems to depend on many factors, including reaction temperature, the core structure of the glycosyl phosphite, and the properties of acceptors and the promoters. Glycosylation using Glucosyl Phosphites with a Benzyl Group at C-2 Glycosylution with 0-selectivity Glucosylation of a primary hydroxyl acceptor with perbenzylated glucosyl phosphite, promoted by TMSOTf at -78"C, gave nearly exclusively the P-product 8 (66%, a : P = 1 :99 in CH2C12; and 78%, a : P = 2: 99 in EtCN). The P-selectivity decreased, giving the anomeric mixture 9 (83%, a : P = 32: 68 in CH2C12; and 65%, a : f3 = 17:83 in EtCN) when a secondary alcohol acceptor was used [17, 211. Of the many promoters studied, BF3.Et20 gave products in the highest yield and Pselectivity, even with secondary hydroxyl acceptors (a:P = 5 :95) [ 171. ZnC12-promoted glucosylations [22] gave lower 0-selectivity than the lowtemperature procedure (TMSOTf or BF3.EtzO), most probably because of the higher reaction temperature. ZnC12-AgC104 [22] was, however, successfully employed in the total synthesis of paeoniflorin reported by Corey and Wu [37]. In this instance glycosylation of the very hindered acceptor 22 (a mixture of enantiomers) with perbenzylated glucosyl phosphite 21 gave the enantiomerically pure 23 (1 8%,
5.3 Glycosylation using Glycosyl Phosphites
'& B
TIP93
22
OP(OMe)2
BnO
BB *
ZnC12-AgClO4
21
x
Y
@
BnO
M
125
e TIPS3
23 18% enantiomeric pure p
Figure 8.
see Figure 8). This process was more successful than any other glycosylation method, including the TMSOTf-catalyzed phosphite procedure. Glycosylation with a-selectivity From the results discussed above it seems that glycosyl phosphites are not adequate glycosyl donors for the formation of a-glycosyl linkages. Despite this, glycosylations using the perbenzyl glucosyl phosphite donor, in the presence of I2 at room temperature, gave the a-glycosylated compounds 7 and 24 as major products. Glucosylation of a hindered acceptor with the glucosyl phosphite, under ZnCl2AgC104 conditions, gave 9 (88%, a : p = 7 : 3, see Figure 9) with lower selectivity [22b]. The best a-selectivity was achieved under DTBPI-Bu4NI conditions to give product 9 in a 95 : 5 a : p ratio. Glycosylation using Galactosyl and Fucosyl Phosphites with a Benzyl Group at C-2
0-Galactosylation using perbenzylated galactosyl dibenzyl phosphite, in the presence of TMSOTf or BF3.Et20 at -78"C, gave nearly pure products [17, 211. In addition, ZnC12-promoted galactosylation of secondary and primary alcohols using galactosyl diethyl phosphite gave the 0 product with selectivity (a: p 1 :4)in the same range as that obtained in glucosylations [22]. To achieve high a-selectivity, 60-acetyl galactosyl dibenzyl phosphite 25 was used to regioselectively glycosylate the 3-0-position of a partially protected acceptor in CH2C12 at - 15"C to give predominantly product 26 in 60% yield (Figure 10) [30]. Fucosylation using fucosyl dibenzyl phosphites has been reported in the synthesis of Lea and Ley catalyzed by TfOH [31]. The phosphite and trichloroacetimidate
s
-
BnO B
O(CH&Ph 7 92%, a l p = 81/19
Figure 9.
24 91%, a@ = 81/19
9 88%, cr/p = 69/31
5 Glycosylution Methods: Use of Phosphites
126
TMSOTf
(0.2equiv) -15"C
B
oP(ow2
25
BB g E # %
b
OBl
60%
HO 26 cL/p = 85/15
Figure 10.
methods have been compared for the synthesis of Lea (Figure 1 1 ) [35].It was concluded that 0-glycosyl phosphites nicely complement 0-glycosyl trichloroacetimidates, especially in the higher reactivity range where trichloroacetimidates tend to be less stable. In the course of development of sialyl LewisXmimetics, the Wong group conducted extensive studies on the fucosylation of more than ten different N-acylated p-hydroxyl amino acids. They found that fucosylation of benzyl-protected amino acids with perbenzylated fucosyl phosphite gave a greater yield and better aselectivity than the corresponding ethyl-protected amino acids [30, 311. Figure 12 depicts representative syntheses of these compounds. In addition, the DTBPIBWNI-promoted fucosylation reported by Hashimoto's group also gives excellent a-selectivity [ 17b]. Glycosylation using other Glycosyl Phosphites with a Benzyl Group at C-2
The perbenzylated glucuronyl phosphite has shown preference for 1,2-truns-Pselectivity, similar to that of glucosyl phosphite, when coupled with primary and secondary alcohols in the presence of BF3.Et20 at -65°C [22]. Synthesis of the fi-mannosyl linkage is a major problem in glycosylation reactions. Many efforts have been made to improve the p-selectivity of mannosylation using, for example, insoluble Ag silicate in glycosylation [38],inversion at the C-2 of p-glucosyl derivative after glycosylation [391, and intramolecular glycosylation methods [40].Recently, p-mannosylation was also acheved at low temperature through the triflate intermediate which is generated by different activation methods [27](see Section 5.3.2). A similar result might be expected from activation of mannosy1 phosphite with TMSOTf or TfOH, because it might proceed through the
A Ac
E
x TMSOTf
____)
Et20 A
AcO 27 X = OP(OCH$CI& 28 X = OC(NH)CC13
Figure 11.
sRq
OBl
TMSOTf
___)
OTDS EtpO
opc 29 64%
88%
N3
AC
Opc
30 88% 78%
5.3 Glycosylution using Glycosyl Phosphites
127
3 2 H 2 1 L
-
BocH
31
CHJ
TfOH, CH2Cl2, -15 "C
OP(QW2
33 84% R= Bn, c(/p=99/1 34 72% R= Et,dD=71/29
Figure 12.
glycosyl triflate intermediate. Indeed, the Hashimoto group recently showed that 2,3-di-O-benzyl-4,6-O-benzylidine-mannosyl diethylphosphite preferentially gives P-selectivity [ 17~1,although ZnCl2- or ZnClz-AgC104-catalyzed mannosylations from the glycosyl phosphites have been reported to afford a- or P-selectivity. When the perbenzylated mannosyl phosphite is glycosylated with cyclohexanol under ZnCl2 conditions, slight preference for the p-anomeric linkage (83%; a : P 2 : 3 ) is obtained. When the glycosylation is performed with methyl 2,4,6-tri-O-benzyla-glucopyranoside and ZnClz-AgCIO4 activation, the product is pure a-linked disaccharide in 76% yield [22]. The phosphite method has also been used in the synthesis of di-mannosylated phosphatidylinositol with two a-glycosyl linkages, as shown in Figure 13. In the first glycosylation, ZnClz-AgC104 was used to activate perbenzylated glycosyl phosphite to give a 2: 1 alp mixture of 36a. The same conditions gave the pure a product 36b in 70% yield when a 2-OAc mannosyl phosphite was used as donor. Interestingly, at room temperature TMSOTf-catalyzed mannosylation using the perbenzylated phosphite gave a-glycoside 37 in 74% yield [41]. It is not clear how the temperature affects the stereoselectivity of glycosylations employing benzylated mannosyl phosphite with TMSOTf or TfOH as promoter. N
5.3.6 Glycosylation with 2-Deoxy Glycosyl Phosphites The synthesis of 2-deoxy oligosaccharides with controlled a- or P-selectivity remains a difficult problem [lOc, 421 and many methods have been developed to solve it.
@-
ZnC12AgC104
P
"'OH Et20
TIP<S
OH RO
35
Figure 13.
oh
36a: R=Bn 65% a:p2:1 36b: R=Ac 70% a only
5 Glycosylation Methods: Use of Phosphites
128
D-A Temp. Yield a:O
38-43 -78 "C 91% 19:81 38-43 -50
"C
87% 29:71
39-43 -94
"c
93% 13187
40-43 -94
"C 97% 1436
41-43 -94 "C 95% 23:77 42-43 -94 "C 96% 67:33 41-44 -94 "C 86% 69:31
38
39
40
Bn9 ,ow B
w (EtOhPO F
41
OH
BBs%q
43
44
B* OMe
Figure 14.
Examples include electrophile-mediated addition of acceptor to glycals [42a], and glycosylation of the C2-substituted glycosyl donor [42b] followed by reductive removal of the C-2 substituent [ lOc]. Direct stereoselective glycosylation using a 2deoxy glycosyl donor is, however, desirable. 'Remote-controlled' [43] activation of the 2-pyridine-thioglycoside by AgOTf [44]provides a very efficient method for the a-selectively controlled synthesis of 2-deoxy glycosides. Use of insoluble silver silicate for the activation of 2-deoxy glycosyl halides provides one of the best methods of preparation of 2-deoxy-0-glycosides [45]. The Schmidt group has studied the glycosylation reaction using 2-deoxy glycosyl phosphites activated by BF3 .Et20 in CH2Cl2-hexane or in Et20 [36]. Preferential a-selectivities were obtained in the formation of both the 2-deoxy galactosyl linkage and the 2-deoxy glucosyl linkage. In the glycosylation of 2-deoxy glycosyl phosphites under TMSOTf activation [12, 131, the Hashimoto group [26] recently found that the reaction temperature is crucially important in the control of P-selectivity (see Figure 14, entries 1-3). The selectivities were not significantly affected by the reactivities of the glycosyl donors (entries 1, 4, and 5; a :P = 10: 90, 13 :87, and 14: 86, respectively). They are, however, affected by other structural properties of the glycosyl donors. For example, compounds 41 and 42 with axial substituents at C-4 gave lower p-selectivities (entry 6, a : P = 23: 77 and entry 7, a : P = 67: 33). The p-selectivity was observed to increase with increasing reactivity of the acceptor alcohol (compare entries 6 and 8). In the synthesis of the hexasaccharide fragment of landomycin A, the desirable p-glycosyl linkage of the 2,6-dideoxy unit was successfully formed by use of the TMSOTf-promoted phosphite method (Figure 15) [ 161.
5.4 Other Applications of Glycosyl Phosphites Besides the 0-,S-, C-glycosylations and 0-glycopeptide synthesis discussed above, other applications of glycosyl phosphites are discussed below.
5.4 Other Applications of Glycosyl Phosphite
129
TMSOTf -94 OC AcO
HO
c,
AcO
47
&*+
OMP
TMSOTf, Toluene,
0 M
48 R' = AC (42%, a:p 5 2 ~ 8 )
A d
Figure 15.
5.4.1 Synthesis of CMP-NeuAc
The synthesis of CMP-NeuSAc remains of interest, because it is a glycosyl donor in glycosyltransferase-catalyzed reactions. Three chemical synthetic methods based on the phosphite strategy have been reported. The Wong group employed a glycosyl phosphoamidate (Figure 16A) [12], whereas the Halcomb group used a nucleoside phosphoamidate in the synthesis of CMP-NeuSAc (Figure 16B) [46a]. The latter method seems preferable, because of its high-yielding deprotection step. The Schmidt group utilized the diethyl glycosyl phosphite and the nucleoside phosphate 53 in its acid form to give P-CMP-NeuAc 54 in 50% yield (Figure 17) [46b].The reaction seems to be mediated by acid catalysis.
5.4.2 Synthesis of GDP-Fucose
GDP-fucose is a glycosyl donor substrate for fucosyltransferases. Although it has been synthesized by many chemical methods [47],the use of fucosyl phosphite in the synthesis seems the most practical [48]. A recent improvement of this method includes the addition of tetrazole to the morpholidate coupling reaction [48b]. This method might also be applicable to the synthesis of other nucleoside diphosphate sugars (Fig-ure 18). Together with the synthesis of CMP-NeuSAc discussed above, glycosyl phosphite chemistry indeed provides a useful alternative for the synthesis of natural and unnatural donor substrates for the study of enzyme-catalyzed glycosylations.
130
5 Glycosylation Methods: Use of Phosphites NHBz 1. HOR 2. BuOOH 12 Yo
50
51
52
Figure 16.
HQ
PH
HAC
CH ,CN/TH FIDMF
0 3
AcO 01% 53
(1:l:l)
-15 "C to rt.,
AcO AcO 54 50%
Figure 17.
1.30% H202 2. H2IPd-C F 0 $ O N a h AcO F z z B n 0 ) 2 3. NaOWH20 HO OH 55 63% 54 cr:p =1:10 tetrazolej Pyr
57 91%
Figure 18.
Ho
OH
OPC
5.4 Other Applications of Glycosyl Phosphite Ac
131
TMSOP(OMe)* TMSOTf CHzCI2
PCH
A d
ACQ 58 63 Yo Ponly
3 Figure 19.
5.4.3 Formation of Glycosyl Phosphonate Glycosyl phosphites can be converted by Arbuzov-type fragmentation to the corresponding glycosyl phosphonates as analogs of glycosyl phosphates [49]. Glycosyl phosphonates are more stable than glycosyl phosphates [50]and can act either as inhibitors [5I] or as regulators in metabolic processes where natural phosphates function. Figure 19 shows the synthesis of a sialyl phosphonate starting from the glycosyl phosphite [52]. 5.4.4 Transformation to other Types of Glycosyl Donor Phosphate Oxidation [I 8, 481 of glycosyl phosphites provides glycosyl phosphates which have been used for the synthesis of oligosaccharides [53] and sugar nucleosides [12, 81 (Figure 20).
rA-OP(ORh
oxidation
8
* a O F ' ( O R h
HOR TMSOTf
OR
Figure 20.
Phosphorimidate More recently, a new glycosylation method has been reported using glycosyl phosphorimidates as glycosyl donors. Thus, compounds were prepared from the corresponding glycosyl phosphites (Figure 21) 1541.
Figure 21.
132
5 Glycosylation Methods: Use of Phosphites
In summary, use of glycosyl phosphites in a new glycosylation method has received much attention for several reasons, including the high reactivity of the glycosyl phosphite at low temperature, the high efficiency of glycosylation with stereo-hindered donors or acceptors, and the high stereoselectivity of the reaction. Applications of glycosyl phosphites in the synthesis of glycosyl phosphates and glycosyl phosphonates are, furthermore, especially useful. Applications and modifications of glycosyl phosphite chemistry will surely be of interest to many chemists in the future.
References 1. Wong, C.-H., Ye, X.-S., and Zhang, Z . J. Am. Chem. SOC.1998,120, 7131. 2. T. Wubberg, C. Kallus, T. Opatz, S. Henke, W. Schmidt, H. Kunz, Angew. Chem. Int. Ed. Engl. 1998,37, 2503-2505. 3. Random glycosylation: (a) 0. Kanie, F. Barresi, Y. Ding, J. Labbe, A. Otter, L. S. Forsberg, B. Ernst, 0. Hindsgaul, Angew. Chem. Znt. Ed. Engl. 1995, 34, 2720. (b) Y. Ding, J. Labbe, 0. Kanie, 0. Hindsgaul, Bioorg. Med. Chem. 1996, 4, 683. Latent-active glycosylation: G.-J. Boons, B. Heskamp, F. Hout, Angew. Chem. Znt. Ed. Engl. 1996,35,2845. Solid-phasemethod: R. Liang, L. Yan, J. Loebach, M. Ge, Y. Uozumi, K. Sekanina, N. Horan, J. Gildersleeve, C. Thompson, A. Smith, K. Biswas, W. C. Still, D. Kahne, Science 1996,274, 1520. 4. (a). D. R. Mootoo, P. Konradsson, U. Udodong, B, Fraser-Reid, J. Am. Chem. SOC.1988,110, 5583; (b). B, Fraser-Reid, Z. Wu, U. D. Udodong, H. Ottosson, J. Urg. Chem. 1990, 55, 60686070. 5. Y. Ito, T. Ogawa, J. Am. Chem. SOC.1994,116, 12073. 6. (a). S. Raghavan, D. Kahne, J. Am. Chem. SOC.,1993,115, 1580. (b) H. Yamada, T. Harada, H. Miyazaki, T. Takahashi, Tetrahedron Lett., 1994, 35, 3979. (c) H. Yamada, T. Harada, T. Takahashi, J. Am. Chem. SOC.1994, 116, 7919. (d) H. K. Chenault, A. Castro, Tetrahedron Lett., 1994, 35, 9145. (e) R. Geurtsen, D. S. Holmes, G.-J. Boons, J. Urg. Chem. 1997, 62, 8145. 7. (a) S. V. Ley, H. W. M. Priepke, Angew. Chem. Int. Ed. Engl. 1994,33,2292. (b) P. Grice, S. V. Ley, J. Pietruszka, H. W. M. Priepke, E. P. E. Walther, Synlett 1995, 781. (c) P. Grice, S. V. Ley, J. Pietruszka, M. I. Osborn, W. M. Henning, H. W. M. Priepke, S . L. Warriner, Chem. Eur. J. 1997, 431. (d). N. L. Douglas, S. V. Ley, U. Lucking, S. L. Warriner, J. Chem. Soc. Perkin Trans. 1, 1998, 51. (e). L. Green, B. Hinzen, S. J. Ince, P. Langer, S. V. Ley, S . L. Warriner, Synlett. 1998, 4, 440. 8. Z. Zhang, I. R. Ollmann, X.-S. Ye, R. Wischnat, T. Baasov, C.-H. Wong, J. Am. Chem. SOC. 1999, 121, 734. 9. Solid-phase synthesis: For review see, (a). P. H. Seeberger, S. J Danishefsky, Acc. Chem. Res. 1998, 31, 685. (b). J. S. Fruechtel, G. Jung, Angew. Chem., Int. Ed. Engl. 1996, 35, 17; For general articles see, (c) M. J. Sofia, Comb. Chem. Mol. Diversity Drug Discovery, Eds: E. M. Gordon, J. F. Kenvin Jr., Wiley-Liss, New York, 1998, 243-269; (d). R. R. Schmidt, Pure Appl. Chem. 1998, 70, 397-402; (e). Z.-W. Guo, Y. Nakahara, Y. Nakahara, T. Ogawa, Angew. Chem., Znt. Ed. Engl. 1997, 36, 1464; (f). R. Rodebaugh, S. Joshi, B. Fraser-Reid, H. M. Geysen, J. Org. Chem. 1997, 62, 5660; (8). S. J. Danishefsky, K. F. McClure, J. T. Randolph, R. B. Ruggeri, Science 1993,260, 1307; For enzymatic methods see, (h). M. Schuster, P. Wang, J. C. Paulson, C.-H. Wong, J. Am. Chem. SOC.1994,116, 1135. 10. For the recent review articles on oligosaccharide synthesis see: (a) H. Paulsen, Angew. Chem. Int. Ed. Engl., 1990, 29, 823; (b) J. Banoub, Chem Rev. 1992, 92, 1167; (c) K. Toshima, K. Tatsuta, Chem. Rev. 1993, 93, 1503. (d) R. R. Schmidt, W. Kinzy, Adv. Carbohydr. Chem. Biochem. 1994, 50, 21. (e) S. J. Danishefsky, M. T. Bilodeau, Angew. Chem. Int. Ed. Engl., 1996,35, 1380. 11. H. Paulsen Angew. Chem. Int. Ed. Engl., 1982,21, 155.
References
133
12. H. Kondo, Y. Ichikawa, C.-H. Wong, J. Am. Chem. SOC.1992,114, 8748. 13. T. J. Martin, R. Schmidt, Tetrahedron. Lett. 1992, 33, 6123. 14. (a). L. I. Smirnova, M. A. Malenkovskaya, D. A. Predvoditelev, E. E. Nifantev, Zh. Org. Khim. 1980, 16, 1170; (b). J. W. Perich, R. B. Johns, Tetrahedron Lett. 1987,28, 101; (c). K. L. Yu, B. Fraser-Reid, Tetrahedron Lett. 1988, 98, 979; (d). R. L. Pederson, J. Esker, C.-H. Wong, Tetrahedron, 1991, 47, 2643; (e). S. Aoki, H. Kondo, C.-H. Wong, Methods in Enzymology, 247, 193; (f). Qiao, B. W. Murray, M. Shimazaki, J. Schultz, C.-H. Wong, J. Am. Chem. SOC. 1996, 118, 1653. 15. H. Paulsen, H. Tietz, Carbohydr. Res. 1984, 125, 47. 16. Y. Guo and G. A. Sulikowski, J. Am. Chem. SOC.1998,120, 1392. 17. (a). S. Hashimoto, K. Umeo, A. Sano, N. Watanabe, M. Nakajima, and S. Ikegami, Tetrahedron Lett., 1995,36, 2251; (b). H. Tanaka, H. Sakamoto, A. Sano, S. Nakajima, S. Hashimoto, Chem. Commun., 1999, 1259; (c). unpublished result, personal communication with Prof. Hashimoto. 18. M. M. Sim, H. Kondo and C.-H. Wong, J. Am. Chem. SOC.1993,115,2260. 19. R. R. Schmidt, B. Wegmann, K.-H. Jung, Liebigs Ann. Chem. 1991, 191, 121. 20. S. Sabesan, S. Neira, Carbohydr. Res. 1992, 223, 169. 21. H. Kondo, S. Aoki, Y. Ichikawa, R. L. Halcomb, H. Ritzen, C.-H. Wong, J. Org. Chem. 1994, 59, 864. 22. (a). Y. Watanabe, C. Nakamoto, S. Ozaki, Synlett, 1993, 115-116; (b). Y. Watanabe, C. Nakamoto, T. Yamamoto, S. Ozaki, Tetrahedron, 1994, 50, 6523. 23. H. Schene, H. Waldmann, Eru. J. Ory. Chem. 1998, 1227. 24. Lemieux Abstr: Pap. Am. Chem. SOC.1958. 133, 31N; (b) J. P. Praly, R. U. Lemieux, Can. J. Chem. 1987,65,213. 25. (a). R. R. Schmidt, M. Behrendt, A. Toepfer, Synlett, 1990, 694; (b). A. J. Ratcliffe, B. FraserReid, J. Chem. Soc., Perkin Trans. 1 1990, 747; (c). P. Sinay, Pure Appl. Chem. 1991, 63, 519. 26. S. Hashimoto, A. Sano, H. Sakamoto, M. Nakajima, Y. Yanagiya, S. Ikegami, Synlett., 1995, 1271. 27. (a). D. Crich, S . Sun, J. Ory. Chenz. 1996, 61, 4506-4507: (b). D. Crich, S . Sun, J. Am. Chem. SOC.1997, 119, 11217; (c). D. Crich, S. Sun, J. Am. Chem. SOC.1998, 120, 435. 28. (a). D. Kahne, S. Walker, Y. Cheng, D. V . Engen, J. Am. Chem. SOC.1989, 111, 6881; (b). L. Yan, D Kahne, J. Am. Chem. SOC.1996, 118, 9239. 29. (a). V. Martichonok, G. Whitesides, J. Am. Chem. SOC.1996, 118, 8187; (b). V. Martichonok, G. Whitesides, J. Org. Chem. 1996, 61, 1702. 30. C.-C. Lin, M. Shimazaki, M.-P. Heck, S. Aoki, R. Wong, T. Kimura, H. Ritzen, S. Takayama, S.-H. Wu, G. Weitz-Schmidt, C.-H. Wong, J. Am. Chem. SOC.1996, 118, 6826. 31. S.-H. Wu, M. Shimazaki, C.-C. Lin, L. Qiao, W. J. Moree, G. Weitz-Schmidt, C.-H. Wong, Angew. Chem. Int. Ed. Engl., 1996, 35, 88. 32. For sialylations see ref. 12, and 13 and references therein. 33. N. E. Nifantev, Y. E. Tsvetkov, A. S. Shashkov, L. 0. Kononov, V. M. Menshov, A. B. Tuzikov, a n d N . V. Bovin, J. Carbohydr. Chem. 1996, 15, 939. 34. U. Sprengard, G. Kretzschmar, E. Bartnik, C. Hiils, and H. Kunz, Angew. Chem. lnt. Ed. Engl. 1995, 34, 990. 35. T. Muller, G. Hummel, R. R. Schmidt, Liebigs Ann. Chem. 1994, 325. 36. T. Miiller, R. Schneider, R. R. Schmidt, Tetrahedron Lett. 1994,35,4763. 1993, 115, 8871. 37. E. J. Corey, Y.-J. Wu, J. Am. Chem. SOC. 38. H. Paulsen, 0. Lockhoff, Chem. Ber. 1981,114, 3102. 39. H. Kunz, W . Giinther, Angew. Chem. Int. Ed. Engl. 1988, 27, 1086. 40. (a). F. Barresi, 0. Hindsgaul, J. Am. Chem. SOC.1991, 113,9376; (b). G. Stork, G. Kim, J. Am. Chem. SOC.1992, 114, 1087; (c). Y. Ito, T. Ogawa, J. Am. Chem. Soc. 1994, 116, 12073. 41. (a). Y. Watanabe, T. Yamamoto and S. Ozaki, J. Org. Chem. 1996,61, 14-15; . (b). Watanabe, T. Yamamoto and S. Ozaki, Tetrahedron, 1997, 53, 903. 42. (a). J. Thiem, W. KlaKke, Top. Curr. Chem. 1990, 154, 870; (b). William Roush and Chad E. Bennett J. Am. Chem. SOC. 1999, 121, 3541. 43. S. Hanessian, C. Bacquet, N. Lehong Carbohydr. Res. 1982, 80, c17. 44. Z. Zhang, G. Magnusson, Carbohydr. Res. 1994,262, 79.
134
5 Glycosylation Methods: Use of Phosphites
45. (a). P. J. Garregg, S. Kopper, P. Ossowski, J. Thiem, J. Carbohydr. Chem. 1986, 5, 59; (b). R. W. Binkley, D. J. Koholic, J. Org. Chem. 1989, 54, 3577; (c). Z. Zhang, G. Magnusson, J. Org. Chem. 1996,61, 2383. 46. (a). M. D. Chappell, R. L. Halcomb, Tetrahedron 1997, 53, 11109; (b). T. J. Martin, R. R. Schmidt, Tetrahedron Lett. 1993, 34, 1765. 47. U. B. Gokhale, 0. Hindsgaul, M. M. Palcic, Can. J. Chem. 1990, 68, 1063; (c). R. R. Schimidt, B. Wegmann, K.-H. Jung Liebigs. Ann. Chem. 1991,191, 121. 48. (a). Y. Ichikawa, M. M. Sim, C.-H. Wong, J. Org. Chem. 1992, 57, 2943-2946; (b). V. Wittmann, C.-H. Wong J. Org. Chem. 1997, 62, 2144. 49. R. Meuwly, A. Vasella, Helv. Chim. Acta. 1986, 69, 25. 50. (a). J. Gao, V. Martichonok, G. M. Whitesides, J. Org. Chem. 1996, 61, 9538; (b). T.-H. Chan, Y.-C. Xin J. Org. Chem. 1997, 62, 3500; (c). B. Miiller, T. J. Martin, C. Schaub, R. R. Schmidt, Tetrahedron Lett. 1998, 39, 509; (d). M. J. Ruira, M. J. Pkrez-Pkrez, Jan. Balzaerini, M. Camarasa, Synlett. 1998, 177. 51. C. L. White, M. N. Janakiraman, W. G. Laver, C. Philippon, A. Vasella, G. M. Air, M. Luo, J. Mol. Biol. 1995,245, 623. 52. M. Imamura, H. Hashimoto, Tetrahedron Lett. 1996, 37, 1451. 53. S. Hashimoto, T. Honda, S. Ikegami, J. Chem. Soc. Chem. Commun. 1989, 685. 54. (a). S. Pan, H. Li, F. Hong, B. Yu, K. Zhao, Tetrahedron. Lett. 1997, 38, 6139; (b). H. Li, M. Chen, K. Zhao, ibid., 1997,38, 6143.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
6 Glycosylation Methods: Use of n-Pentenyl Glycosides Bert Fraser-Reid, G. Anilkumar, Mark R. Gilbert, Subodh Joshi, and R a y Kraehmer
6.1 Introduction The use of protecting groups in syntheses of complex oligosaccharides is currently inevitable. Electronic or steric factors may enable regioselectivity between competing hydroxyl groups, but for the foreseeable future such interventions will be exceptions to the rule. If protecting groups are to be necessary, it would be advantageous for them to serve additional roles. In our laboratory this concept was first appreciated with the development of n-pentenyl glycosides (NPG), in which the pentenyl group protects the sensitive anomeric center, and survives (most of) the chemical transformations tolerated by normal alkyl glycosides. Treatment with an electrophile converts the pentenyl moiety into a leaving group, however, thereby enabling replacement reactions to occur at the anomeric center. This dual protection/activation role is a fundamental property of NPGs, and this duality was heavily exploited in the synthesis of the rat brain Thy-1 glycosylphosphatidylinositol membrane anchor by the retrosynthetic plan outlined in Scheme 1 [I]. NPGs have, however, also been valuable in mechanistic investigations designed to probe events at the anomeric center. For example, NPGs were instrumental in the demonstration that protecting groups can profoundly alter reactivities of glycosyl donors-and glycosyl acceptors. These observations indicated the importance of fine-tuning reactivity of donor/acceptor partners, and efforts to contribute to this insight continue in our laboratory.
6.2 Fundamental Reactions A summary of (most of) the fundamental reactions of n-pentenyl glycosides is presented in Scheme 2 [ 2 ] .Being simple alkyl glycosides, NPGs can be readily prepared
6 Glycosylation Methods: Use of n-Pentenyl Glycosides
136
V
0
HO
HO-
-
Thy1 Anchor from Rat Brain
OBn BnO
Scheme 1. Retrosynthetic plan for rat brain Thy-1 GPI membrane anchor.
by standard Fischer glycosidations, e.g., 4 4 5 . Where one or other anomer is specifically required, however, the standard procedures used for simple alkyl glycosides can be applied as summarized in Scheme 3. The role as glycosyl donor proceeds through the standard oxocarbenium intermediate 2, which can be trapped by nucleophiles. In aqueous medium the product is an aldose (1, Nu = OH). When such oxidative hydrolyses were probed it was found that with C2-OAcyl substrates the reaction occurs much more slowly than with C2OAlkyl. This observation has been introduced into a chemoselective coupling [3] protocol that has come to be known as the 'armed-disarmed strategy.' [4]Thus as illustrated in Scheme 4a, the partners 10 and 11 gave disaccharide 12a, with no evidence of the product of self-coupling of 11, nor of further reaction of disaccharide 12a with the acceptor 11, both of which would have occurred at disarmed anomeric
RN"& p-1 Y
1 Nu = OR, OSugar, Br, NHCOPeptide
/
6.2 Fundamental Reactions
p!
Y
Y
2
3 furanylium ion
glycosyl donor
137
J
6
free sugar
NPG
I disaccharide or higher
Zn or Smlp or Nal
glycosyl acceptor
cyclic halonium
I
+
XsNu'
9 bromohydrin Nu=OH dibromide Nu=Br
Scheme 2. The chemistry of n-pentenyl glycosides (NPG).
centers. Transformation to the armed counterpart 12b, however, enabled ready formation of trisaccharide 13 [3]. Glycoside hydrolysis is of immense mechanistic interest because of the insight it provides into enzymatic cleavage or assembly of oligosaccharides [5, 61. The option of oxidative hydrolysis meant that the anomeric center of NPG could be cleaved without the use of acids and, furthermore, that acid-labile protecting groups could be employed to enhance mechanistic insight. In the course of such investigations, it became clear that frequently used cyclic protecting groups could also deactivate glycosyl donors [7]. Thus, the advantage of simultaneous protection of two hydroxyl groups might be offset by the torsional constraints imposed upon the bicyclic array. Theoretical calculations supported this finding [81.
138
(Hqn-k>0H
6 Glycosylution Methods: Use of n-Pentenyl Glycosides
camphor Pent-OH
(HO)n{>O(/
sulfonic acid
I
OPent
Scheme 3. Preparative routes to NPG.
The reality of torsional armed-disarmed effects was demonstrated (Scheme 4b) with the glucosides 14 and 15, which gave the product of cross coupling, 16, but with no evidence of 17, the product from self-coupling of 15 [9].
6.3 Determination of Relative Reactivities The evidence in Scheme 4 makes it clear that success in oligosaccharide coupling can be dependent on protecting groups present on the reactants. A procedure for direct comparison of glycosyl donor reactivity would be advantageous, and such a methodology was therefore developed [lo]. As shown in Figure 1, eq. (l), equivalent amounts of the two NPGs, S1 and S2, compete for one equivalent of NBS in aqueous solvent. As eq. (2) shows the method requires that the NBS must be completely consumed, and so the reaction must continue until a negative test is obtained with potassium iodide-starch paper, shown independently to be accurate to within 2%. Because both anomers are in the same reaction medium, the NBS terms in the rate expressions cancel out, leading eventually to eq. (3). After work-up, the ratio of unreacted NPGs in the crude reaction product is determined, eq. (4). For our purposes, HPLC was convenient for these determinations. All the terms in eq. ( 5 ) are now known or knowable. And so the relative reactivity, kl/k2, of the two NPGs can be obtained by solving eq. (3). Some comparisons of reactivities, as determined by the procedure in Figure 1, are assembled in Scheme 5. That the choice of anomeric configuration of the donor might be important is apparent from the results for 18 and 19. In both the anomer
6.3 Determination of Relative Reactivities
k M
J+
0
,""I?,..
b T a
9
139
6 Glycosylation Methods: Use of n-Pentenyl Glycosides
140
S1
+ S2 + NBS
H20 ,Products CH,CN
(1 eq. of each) [end point determined by negative KI-starch test paper]
Figure 1. Calculating the relative rates of two glycosyl donors.
reacts more quickly, the higher ratio in 19 being evidence of anchimeric assistance resulting from neighboring-group participation in the cleavage reaction [ 1I]. The classical electronic armed-disarmed situation (Scheme 4a) is seen with the p glycosides 20a and b. Notably, 20b is less reactive than 20a despite the anchimeric assistance which must be occurring in the ester (cf 19). The results for the p glucoside pairs 20 and 21 confirm that the benzylidene ring can be disarming-much more so for acyl protecting groups (21, R = Ac compared with R = Bn). Couplings involving benzylidinated or acetonated donors might, therefore, occur less readily than with their torsion-free counterparts.
BnO
OPent
AcO
,OAc
OBn 18
OPent
R:)ope,,
= 1.59
OR
20
19
18p/ M a = 1.70
20d20b
\
a R=Bn
19p/ 19a= 5.19
b R=Ac R RO o R ~ O F ’ e n pl
~
~
~
~2 0 d 2 l a O = 1.6
\
OR
OK
20
21
a R=Bn
a R=Bn
b R=Ac
b R=Ac
Scheme 5. Relative reaction rates for some glycosyl donors.
F20b/ 2lb m= 10.4
6.4 n-Pentenyl Orthoesters as Glycosyl Donors
141
Although these results are based on NPG, they should apply equally to any pair of donors, irrespective of the anomeric leaving group. This assumption is based on general relevance of armed-disarmed factors across the spectrum of glycosyl donors 1121.
6.4 n-Pentenyl Orthoesters as Glycosyl Donors Alkyl orthoesters, 23, like cyclic acetals, also simultaneously protect two hydroxyl groups. Their use in carbohydrate synthesis [13] is greatly enhanced by a regioselective, acid-catalyzed rearrangement, whereby the resulting ester is axial rather than equatorial [ 141. Glycosyl (or 1,2-) orthoesters are readily prepared by Lemeiux’s procedure, in which acylated glycosyl bromides are treated with a hindered base, 22 23 [ 151 (Scheme 6). Mild acid treatment causes a rearrangement, 23 -, 26, in which the alkoxy unit is transferred to the anomeric center [16] (Scheme 6). The group so transferred can vary from simple alkoxy to an oligosaccharide [ 171. n-Pentenyl orthoesters (23, S = Pent) have an additional mode of reaction. Thus, treatment with an electrophile leads to furanylium ion 27, and thence to the same dioxolenium intermediate 25 (Scheme 6). The discharged entity, 28, however, is not nucleophilic and so a nucleophile present in the reaction medium faces no competition for reaction with 25 leading to 29. Notably, 26c and 29a are both higher saccharides, but the strategies for their preparation offer a choice between protonand electrophile-driven reactions. Again, n-pentenyl orthoester 23 (S = Pent) and NPG 26b are both glycosyl donors, but they have very different properties, notably towards acids. Further donor versatility comes from the fact that titration with bromine affords bromide 29b, the classic glycosyl donor, ready for reaction with silver triflate [ 181. To go from one glycosyl bromide to another, 22 +29b, might seem redundant, but the advantage of this possibility is readily appreciated from Scheme 7, which summarizes two approaches to trimannan 36, a building block for a synthesis of the Thy-1 GPI rnembrane (see Scheme 1) [19]. The tetrabenzoyl mannosyl bromide 30 was converted into orthoester 31a [15]. One portion was silylated [20] and then benzylated to give 31b, which upon treatment with bromine led to the glycosyl bromide 32. The remainder of 31a was completely benzylated, and one portion of the resulting material, 31c, was processed to give the acceptor 33a whereas the other was brominated to give glycosyl bromide 35. Coupling of 32 and 33b, led to 34a and debenzoylation to acceptor 34b which reacted with 35 to give trimannan 36 P11. Glycosyl bromides 32 and 35, being unstable, are best generated and used in situ. An approach to the same trimannan in which all reacting participants are stable is, however, outlined in Scheme 7b. The pathway takes advantage of some of the unique aspects of n-pentenyl glycosides. Thus, a major development in NPG chemistry emanated from mechanistic investigations which showed that intramolecular reaction of the cyclic halonium compound leading to the furanylium ion, --f
142
6 Glycosylation Methods: Use of n-Pentenyl Glycosides
v1
c
x 8 *
8
%
Scheme 7. Two routes to a trimannan from an n-pentenyl orthoester. (i) Pent-OH, 2,6-lutidine, CHzC12; (ii) tBuPh2SiCl; imidazole, THF; (iii) BnBr, NaH, B U ~ N ITHF; , (iv) 31b or c; Br2, CH2C12, 0 ° C ; (v) 31c CSA, CH2C12, 5OoC, 6 h; (vi) NaOMe; (vii) AgOTf, CHzCl,; (viii) TBAF, THF then Ac20, DMAP, CHzC12; (ix) Br2, Et4NBr, CH2C12; (x) NIS, Et&OTf, CH2Clz;(xi) NH3, MeOH; (ClAc)20, Et3N, CH2CI2; (xii) Zn, BQNI, EtOH then (ClAc)20.
U
144
6 Glycosylation Methods: Use of n-Pentenyl Glycosides
(Scheme 2, 6-+3), could be offset by the bimolecular process leading to 9, in the presence of excess nucleophile. For example, added bromide ion led to a vicinal dibromide; because, however, reductive elimination readily restored the double bond, dibromides 7 and 8 represent ‘sidetracked’ forms of an NPG. Nevertheless, the sidetracked material enables an hydroxyl group to be liberated, thereby creating a glycosyl acceptor 8 capable of reacting with a donor to give 7. The utility of the ‘sidetracked’ capability is illustrated in Scheme 7b. A portion of NPG 33a was debenzoylated to 33b and then ‘sidetracked’ to give acceptor 38. Coupling with orthoester 37 then gave dimannan 39a, which underwent routine protecting-group alterations to produce disaccharide acceptor 39b. Coupling with the remainder of 33a now produced 40. The major process for obtaining the previously described 36 now involved reductive debromination to regenerate the pentenyl moiety [22].
6.5 n-Pentenyl Orthoesters as Latent C2 Esters It is apparent from Scheme 7 that proton- or electrophile-driven reaction leads to specifically C2-0-acylated products. C2-Esters are important for controlling stereoselectivity in anomeric replacement reactions [ 1 11, and so the use of orthoesters as specific latent C2-esters has been explored in the context of a protected form of the ‘linkage region’ of proteoglycans, which contains four 1,2-trans diequatorial linkages (Scheme 8). For the C2-acylated galactoside acceptor, a simple route begins with galactosyl bromide 41 (Scheme 8a). Orthoester formation required the use excess pentenyl alcohol and tetrabutylammonium iodide as promoter to obtain 42a and thence 42b. Acid-catalyzed reaction of the latter in the presence of excess pentenyl alcohol, then benzylidination gave acceptor 43a. The xylosyl acceptor 46b (Scheme 8b) was obtained similarly from bromide 44 via the n-pentenyl orthoester 45. Diol 46a was subjected to selective silylation at C4, paving the way to 46b. It was expected that the uronate moiety would be problematic because such 2-0acyl donors usually lead preponderantly to orthoester products. Of several donors that were tested, trichloroacetimidates were found to give the least amount of orthoesters. Donor 49 was therefore prepared from commercially available benzyl P-glucoside, 47a, via methyl ester 48 as summarized in Scheme 8c. Coupling with galactoside 43a gave the disaccharide donor 50 as the major product, the orthoester (51) being minimized at 25%. The disaccharide acceptor 52b was obtained by coupling the ‘sidetracked’ xyloside 46b to the n-pentenyl galactoside 43b, followed by dechloroacetylation. The latter reaction was problematic and required use of the van Boeckel procedure [23]. The two disaccharides were then coupled to give 53a. Iodide-induced debromination [24], a milder alternative to the zinc reduction used for 42 -+ 38 in Scheme 7, afforded 54b. Finally, coupling with the protected serine 54 [25] occurred smoothly to give the desired ‘linkage region’ construct 55 [26].
6.5 n-Pentenyl Orthoesters as Latent C2 Esters
145
146
6 Glycosylution Methods: Use of n-Pentenyl Glycosides
6.6 Protecting Groups The presence of one free amine and four amide functions in the Thy-1 anchor (Scheme 1) sensitized us to the need for new, easily removable amine protecting groups. The n-pentenyl based protecting groups for alcohols and vicinal diols shown in Scheme 9a-c are outgrowths of NPG chemistry [27], all of which are cleaved or installed with halogen electrophiles. Notably, the acetalating agents in Scheme 9d can be put in place without disturbing acid-sensitive groups already present [28]. Glycosyl n-pentenoyl esters (Scheme 9e) have been found to be good glycosyl donors, a circumstance that provides evidence for ready loss of the pentenoyl group [29]. In the light of these precedents, amine protection via n-pentenoylation was examined. As shown in Scheme 9f installation for primary and secondary amines is routine, and cleavage occurs readily with iodine. C2-N-acylated sugars are, however, prone to give oxazolines as shown in Scheme 9g. Lemieux showed that this problem could be obviated by employing phthaloyl protecting groups [30], de-Nphthaloylation being effected by prolonged treatment with hydrazine or ethylenediamine. It has been shown [31, 321 that electron-withdrawing groups on the aromatic ring facilitate cleavage remarkably, as indicated by the difference for X = C1 and X = H in Scheme 9h. Cleavage of pentenoyl or tetrachlorophthaloyl (TCP) groups is therefore seen to occur very readily, and the very different procedures employed enable these protecting groups to be used orthogonally. This aspect was explored in the context of lipid A, 56 [33] (Scheme lo), a molecule in which all sites on the disaccharide need to be differentiated. The glucosaminide functions would be protected by either npentenoyl or TCP; but use of the former on the acceptor rather than the donor was dictated by the oxazoline problem noted in Scheme 9g. In the event Koenigs-Knorr coupling of 57 or 58 produced 59 in good yield. Comparison with lipid A, 56, shows that differentiation between the three acetylated sites of 54 would be necessary for a more useful precursor. Deacetylation with base would not be tolerated by the TCP group; but acid-catalyzed deacetylation was successful and benzylidination led to the more attractive counterpart 60 with the five critical sites being differentially accessible. With respect to the protected amines, treatment with ethylenediamine liberated one and iodine the other, giving 61 and 62 respectively [34]. Notably, cleavage of the benzylidine group with sodium cyanoborohydride [35] is tolerated by TCP [36].
6.7 Solid-Phase Iterative Couple-Deprotect-Couple Strategy Scheme 2 shows that NPGs have the potential to function as glycosyl donors and also as glycosyl acceptors via sidetracked intermediates (e.g. 8). The latter property
6.7 Solid-Phase Iterative Couple-Deprotect-Couple Strategy
h I
QfB I
1 (0 0
+ I '
8
q)==J 0
I
\
141
148
6 Glycosylation Methods: Use of n-Pentenyl Glycosides
Lipid A /OAC
AcO AcO BzO
Ad3
BzO
Oknt
-
57
DIFFERENTIALLY ACCESSIBLE SITES
Scheme 10. Orthogonal protecting groups for a lipid a precursor. (i) AgOTf; (ii) Me2CO/H20/HCl, 60"C, 20 h; PhCH(OMe)2/PF'TS/dioxane, reflux; (iii) ethylenediarnine, 60°C 2 h; (iv) 12 (2 equiv.), HzO/THF, 8 min.
was instrumental in a process used to assemble the high-mannose nonamannan component via an iterative deprotect-couple-deprotect strategy [37]. A solid-phase version of this procedure would have the advantage of minimizing transformations on the bound saccharide(s). An attempt to develop such an iterative strategy, shown in Scheme 11, used the known [38], bound pentenyl alcohol 63 for a KoenigsKnorr reaction with 64, by following the procedure developed by Frechet and Schuerch [39]. To test the viability of the cleavage reaction, compound 65 was treated with benzyl alcohol and NIS/TESOTf in wet dichloromethane to afford the
R=Bn
b)
OPent
+
BnO
ii
70
BnO
BnO
65
BnO
66
BnO
71
BnO
67
Scheme 11. A solid-phase version of NPG. (i) THF; (ii) pyridine, CH2C12; (iii) BnOH, NIS, TESOTf, wet CH2C12, 2 h; (iv) NaOMe/MeOH/THF; Br2, Et4nBr, CH2C12; (v) NIS, TESOTf, CHzC12, 2 h; (vi) 0.1 M SmI2, THF, 30 mix; (vii) thiourea, methoxyethanol, 8 0 T , 6 h.
68 R=Ac
a)
Bn?% BnO
63
OH
u
P
150
6 Glycosylation Methods: Use of n-Pentenyl Glycosides
benzyl glycoside 66 in 60% yield and, presumably, the bound iodomethyltetrahydrofuran 67. For a more elaborate test case, the sidetracked acceptor 69 was prepared and coupling with 68a proceeded smoothly to give a dibromide. Zinc could not be used to restore the double bond, because of the solid-solid interaction that would be involved. A one-electron reductive elimination with samarium I1 iodide [24] proceeded smoothly, however, to give 70. Unfortunately, efforts to cleave the disaccharide from the solid support to produce 71 were unsuccessful. FTIR data suggested that addition to the double bond overwhelmed the cyclization reaction. The above results meant that the oxidative cleavage worked sometimes (65-+ 66), but not always (70 -+ 71). The use of sidetracked acceptors such as 69 might, therefore, be unreliable. Nevertheless, use of NPG as donors was still an option and so the oxidative cleavage aspect was replaced by a photocleavable alternative (Scheme 12). Thus, the Rich-Guwara linker 72A [40]was investigated. Success was had with a linear trimannan, and so a branched trimannan was tested, because this would be more demanding in that chemoselectivity deprotection would be required. This study is reported in Scheme 12. The Rich-Guwara photolinker was successfully coupled with diester 73, and chemoselective deacylation of the bound monosaccharide, 74a +74b, was found to proceed smoothly. Although coupling with donor 68b gave 15a in only moderate yields, ranging from 58-76'3'0, subsequent deprotection of the C6 acetate and iterative coupling with donor 68b proceeded without incident to give the resin-bound trisaccharide 76. Irradiation of the A-series trisaccharide required three days for recovery of optimum (10%) amounts of trisaccharide 77. On the assumption that this low yield was because of problems with the photocleavage reaction, a new linker, 72B, was designed. Advantages over 72A were (a) that linkage to the sugar was through a secondary, rather than primary, hydroxyl, and (b) that the electron-withdrawing amide carbonyl was remote from, rather than attached to, the benzene ring, where its deactivating effect would be irrelevant to the photocleavage event [41]. Indeed with 76B, the cleavage was very much faster and the overall yield of 77 was very much better [41]. An effort has also been made to synthesize polymer bound, fully deprotected saccharides by the couple-deprotect-couple iterative strategy [42]. A crucial problem with this approach was, however, that normally used benzyl groups, as in Scheme 12, would be problematic, because hydrogenolytic cleavage would involve a solid-solid interaction. Dissolving metal options were examined, but were also problematic in that the resin backbone apparently acted as an electron sink. Ester protecting groups were the only other option, and so concerns about their deactivating affect on the NPG donors were dispelled by success with preliminary experiments. The plan in Scheme 13 was therefore tested. The known NPG 78 [43] was converted into the differentially protected counterpart 79, and coupling with the polystyrene resin, A, led to 80a which reacted with 81 to give disaccharide 82a. Another decouple-couple iteration led to trisaccharide 83. Global deprotection by treatment with ethylenediamine in methoxyethanol at 70 "C for 4 h gave the fully deprotected bound trisaccharide 84.
6.7 Solid-Phase Iterative Couple-Deprotect-Couple Strategy
wI . -
P OD
\ .-
'-
I
+
Q
t
15 1
152
6 Glycosylution Methods: Use of n-Pentenyl Glycosides
..-
.-.-
;.i d
4 >
P
d 0 \
?
References
153
The sequence could be repeated with the tentage1 macrobeads, B, and the grafted crown linkers, C. The last was particularly valuable in that acetylation followed by photocleavage afforded 85, the identity of which was verified by independent synthesis [42].
References 1. For a concise review see: A. J. Bridges in Chem Tracts-Organic Chemistry, 1996, 9, 215-223. 2. B. Fraser-Reid, U. E. Udodong, Z. Wu, H. Ottosson, R. Merritt, C. S. Rao, C. Roberts and R. Madsen. Synlett, 1992, 927-942. 3. D. R. Mootoo, P. Konradsson, U. Udodong and B. Fraser-Reid. J. Am. Chem. SOC.,1988, 110, 5583-5584. 4. G. J. Boons, Tetrahedron, 1996, 52, 1095-1121. 5. M. L. Sinnott. Advances in Physical Organic Chemistry. 1988, 24, 113-204. 6. P. Deslongchamps. Stereoelectronic Efsects in Organic Chemistry. Pergamon, Oxford. 1983. 7. A. J. Ratcliffe, D. R. Mootoo, C. W. Andrews and B. Fraser-Reid. J. Am. Chem. Soc., 1989, I l l , 7661-7662. 8. C. W. Andrews, J. P. Bowen and B. Fraser-Reid. J. Chem. SOC. Chem. Comm., 1989, 19131916. 9. B. Fraser-Reid, Z. Wu, C. W. Andrews, E. Skowronski and J. P. Bowen. J. Am. Chem. Soc., 1991, 113, 1434-1435. 10. B. G. Wilson and B. Fraser-Reid. J. Org. Chem, 1995, 60, 317-320. 11. H . L. Frush and H. S. Isbell, J. Res. Nutl. Bur. Stds, 1941, 27, 413. 12. S. Oscarson and U. Tedebark. J. Carbohydr. Chem., 1996, 15, 507-512. 13. G. J. Boons, Contemp. Org. Syn., 1996, 3, 173-200. 14. J. F. King and A. D. Allbutt. Can. J. Chem., 1970, 48, 175441767, 15. R. U. Lemieux and N. J. Chu. Abstracts of Papers. Am. Chem. SOC., 1958,133, 31N. 16. A. F. Bochkov and G. E. Zaikov. Chemistry of the 0-Glycosidic Bond, Chapter 2, Pergamon Press, Oxford, 1979. 17. For some recent examples see: W. Wang and F. Kong. J. Org. Chem. 1998, 63, 5744-5745. L. Snaidman, C. Johnson, C. Crasto and S. M. Hecht. J. Org. Chem., 1995,560,3942-3943. 18. S. Hanessian and J. Banoub. Carbohydr. Res. 1977, 53 C13-Cl6. 19. A. S. Campbell and B. Fraser-Reid. J. Am. Chem. SOC.,1995,117, 10387-10388. 20. S. Hannesian and P. Lavallee, Can. J. Chem. 1975, 53,2975-2978 21. C. Roberts, C. L. May, and B. Fraser-Reid. Carbohydr. Lett., 1994, 1, 89-93. 22. C. Roberts, R. Madsen and B. Fraser-Reid. J. Am. Chem. Soc., 1995,117, 1546-1553. 23. C. A. A. van Boeckel and T. Beetz. Tetrahedron Lett. 1983,24, 3775. 24. J. R. Merritt, J. S. Debenham and B. Fraser-Reid. J. Carbohydr. Chem., 1996, 15, 65-72. 25. M. Meldal and K. J. Jensen. J. Chem. SOC.,Chem. Commun., 1990,483-484. U. K. Saha and R. R. Schmidt. J. Chem. Soc., Perkin I, 1997, 1855-1858. 26. J. G. Allen and Bert Fraser-Reid. J. Am. Chem. Soc., 1999, 121, 468-469. 27. Z. Wu, D. R. Mootoo and B. Fraser-Reid. Tetrahedron Lett., 1988, 29, 6549. 28. R. Madsen and B. Fraser-Reid. J. Chem. SOC.,Chem. Commun., 1994, 749-750. R. Madsen and B. Fraser-Reid. J. Org. Chem., 1995, 60, 772-779. 29. J. C. Lopez and B. Fraser-Reid. J. Chem. SOC.Chem. Comm., 1991, 159-161. 30. R. Lemieux, T. Takeda, B. Chung. Synthetic Methods for Carbohydrates (Ed. H. S. El Khadem, ACS Symposium Series, Washington, D. C., 1976, vol. 39, pp. 90-115. 31. J. S. Debenham, R. Madsen, C. Roberts, B. Fraser-Reid. J. Am. Chem. Soc., 1995,117, 33023303. 32. J. C. Castro-Palomino, R. R. Schmidt, Tetrahedron Lett. 1995, 36, 5343-5346. H. Shimizu, Y . Ito, Y. Matsuzaki, H. Iijima, and T. Ogawa. Biosci. Biotech. Biochem, 1996, 60, 73-76. 33. C. R. H. Raetz. J. Bacteriology, 1993, 175, 5745-5753. 34. S. L. Griffiths, R. Madsen, and B. Fraser-Reid. J. Org. Chem., 1997, 62, 365443658,
154
6 Glycosylation Methods: Use of n-Pentenyl Glycosides
35. P. T. Garegg. Pure Appl. Chem., 1984, 56, 845-858. 36. J. S. Debenham, S. D. Debenham, and B. Fraser-Reid. Bioorg. Med. Chem., 1996, 4, 19091918. 37. J. R. Merritt, E. Naisang and B. Fraser-Reid. J. Org. Chem., 1994,59, 4443-4449. 38. M. Bernard and W. T . Ford. J. Org. Chem., 1983, 48, 326-332. 39. J. M. Frechet and C. Schuerch. J. Am. Chem. Soc., 1972, 94,604-609. 40. D. H. Rich and S. K. Guwara. Tetrahedron Left., 1975, 16, 301-304. 41. R. Rodebaugh, B. Fraser-Reid and H. M. Geysen. Tetrahedron Lett., 1997,38,7653-7656. 42. R. Rodebaugh, S. Joshi, B. Fraser-Reid and H. M. Geysen. J. Org. Chem., 1997, 62, 56605661. 43. J. S. Debenham, R. Rodebaugh and Bert Fraser-Reid. Liebigs AnnJRecueil, 1997, 796-802.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
7 Glycosylidene Diazirines Andrea Vasella, Bruno Bernet, Martin Weber, and Wolfgang Wenger
7.1 Introduction Glycosylidene diazirines are useful as precursors of glycosylidene carbenes [I]. These cyclic, amphiphilic singlet alkoxycarbenes enable the formation of two bonds at the anomeric center. Although many aspects of the chemistry of pyranosylidene diazirines have been reviewed [2-51, furanosylidene diazirines [ 61 are hardly known because of their high reactivity and concomitant poor stability. Furanosylidene carbenes or carbenoids have been generated from the readily available lactone sulfonyl hydrazones [7].Whereas diazirines generate carbenes either thermally or photochemically under mild conditions, and simultaneously only produce dinitrogen, sulfonyl hydrazones, however, also generate potentially reactive side-products [7, 81. Glycosylidene carbenes, like other intermediates, are reactive and versatile. Their precursors-glycosylidene diazirines (= 1-azi-l,5-anhydroglycitols)-are thus expected to be useful. They have been devised primarily to serve as universal, highly reactive glycosylating reagents responding to the kinetic acidity of glycosyl acceptors, irrespective of their nucleophilic properties and of steric hindrance, and requiring no promoter. Their advantages will be discussed, as will their two disadvantages, uiz. their restricted stability, and their preparation which requires five (generally high-yielding) steps from hemiacetals.
7.2 Synthesis of Glycosylidene Diazirines The original route, depicted in Scheme 1, for the synthesis of the benzylated glucosylidene diazirine 7 [6, 91, is still used with minor variations, as evidenced by the preparation of the GlcNAc-derived analog 14 [3]. Details have been published, and some comments will prove useful.
156
7 Glycosylidene Diuzirines
BnO% BnO
i_ 1
B BnOn
B BnO n O
6 OBn N O
B BnO .
H
0
6 NHOH
BnO OH
O
h
2
%,
y
v
BnO BnO
BnO
Brio
7
iv
BnO& BnO BnO
NH
6
vi
BnO% BnO AcHN
NH
B BnO n 0
6
iii
N
O
H
AcHN
OH
8
/OR
E45 RR==MHs
B BnOn
~
N
O
6NHOH
AcHN
10
9
vii
c xF*n:niE
BnO% BnO y
ix
AcHN
AcHN
14
13
NH
&: ;B AcHN
/OR N
Scheme 1. Reagents: (i) Na, 96% EtOH, then NH3OH C1 and 1 , 7 h at 60°C, quant. (variable ratio of 2/3, ( E / Z )cu 65:35); (ii) NaOAc, NaI04, EtOH/H20, 19 h at 70°C, 79% from 1; (iii) pyridine, MsCl, CH2C12, 12 h at 0 ---t room temp., 87%; (iv) sat. NH3 in MeOH, CH2C12, 5 days at room temp., 76%; (v) MqN, 12, CH2C12, 2-5 h at -5O"C, 83%; (vi) Na, MeOH, then NH30HCl and 8, 33 h at 60"C, quant. (variable ratio of 9/10); (vii) NaOAc, NaI04, EtOH/H20, 18 h at 50°C, 70% from 8; (viii) Et3N, TsCl, CHzC12, 3 h at O T , 100%; (ix) sat. NH3 in MeOH, MeOH, 7 days at -2O"C, ca 70%; (x) Et3N, 12, CH2C12/MeOH, 3 h at O T , 68% from 12.
Formation of the oximes-generally obtained as ( E / Z )isomers and, most clearly [ 10, 111 (see also Ref. [ 121) and 2-acetamidofor 2,3,4,6-tetra-0-benzyl-~-glucose 3,4,6-tri-O-benzyl-2-deoxy-~-glucose oximes [3], as a mixture with the cyclic p-Dpyranosyl hydroxylamines (3 and 10, respectively)-proceeds readily at a pH of ca 6. Yields are high. The best procedure for the oxidation to the hydroximolactones depends on the structure of the oximes. Several oxidants have been tested. MnOz is generally reliable when prepared according to Attenburrow [ 131; commercial Mn02 has led to low yields [ 141.Oxidation is slightly exothermic. Fresh batches of MnOz might have to be added during the oxidation of large batches. Starting material and product have very similar RF values, but stain differently (H2S04, mostain) on the TLC
7.2 Synthesis of Glycosylidene Diazirines
157
plate. If the structure of the starting material enables formation of 1,4- and 1,5hydroximolactones, both might be formed [ 141. The relative amounts occasionally depend upon the oxidant (e.g. Hg(OAc)2 us 02/CuC12 in pyridine). NCS in the presence of DBU at low temperatures (ca -40°C) is a faster oxidant than MnO2. Low temperature is required for the oxidation of 0-acetyl oximes to avoid migration of the acetyl group from OC(4) to OC(5) [15]. Occasionally, NaI04/NaOAc has proven useful. 0-Sulfonylation of the hydroximolactones is unproblematic. Sulfonyl chlorides or anhydrides in the presence of base react rapidly at low temperature. The products often crystallize readily. A range of sulfonates has been tested [3]. Mesylates are often well suited; triflates are sometimes too unstable, although Beckmann rearrangement does not readily occur [ 141. Formation of the diaziridine requires a protic solvent; MeOH has been used as a standard. The concentration of NH3 required for the transformation depends on the nature of the sulfonyl group and on the acceptor properties of the 0-protecting groups. For the transformation of benzyl-protected sulfonates, MeOH must be saturated with NH3 at 0 "C in a thick-walled flask (the volume increases by ca 30% and the solution contains ca 24.5 g NH3 in 100 mL MeOH; it is stable for months at -20 "C); the reaction is performed at 22-26 "C, and the pressure is maintained by closing the flask with a septum; alternatively an autoclave may be used. The 2,3,4,60-pivaloylated glucose-derived hydroximomesylate reacts much faster [3]. The 2,3,4,6-0-acetylated glucose-derived hydroximotriflate reacted with half-saturated NH3/MeOH/CH*C12 at -20 "C without deacetylation [ 151. The diaziridines are ninhydrin-positive, stain dark blue with mostain, and oxidize KI to 12 in the presence of acid. They often crystallize from EtzO, and their purification by crystallization leads to improved overall yields. They are acid-labile; chromatography on silica gel must be rapid. It has led to slight losses even in the presence of base (Et3N); acetone is often a suitable (co)solvent. Crystalline diaziridines can be kept for months at -2O"C, and their solutions are stable in the absence of acid and oxygen. At 25°C and in the air, the diazirines turn red, presumably by forming increasing amounts of tetrazines. The diaziridines are usually obtained as mixtures of the two trans diastereoisomers, readily equilibrating in the presence of traces of acids. The ratio of the isomers depends on the glycosylidene residues. Oxidation of the diaziridines to diazirines requires some skill; anhydrous conditions are mandatory. Oxidation with I2 in the presence of a tertiary amine is the standard procedure. It was originally performed in MeOH and in the presence of 1-azi-glucose 7 crysEt3N. Under these conditions, the 2,3,4,6-tetra-O-benzylated tallizes from solution and can be isolated by filtration under anhydrous conditions [9]. For some diazirines, however, it is advantageous to perform the oxidation in CH2C12, using I2 and Me3N. The reaction corresponds to a titration, it is stopped when the solution remains slightly yellow. Because excess 12 is not readily removed, it is best to add a small amount of the starting diaziridine to discharge the color of the solution. The product is purified by filtration through a short Si02 column and thoroughly dried under high vacuum; a low boiling solvent (CH2C12) is recommended. On TLC, the diazirines stain purple with a solution of 4-(4-nitro-
158
7 Glycosylidene Diazirines
benzy1)pyridine; this is a specific and useful way of detection. Acyl-protected diazirines are more stable than their benzylated analogs and can be purified by flash chromatography at 0°C; solvents should be low boiling. The fractions are cooled, and condensation of water must be avoided [3, 151. The diazirines remain in the crystalline form or in solution for several weeks at -78 "C; 0-acyl protected diazirines can be stored at -20 "C.
7.3 Stability of the Glycosylidene Diazirines The stability of l-azi-l,5-anhydrohexitols depends on the substituents (protecting groups), the configuration, and the conformational flexibility. It has been quantified by thermolysis of the glycosylidene diazirines in methanol at 298 K [3, 15, 161; the results are given in Table 1. Their relative stability has been rationalized by postulating sequential (non-synchronous) heterolytic breakage of the C (1),N bond, leading to a cationic intermediate and requiring a conformational change of the pyranose ring whereby the pseudoequatorial C-N bond becomes pseudoaxial. Hence, acceptor substituents should increase the stability, and so should conformational locking, as by an intramolecular NH. . .OC(1) hydrogen-bond in the N-acetylallose derivative 25.
7.4 Glycosidation by Glycosylidene Diazirines 7.4.1 General Aspects Deprotonation of the glycosyl acceptor by a glycosylidene carbene ought to lead to a highly reactive, initially unsolvated ion pair, where both donor and acceptor are activated and should react readily with each other. Stereoelectronic effects dictate that deprotonation of the acceptor occurs in the a-plane of the carbene (more or less equivalent to the plane of the pyranoside ring), whereas addition of the oxyanion to the oxycarbenium cation has to occur in the mplane, i.e. more or less perpendicular to the a-plane, and preferentially from the axial direction (illustrated in Scheme 2 by the carbene derived from the benzylated glum-diazirine 7). Glycosidation by diazirines has been studied by using equivalent amounts of glycosyl acceptor and donor. It is expected that yields of the products increase with excess diazirine; occasionally this was shown to be so. Solvents have not systematically been optimized, but toluene, THF, and dioxane seem well suited; early experiments were mostly performed in dichloromethane. The course of the glycosidation of hydroxy compounds by glycosylidene diazirines is mainly dictated by four factors:
159
7.4 Glycosidution by Glycosylidene Diazirines Table 1. Kinetic parameters for the thermolysis of 1-aziglycosesin MeOH.
Diazirine
Compound
logA
15 R = Piv 16 R = AC 17 R = 4-F-Bn
23.0 25.0 21.6 23.7
13.4 14.1 11.6 13.8
33 202 181 43
22.4 24.0 21.0 23.1
1.7 4.8 -7.5 2.7
18
22.2
12.4
110
21.7
-3.2
19 R = B n 20 R = A c
23.2 20.5
14.2 11.4
I 45
22.6 19.9
5.5 -8.2
20.0
11.4
23
19.5
-8.2
-3.1
7 R = Bn
N
MeophT%r
BnO
AH= AS= (298 K) (298 K) [kcal [cal mol-' mol-'1 K-'1
E, [kcal mol-'1
z
(298 K) [min]
N
r *FCR
N
*::kS+yr
ND"
ND"
23
ND"
ND"
24
ND"
ND"
l4
22.6
12.6
112
22.0
25
28.1
15.1
4159
27.2
26 R = B n
20.8 22.3
11.4 12.1
88 202
20.2 21.7
BnO N OPiv PivO
Bn$Ss-,,
N
AcHN N
8.1
N
//"
RO OR
Ro
N
27 R = A c
-8.4 -5.0
160
7 Glycosylidene Diuzirines
Table 1 (continued) Diazirine
Compound
B n O - ' BnO f $ O a , N OBn BnO BnO
logA
21.8
12.2
T
(298 K) [min]
79
BnO BnO
OH
AS= (298 K) [cal mol-I K-'1
21.2
-4.8
- 100°C.
&:]
BnO% BnO
AH= (298 K) [kcal mol-'1
28
"Not determined. bVery unstable; slow decomposition even at
BnO
E, [kcal mol-l]
-
BnO
p-D
1
11 (OBn
(OBn
7
I
1
/OBn
I
OBn
BnO BnO
30
BnO
Scheme 2. Glycosidation of alcohols by gl ycosylidene carbenes derived from diazirines.
7.4 Glycosidution by Glycosylidene Diazirines
161
1) The kinetic acidity of the hydroxy group (inversely proportional to its basicity and more or less proportional to its nucleophilic properties). A sufficiently high kinetic acidity ensures rapid protonation of the carbene. Even an oxyanion derived from a poorly nucleophilic alcohol reacts readily with the simultaneously generated oxycarbenium cation. This property constitutes a major advantage of glycosylidene diazirines in the glycosidation of acids [ 171, phenols [9], highly fluorinated alcohols [ 181, hydroperoxides 1191, and hydrated Ti02 [ 151. Slow protonation of the carbene by a hydroxy compound of low kinetic acidity will lead to increasing amounts of azines (30 in Scheme 2), resulting from the interaction of the carbene with the starting diazirine. The azines are formed as a mixture of diastereoisomersthat are converted, by mild acid catalysis, mostly to the ( Z / Z )isomer [3]. 2) Hydrogen-bonding (a) Hydrogen-bonding influences the kinetic acidity of hydroxy groups. H-bond acceptors (that are not simultaneously hydrogen-bond donors) have enhanced kinetic acidity, whereas H-bond donors have reduced kinetic acidity; intramolecular hydrogen-bonding might thus determine the regioselectivity of deprotonation by the glycosylidene carbene-one of the conditions to be fulfilled for regioselective glycosidation. This effect has led to the regioselective glycosidation of dihydroxybenzene derivatives, where one of the phenolic OH groups functions as an intramolecular H-bond donor to a carbonyl group and is thereby glycosylated more slowly (Figure la) [9]. (b) Intra- or intermolecular hydrogen-bonding can also influence or determine the regio- and stereoselectivity of the ion pair combination resulting from deprotonation of the glycosyl acceptor. Whereas the kinetically most acidic OH group is deprotonated in the o-plane, a second OH group, hydrogen-bonded to the deprotonated OH group (and now linked by a H-bond to the newly generated oxyanionic center, Scheme 2) might be located in or close to the n-plane and attack the intermediate oxycarbenium ion. This effect has been demonstrated for a range of diols [20-251 and triols [26-291. It has also been evidenced for N-acetylglucosamine-deriveddiazirines, for which hydrogen-bonding to the NH group lead to the predominant formation of a-D-glycosides of sufficiently strong hydrogen-bond-accepting alcohols [301. 3) Solvation of the intermediate oxycarbenium ion. Solvation of the initially unsolvated or at least not optimally solvated oxycarbenium ion might involve the solvent, the glycosyl acceptor, or the C(2) substituent of the carbenium ion [18]. In an inert solvent, such as dichloromethane, solvation will occur by the (nucleophilic) glycosyl acceptor (inter- or intramolecularly) if it is sufficiently nucleophilic, or by the C(2)O-benzyl group for the less nucleophilic glycosyl acceptors such as phenols (as evidenced by studying the 2-deoxy analog 24 [31]). THF seems to solvate the carbenium ion well, and preferentially from the axial direction, leading preponderantly to equatorial glycosides. 4) In contradistinction to other methods of glycosidation, steric hindrance does not seem to be an important factor, as long as the hydroxy group is sufficiently acidic [ 18, 291. The following transformations illustrate these points.
162
A
7 Glycosylidene Diazirines
a)
b)
C)
F
3
C
Y
OGlcBn.,
/ OGlcBn, toluene: 79%, 8915
,,11..'
'...,,,
OGlcBn( toluene: 79%. 8416
CHzCI2: 34% (hv: 55%), 5050
dioxane, 25 "C: 76%. 95: 5 DME,25 "C 80%. 98: 2 EtCN, 25 "C: 5446,9010(16% of 31) EtCN, hv, -60 "C: 25%. 8713 (55% of 31)
31
e) CF3(CF2)5CH2CH~-OGlcBn, dioxane, 25 "C: 68%. 76:24
CHF~(CFZ)&H~~GICBQ toluene: 73% 6733
dioxane: 90%. 1:l mixture of p-D-isomers only
dioxane, 25 "C: 79%,78:22
CICH2CH2CI: 62%. 65:35 (4% of regioisorner, 4555)
Figure 1. Products of the reactions of the diazirine 7 with mono- and dihydroxy compounds: solvents, total yields, and p-D/a-D ratios (GlcBm = 2,3,4,6-tetra-0-benzyl-~-glucopyranosyl).
7.4.2 Glycosidation of Strongly Acidic Hydroxy Compounds Glycosidation of Phenols Glycosidation by the 1-azi-tetra-0-benzylglucopyranose7 proceeds in yields of approximately 75-80%, and leads mostly to the P-D-anomers [9]; presumably, the use of THF will increase the anomeric selectivity (Figure la, b). The analogous I -azimannose 19 yielded mostly a-D-mannosides, but has not been thoroughly studied [9].
7.4 Glycosidation by Glycosylidene Diazirines
163
The influence of intramolecular hydrogen-bonding is evident from the regioselective monoglycosidation of methyl orsellinate (Figure 1a) [9]. Upon addition of a second equivalent of diazirine, the chelated OH group is also glycosylated (67%,),although with less anomeric selectivity, as also observed for 4-nitrophenol. The insensitivity to steric hindrance is seen from the result of glycosylating 2,6-di(tert-butyl)-4-methylphenol(Figure 1b) [9]. Thiophenols react similarly to phenols; 4-methoxythiophenol yielding 70% of a D of thioglucosides [32]. 43 : 57 C X - D / ~ - mixture Glycosidation of Fluorinated Alcohols The influence of pK and the insensitivity to steric hindrance is seen from the glycosylation of fluorinated alcohols (Figure lc) [ 181. Glycosidation of trifluoroethanol, hexafluoro-2-propanol, and hexafluoro-tert-butanol by one equivalent of the diazirine 7 in dioxane yielded the anomeric glycosides in yields of 70-74% and in ~-D/.-D ratios ranging from 77:32 to 95:5. The solvent has an influence on the anomeric ratio, which changes for hexafluoro-tert-butanol from ~ - D / C X -= D 84 : 16 (75%) in dichloromethane to 98:2 (80%) in DME. The formation, upon glycosidation in propionitrile at -60°C, of the imino ether 31 (55%) besides the glycosides evidences the formation of an oxycarbenium cation, its solvation, and the preferred axial attack of the solvent. 3,3,4,4,5,5,6,6,7,7,8,8,-tridecafluorooctanoland of Glycosidation of CHF2(CF2)&H*OH by 7 in dioxane proceeded similarly and yielded 68 and 79% of the glucosides (P-D~CX-D = 76: 24 and 78 :22).
7.4.3 Glycosylation of Weakly Acidic Hydroxy Compounds Glycosidation of Monovalent Alcohols In contradistinction to the glycosylation of hexafluoro-tert-butanol, tert-butanol is glycosylated in only 34% yield under similar conditions (Figure 1d). Glycosylation at low temperature (photolytic generation of the carbene) doubled the yield, but had no influence on diastereoselectivity (IX-D/~-Dca 1 : 1). This dependence on the mode of generating the carbene is not general, and might reflect the temperaturedependence of the association of tert-butanol. Monoalcohols with o-acceptor substituents (such as partially protected monosaccharides) are expected to be more highly acidic and, hence, be glycosylated in higher yields. Indeed, 1,2: 5,6-di-0isopropylidene-a-D-glucofuranose yielded 75% of a 2 : 3 mixture of the corresponding anomeric disaccharides (Figure 1e) [ 181. The influence of acidity is also seen in the successful glycosidation of (hydrated) Ti02 [15]. The OH groups of hydrated Ti02 are characterized by pK values of 3.8 and 6.6. Exposure of Ti02 to benzylated or acetylated azi-glucoses 7 and 16 led to formation of a monomolecular layer of strongly (presumably covalently) bonded glucosyl residues (density 1.5 0.9 glucosyl moieties nm-2). The acetylated glucosyl moieties were deacetylated without removing the glucosyl moiety.
164
7 Glycosylidene Diazirines
Glycosidation of Diols and Triols Efsects of hydrogen-bonding The enhancing influence of an (intramolecular) hydrogen-bond on the kinetic acidity of the H-bond-accepting hydroxy group is seen from the (concentration independent!) result of the glycosidation of the myo-inositol-derived orthoester (Figure I f ) [26]. The two diastereoisomers derived from monoglycosylation of one each of the two enantiotopic hydrogen-bonded OH groups were obtained regio- and diastereoselectively in high yields. The low reactivity of the isolated equatorial OH group is because of a (bifurcated) hydrogen-bond. This effect also operates in the regioselective glycosidation at OC(2) of methyl 4,6-O-benzylidene-a-~-altropyranoside, yielding 62% of a 2: 1 mixture of anomers in dichloroethane (Figure lg) [20]. As expected, the intramolecular hydrogen-bond reduces the kinetic acidity of HOC(3), but enhances its nucleophilicity and leads to glycosylation at OC(3) bromide under Lemieux halogenwith 2,3,4,6-tetra-O-benzyl-a-~-glucopyranosyl exchange conditions, resulting in 68% of the a-D-configured, 1,3-1inked disaccharide (and 10% of the anomeric regioisomers). Combined efsects of intramolecular hydrogen-bonding and of stereoelectronic control The intramolecular hydrogen-bond between HOC(3) and the anomeric methoxy group in methyl 4,6-0-benzylidene-a-~-allopyranoside is expected to lead to protonation of the carbene mostly by the kinetically more highly acidic HOC(2) (Figure 2a) [22]. This is presumably taking place. In contradistinction to the altro-diol, however, the two OH groups are cis-oriented. Stereoelectroniccontrol requires that protonation occurs in the o-plane of the carbene and attack of the nucleophile on the ensuing oxycarbenium cation proceeds in the n-plane. HOC(3) is much closer to the x-plane than -0C(2). Glycosylation in toluene at 70 "C or in dioxane at 24 "C indeed leads mostly to the a-D-anomer of the 1,3-linked disaccharide [22]. The predominant a-D-configuration reflects the stereoelectronic control. Replacing these solvents by the more nucleophilic THF at -85°C leads to participation of the solvent. THF competes with
toluene: 79%.
C(2)0/C(3)0 2080 (68:32and 28:72)
CH2C12,-85", hv: 81%, C(2)0/C(3)O/C4)05:81:14(60:40,23:77, and 3664)
THF, -85", hv: 79%, C(2)0/C(3)0 7228 (9010and 3367)
Figure 2. Glucosylation of (a) methyl 4,6-O-benzylidene-a-~-allopyranoside and (b) benzyl ribopyranoside by the diazirine 7:solvents, total yields. regioselectivity, and p-D/a-Dratios.
p-D-
7.4 Glycosidution by Glycosylidene Diuzirines
165
Scheme 3. Preferred formation of the 3-O-P-~-glucosylateddimer in the reaction of 7 with benzyl (3-D-ribopyranoside.
HOC(3) in attacking the intermediate oxycarbenium ion, again from the axial di~ of the 1,2-1inked disaccharide! rection, and the main product is now the p - anomer Similarly for benzyl P-D-ribopyranoside, preferential protonation of the carbene ought to occur by either HOC(2) or HOC(4), but HOC(3) is in a better position to attack the carbenium ion from an axial direction. The main product, isolated in 65% yield, is the a-Danomer of the 1,3-linked disaccharide (Figure 2b) [28]. The rationalization of that reaction is illustrated in Scheme 3. In THF at -78 "C, however, the main products are the p-D anomers of the 1,2- and 1,4-linked disaccharides. More examples of regioselective glycosidations resulting from the combination of these factors have been published and a particularly striking effect of hydrogenbonding is provided by the glycosidation of the N-phthaloylated allosamine derivatives. The dominant product of the a-D-configured diol is the 1,3-linked disaccharide (Scheme 4a) [23]. The anomeric configuration suggests that the regioselectivity is the result of a protonation by HOC(4) and an interception of the carbenium ion by HOC(3); indeed, ' H NMR analysis showed the presence of a very strong hydrogen-bond between HOC(3) and the phthaloyl group, and a weaker hydrogen-bond between HOC(4) and HOC(3). The former hydrogen-bond strongly reduces the kinetic acidity of HOC(3), the latter orients the protonating HOC(4) close to OC(3). In agreement with this, the a-D-configured monoalcohol is unreactive (Scheme 4b), whereas its anomer is glycosylated (Scheme 4c), because the N-phthaloyl group now adopts a different conformation; also the anomeric diol is no longer regioselectively glycosylated (Scheme 4d). The results of the glycosylation of the a-D-configured trio1 (Scheme 4e) evidences that the weakest hydrogen-bond is that from HOC(4) to HOC(3)! Intramolecular solvation of the intermediate oxycarbenium ion has also been postulated to explain the high-yielding glycosylation of the tertiary hydroxy group
7 Glycosylidene Diazirines
166 a)
80%, 90:lO
no reaction
C)
*Thp
OAll Bn4Glc0
NFt
66%,2 1 3 9
4
CHIOBn H
o ~ HO
O
A
l
l
B n 4 G l f : N OAll + OH BqGlcO
29%.a 6 5
33%,39:61
B n 4 G l c ? !
HO FNOAll
HO FtNOAll 9%,(only p-D)
OAll NFt
+
E N Bn4Glc0 FtN OAll 51%. 72:28
Scheme 4. Glucosylation of anomeric N-phthaloylatedallosamine derivatives by one equivalent of the diazirine 7 in dioxane at room temperature: total yields and a-D/p-D ratios.
of 10-0-acetylginkgolide A by a lactose-derived diazirine (Scheme 5a) [29]; intramolecular participation of the carbonyl group of the adjacent lactone ring is presumably playing an important role also in the glycosylation of ginkgolide B, where reaction with 3.5 equivalents of 7 in THF yielded 42% of the all-P-D-configured trisaccharide (Scheme 5b) [29]. Thus, regioselective glycosidation of diols and triols by diazirines is feasible and often strikingly successful. Its course can be predicted, even if the factors are complex, if the hydrogen-bonds in the glycosyl acceptor are known. Analytically, the results of glycosylation provide important clues to the location of hydrogen-bonds (Figure 3a), e.g. evaluation of the influence of weak intramolecular OH. . .F-C hydrogen-bonding in apolar solvents (Figure 3b) [24, 251. There is yet another factor, operative in carbenes derived from 1-azi-2-acetamido-2-deoxy sugars that can occasionally be exploited. Efect of intermolecular hydrogen-bondingfrom the diazirine to the acceptor Surprisingly, thennolysis of the N-acetylglucosamine- and N-acetylallosamine-derived diazirines 14 and 25 in methanol led predominantly to the 1,2-cis-configured
7.4 Glycosidation by Glycosylidene Diuzirines
Me
167
28 (1.3 equiv.)
HO 3
THF, 25°C
0 92% of B-D-anomer
b'
0
N
Me
-
Me
7 (3.5 equiv.) THF, 25°C
42%
R = GlcBn, p+l p+3 p-10
15% p+l p-3 a+10 13% a+l p+3 p-10 24% diglucosides no rnonoglucosides
Scheme 5. (a) Lactosylation of 10-0-acetylginkgolide A by 28. (b) Glucosylation of ginkgolide B by 7.
a-r>-glycosides; similarly, glycosidation of 2-propanol (2 equivalents) by 14 at -84 "C yielded 70% of the a-D-glycoside (no 0-D-anomer observed) and 13% of the dihydrooxazole [30]. At 26 "C, the dihydrooxazole (resulting from protonation of the carbene by the acetamido group) became the major product. Under analogous conditions (-84 "C), hexafluoro-2-propanol yielded between 58 and 9 1% glycosides, depending on the excess of alcohol (1 to 10 equivalents), the anomeric ratio varying between 72 :28 and 80 :20. Glycosidation at 26 "C of 2 equivalents of hexafluoro-2propanol led to higher yields of glycosides (72 compared with 59%) and higher anomeric selectivity (.-D/~-D = 88 : 12). Glycosidation of the still more highly acidic
Figure 3. Intramolecular hydrogen-bonding in (a) ally1 2-deoxy-2-phthalimido-a-~-allopyranoside and in (b) L-4-deoxy-4-fluoro-1,3,5-pentyIidyne-myo-inositol.
168
7 Glycosylidene Diazirines
Figure 4. Intermolecular hydrogen-bonding of the carbene derived from the GlcNAc diazirine 14.
4-nitrophenol (2 equiv., 26 "C) reduced the anomeric selectivity to 58 :42 (87% yield). These and analogous observations made by glycosylating the A11NAc-derived diazirine 25 were rationalized by postulating an intermolecular hydrogen-bond from the NHAc group to the glycosyl acceptor (Figure 4). This hydrogen-bond locates the alcohol below the plane of the pyranose ring, well positioned to attack the oxycarbenium and form a-D-glycosides. The hydrogen-bond depends on the conformation of the acetamido group and on the basic properties of the alcohol (higher selectivity with the less acidic, i. e. more strongly basic 2-propanol). The conformation of the acetamido group, in its turn, depends on the configuration (glum or d o ; for a detailed study of the conformational behavior of the NHAc group see Ref. [33]) and the temperature (higher c ~ - D / ~ - Dratio at a higher temperature). The temperature-dependent association of the alcohols explains the much higher yield of the glycosidation of 2-propanol at lower temperature, influenced by the higher basicity of 2-propanol than hexafluoro-2-propanol (better H-bond acceptor) and by the greater acidity of the hydrogen-bond-accepting terminal alcohol unit in a chain of associated alcohols properly located in the o-plane of the carbene (Figure 4). Protonation of the carbene, which depends on the kinetic acidity of the alcohol, is reflected by the higher yields of the hexafluoro-2-propanol- and 4-nitrophenol-derived glycosides. These results confirm the strong influence of hydrogen-bonds on the result of glycosidation and characterize these diazirines as subtle, reactivity-based tools for the investigation of intra- and intermolecular hydrogen-bonds and their influence on regio- and diastereoselective glycosidation.
7.5 Synthesis of Spirocyclopropanes Glycosylidene diazirines react with electrophilic alkenes, e.g. N-phenylmaleimide, acrylonitrile, dimethyl fumarate, dimethyl maleate, and C&-fullerene, to yield spirocyclopropanes with formation of two C-C bonds at the anomeric center [34-371.
7.5 Synthesis of Spirocyclopropanes
R = Bn: So%, 9O:lO R = Piv: 60%. 92: 8
RO
RO
.
,
"'v
169
RO
y0N , P h
Ph
0
22%
BBnO n BnO
70%
Bn ~O- n 0
BnO
52
BnO
BnO Me02C
BBnO n BnO NC
6
,,C02Me
fCo2r"72%
q ..*>CN
CN
Brio%,
C)
O
O OBn CN q BnO
11
31
Brio%. %
Brio BnO
Me BnO BnO
,,C02Me BnO BnO
BnO C02Me
,
C02Me
BnO Me02C
60
40
-
-
29
20
33
1s
le02C
C02Me
[
: 60%
C02Me
d'
z:*,
RRO O+]. RO
'%O
R=Bn:
33%
R=Piv:
72%
Me
24%
'$o
b 9%
14%
Figure 5. Products of the reaction of the benzylated and pivaolyated diazirines 7 and 15 with (a) N-phenylmaleimide, (b) acrylonitrile, (c) dimethyl fumarate and dimethyl maleate, and (d) dihyand 2,3-dihydro-4H-pyran. drofuran, 5-methyl-2,3-dihydrofuran,
Yields depend on the protecting groups; substitution of benzyl by pivaloyl groups reduces yields, in agreement with a reduction of the predominantly nucleophilic character of these ambiphilic carbenes. As illustrated by the cyclopropanation of N-phenylmaleimide, the diastereoisomer with the substituents oriented syn to the glycosyl ring oxygen are formed preferentially (ratio = 90: 10; Figure 5a); cyclopropanation with acrylonitrile confirms this (ratio = 58 :42) and shows that the nitrile function is predominantly located below the average plane of the pyranosyl ring (ratio = 83: 17; Figure 5b). This is confirmed by cyclopropanation of ethyl acrylate [38]; the major isomer, formed in ca 47% yield, has the same configuration as the major isomer resulting from addition to acrylonitrile. The stereoselectivity of cyclopropanation with dimethyl fumarate and dimethyl maleate (Figure 5c) suggests that formation of the two C-C bonds is not concerted; it is rationalized by
170
BnO
7 Glycosylidene Diuzirines
-
BnO BnO
EN pseudoaxial approach
pseudoequatorial approach
Scheme 6. Pseudoaxial and pseudoequatorial. ring closure of the zwitterion obtained upon reaction of acrylonitrile with the carbene derived from 7.
postulating a two-step process with a preferred pseudoaxial trajectory in the formation of the second C-C bond, as illustrated in Scheme 6. In agreement with the ambiphilic character of alkoxycarbenes, glycosylidene carbenes form cyclopropanes also with electron-rich alkoxyalkenes, provided their reactivity is enhanced by ring tension, such as that in dihydrofuran, that leads to 33% (benzylated diazirine 7) or 72% (pivaloylated diazirine 15) of a single isomer (Figure 5d) [39]. Steric hindrance by an additional methyl group reduces the yield. Dihydropyran reacts less readily, and open chain enol ethers lead to low yields only. The 0-benzyl protected diazirines gave lower yields than the 0-pivaolylated analogs. The transition states of the cycloadditions leading to the isomeric cyclopropanes have been calculated (semiempiric program AM1). At the beginning of the reaction, the interaction of the HOMO of the carbene with the LUMO of the enol ether dominates, but in the transition state the interaction between the LUMO of the carbene and the HOMO of the enol ether is more important. The cyclopropanation yields of the pivaloylated diazirine 15 are higher because of the more efficient competing reaction of the 0-benzylated (as compared to the 0-pivaloylated) carbene with the starting diazirine. Azines are isolated as the main by-products. Calculations show that a pseudoaxial attack of the carbene is favored.
7.6 Addition to Aldehydes and Ketones Addition of the benzylated aziglucose 7 to acetone or cyclohexanone, used as solvents, gave 63 and 78%, respectively, of a mixture of anomeric spiroepoxidesin P-D/ a-Dratios of 70 :30 and 65 : 35, respectively (Figure 6a), whereas addition of the A11NAc-derived diazirine 25 to cyclohexanone at 60°C gave 83% of the a-Dconfigured spiroepoxide and a dihydrooxazine formed either directly from the intermediate oxycarbenium cation or by opening the anomeric oxirane (Figure 6b) [40].Similarly, addition of 25 to a threefold excess of benzaldehyde gave a mixture of &,trans-related a-D-spiroepoxides (27%), epimeric dihydrooxazoles (1 2%; formally derived from the epimeric epoxides), and a range of by-products. Such spiroepoxides have been prepared in a convenient way by Nicotra and Panza [41-431.
7.7 Explorutory Use of Diuzirines
171
b)
a)
BnO
78%
83%
35
60
40
Me
Figure 6 . Products of the reactions of (a) the diazirine 7 and (b) the diazirine 25 with cyclohexanone: total yields and product ratios.
7.7 Exploratory Use of Diazirines: Formation of Glycosyl Phosphines, Stannanes, N-Sulfonylamines, Esters, Boranes, and Alanes, and of 1,l-Difluorides The high reactivity of glycosylidene carbenes enables rapid access to the first representatives of intermediates or products for which more convenient routes might subsequently be developed. Thus, the benzylated aziglucose 7 reacted with Ph2PH under thermal conditions to give anomeric glycosyl phosphines that were isolated as the corresponding phosphine oxides in yields of up to 76% (Figure 7a) [44]. The u - D / ~ - Dratios of anomers varied between 5 1 :49 and 67 :33. Generation of the carbene at -78 "C (hv) led in 55% yield to a 74:26 mixture of anomers. Glycosyl phosphine oxides were then prepared from glycosyl acetates by TMSOTf-catalyzed treatment with MeOPPh2 and subsequently reduced to glycosyl phosphines [45].
b)
a)
BnO BnO
PPhZ OBn
16%, 55:45
Bd-0-
SnBuj
SdU3
OBn
70%. 84:16
53%. 4357
C)
."in-&+,
BnO BnO &NH.,.s OBn
50%. 2575
BnO
45%
oyyco"n ri'
NH-Bw
Figure 7. Products of the reactions of (a) the diazirine 7 with diphenylphosphine, (b) the diazirines 7 and 19 with tributylstannane, and (c) the diazirine 7 with 4-toluenesulfonamide and N-Bocasparagine benzyl ester: yields and a-D/p-D ratios.
172
7-
7 Glycosylidene Diazirines
BR3
Scheme 7. Expected primary products of the reaction of diazirine 7 with trialkylboranes.
The benzylated aziglucose 7 and azimannose 19 react with tributyl or triphenyl stannane and yield glycosyl stannanes (Figure 7b) [46]. The 1,2-cis-configured anomers are formed preferentially and the 1-C-stannylated 2-0-benzyloxyglucal is the main by-product. Advantageous preparations of glycosyl stannanes-useful precursors of glycosyl lithium compounds-from glycosyl halides have been described by Kessler et al. [47-501. Exploratory experiments have shown that glycosylidene carbenes also insert into the N-H bond of sulfonamides, whereas carboxamides yield esters, as illustrated by the product obtained from an N-Boc-asparagine benzyl ester (Figure 7c) [32]. Presumably, such esters are formed by hydrolysis of the initially formed imino ether 0-glycosides, suggesting that glycosylidene diazirines may be used for (selective?) cleavage of peptidic bonds. Nucleophilic addition of glycosylidene carbenes to triligated boron compounds followed by migration of a boron substituent to the glycosidic center should lead to anomeric glycosyl boranes (Scheme 7).
Bnb,!,, BBu3:
""
Bnd
45%
20%
62%
-
C>
B-Bu:
1) BEt3.2) : ' H
B BnO n
O
h
E
t BnO
BnO HO' 'Et 1 ) BEt, 2) PPh3.02: 25%
BnO BnO
0
46%
WC'
4
C)
'Et
55%
46
:
BnO
54
Figure 8. Products of the reaction of diazirine 7 with (a) tributylborane and 2-butyl-l,3,2-dioxaborinane, (b) triethylborane followed by acid treatment, (c) triethylborane followed by oxidation, and (d) the 9-BBN-derived phenethylborinate.
7.7 Exploratory Use of Diazirines -0Bn
D*OnB R = Me: R = 'Bu:
BnO
,OBn
BnOR
30% 20%
173
R
30% 25%
Figure 9. Products of the reaction of diazirine 7 with trialkylalanes followed by deuterolysis.
Reaction of 7 with tributyl-, triethyl-, and triphenylborane, followed by oxidation, yielded the C (1)-alkylated hemiacetals and the C(1)-alkylated glycals (Figure 8a) [51, 521. The cyclic 2-butyl-l,3,2-dioxaborinane led only to the hemiacetal. The presence of the intermediate (anomeric?) diethyl glycosyl boranes was evidenced by treating them with trifluoroacetic acid, leading to migration of a second ethyl group to C(l) and formation of a cyclic stable borinate (Figure 8b). Oxidation of the intermediate glycosyl borane by oxygen in the presence of excess triphenylphosphine led to a stable ethylglycosylborinic acid (Figure 8c). We took advantage of the higher resistance to oxidation of borinates, compared with boranes, by treating the aziglucose 7 with the 9-BBN-derived phenethyl borinate (Figure 8d). The configurations of the anomeric borinates was assigned on the basis of NOE data. Trialkylalanes, similarly to trialkylboranes, react with the aziglucose 7 leading to bona jide glycosyl alanes, as was proved by deuteration to yield the axial C-glycoside and the C(1)-alkylated glucal (Figure 9) [52]. Finally, because the 1,l-difluoro-1-deoxy derivative of cellobiose was required for kinetic studies of cellobiohydrolases, we examined the reaction of the corresponding diazirine with XeF2. A method for the synthesis of monosaccharide-derived anomeric difluorides has been described by Praly [53,541, but seems unsuitable for the synthesis of disaccharide-derived analogs. Treatment of the cellobiose-derived diazirine 26 with XeF2 in dichloromethane yielded a maximum of 22% of the desired difluoride and large amounts of azines (Scheme 8) [ 161. In the presence of small amounts of MeOH, these azines were replaced by, mostly, the equatorial monofluoride. These results show that the reactivity of glycosylidene carbenes towards XeF2 is not pronounced, but sufficient to furnish the amounts of the desired difluoride required for the enzymatic tests [ 5 5 ] .
26
R=F R=H
22%
Scheme 8. Reaction of the cellobiose-derived diazirine with xenon difluoride.
174
7 Glycosylidene Diuzirines
Acknowledgments
We thank the Swiss National Science Foundation and F. Hoffmann-La Roche AG, Basel, for continuous generous support.
References 1. A. Vasella, K. Briner, N. Soundararajan, M. S . Platz, . I . Org. Chem., 1991, 56, 4741-4744. 2. A. Vasella, in Topics in Bioorganic and Biological Chemistry (Ed.: S. M. Hecht), Oxford University Press, Oxford, 1999, pp. 56-88. 3. A. Vasella, C. Witzig, C. Waldraff, P. Uhlmann, K. Briner, B. Bernet, L. Panza, R. Husi, Helv. Chim. Acta 1993, 76, 2847-2875. 4. A. Vasella, Pure Appl. Chem. 1993, 65, 731-752. 5. A. Vasella, Pure Appl. Chem. 1991, 63, 507-518. 6. K. Briner, A. Vasella, Helv. Chim. Acta 1989, 72, 1371-1382. 7. S. E. Mangholz, A. Vasella, Helv. Chim. Acta 1995, 78, 1020-1035. 8. S. E. Mangholz, A. Vasella, Helv. Chim. Acta 1991, 74, 2100-2111. 9. K. Briner, A. Vasella, Helv. Chim. Acta 1990, 73, 1764-1778. 10. B. Aebischer, Thesis, Universite de Fribourg No. 854, 1983. 11. D. Beer, Thesis, Universitat Zurich, 1989. 12. P. Finch, Z. Merchant, J. Chem. SOC.,Perkin Trans. I 1975, 1682-1690. 13. J. Attenburrow, A. F. B. Cameron, J. H. Chapman, R. M. Evans, B. A. Hams, A. B. A. Jansen, T. Walker, J. Chem. SOC.1952, 1094-1111. 14. D. Beer, A. Vasella, Helv. Chim. Acta 1985, 68, 225442274, 15. M. Weber, A. Vasella, M. Textor, N. D. Spencer, Helv. Chim. Acta 1998, 81, 1359-1372. 16. M. Weber, Thesis, ETH-Zurich No. 13212, 1999. 17. K. Briner, A. Vasella, unpublished results. 18. K. Briner, A. Vasella, Helv. Chim. Acta 1992, 75, 621-635. 19. K. Briner, Thesis, Universitat Zurich, 1995. 20. E. Bozo, A. Vasella, Helv. Chim. Acta 1992, 75, 2613-2633. 21. P. R. Muddasani, E. Bozo, B. Bernet, A. Vasella, Helv. Chim. Acta 1994, 77, 257-290. 22. P. R. Muddasani, B. Bernet, A. Vasella, Helv. Chim. Acta 1994, 77, 334-350. 23. K. Briner, B. Bernet, J. L. Maloisel, A. Vasella, Helv. Chim. Acta 1994, 77, 1969-1984. 24. A. Zapata, B. Bernet, A. Vasella, Helv. Chim. Acta 1996, 79, 1169-1191. 25. M. A. Biamonte, A. Vasella, Helv. Chim. Acta 1998, 81, 695-717. 26. P. Uhlmann, A. Vasella, Helv. Chim. Acta 1992, 75, 1979-1994. 27. E. Bozo, A. Vasella, Helv. Chim. Acta 1994, 77, 745-753. 28. P. Uhlmann, A. Vasella, Helv. Chim. Acta 1994, 77, 1175-1 192. 29. M. Weber, A. Vasella, Helv. Chim. Acta 1997, 80, 2352-2367. 30. A. Vasella, C. Witzig, Helv. Chim. Acta 1995, 78, 1971-1982. 31. Y. Takahashi, A. Vasella, Helv. Chim. Acta 1992, 75, 1563-1571. 32. T. Rajamannar, A. Vasella, unpublished results. 33. P. Fowler, B. Bernet, A. Vasella, Helv. Chim. Acta 1996, 79, 269-287. 34. A, Vasella, C. A. A. Waldraff, Helv. Chim. Acta 1991, 74, 585-593. 35. A. Vasella, P. Uhlmann, C. A. A. Waldraff, F. Diederich, C. Thilgen, Angew. Chem. 1992,104, 1383-1385. 36. P. Uhlmann, E. Harth, A. B. Naughton, A. Vasella, Helv. Chim. Acta 1994, 77, 2335-2340. 37. U. Jonas, F. Cardullo, P. Belik, F. Diederich, A. Gugel, E. Harth, A. Herrmann, L. Isaacs, K. Mullen, H. Ringsdorf, C. Thilgen, P. Uhlmann, A. Vasella, C. A. A. Waldraff, M . Walter, Chem. Eur. J. 1995, I , 243-251. 38. C. Bluchel, A. Vasella, unpublished results. 39. C. Waldraff, B. Bernet, A. Vasella, Helv. Chim. Acta 1997, 80, 1882-1900. 40. A. Vasella, P. Dhar, C. Witzig, Helv. Chim. Acta 1993, 76, 1767-1778.
References 41. 42. 43. 44. 45. 46. 41. 48. 49. 50. 51. 52. 53. 54. 55.
115
L. Lay, F. Nicotra, L. Panza, G. Russo, G. Sello, J. Curbohydr. Chem. 1998, 17, 1269-1281. L. G. Lay, F. Nicotra, L. Panza, G. Russo, Synlett 1995, 167-168. F. Nicotra, L. Panza, G. Russo, Tetrahedron Lett. 1991, 32,4035-4038. A. Vasella, G. Baudin, L. Panza, Heteroat. Chem. 1991, 2, 151-161. A. Lopusinski, B. Bernet, A. Linden, A. Vasella, Helv. Chim. Acta 1993, 76, 94-112. P. Uhlmann, D. Nanz, E. Bozo, A. Vasella, Helu. Chim. Acta 1994, 77, 1430-1440. F. Burkhart, M. Hoffmann, H. Kessler, Tetrahedron Lett. 1998, 39, 7699-7102. M. Hoffmann, H. Kessler, Tetrahedron Lett. 1997, 38, 1903-1906. M. Hoffmann, H. Kessler, Tetrahedron Lett. 1994, 35, 6067-6070. 0. Frey, M. Hoffmann, V. Wittmann, H. Kessler, P. Uhlmann, A. Vasella, Helu. Chim. Actu 1994, 77, 2060-2069. A. Vasella, W. Wenger, T. Rajamannar, Chem. CommuM. 1999, 2215-2216. W. Wenger, A. Vaseila, Helv. Chim.Acta, submitted. J. P. Praly, L. Brard, G. Descotes, L. Toupet, Tetrahedron 1989, 45, 4141-4152. J. P. Praly, G. Descotes, Tetrahedron Lett. 1987, 28, 1405-1408. D. Becker, K. S. H. Johnson, A. Koivula, M. Schiilein, M. L. Sinnott, Biochem. J. 2000, 345, 315-319.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
8 Glycosylation Methods: Alkylations of Reducing Sugars Jun-ichi Tamuru
8.1 Introduction Methods for glycosylation generally contain a procedure for activation of the anomeric position. Scheme 1 shows two possible procedures for formation of a glycosidic linkage. Conventionally, the specific affinity of a leaving group at the anomeric position and a suitable promoter accelerates a coupling reaction, as described in the previous chapter. As a result a glycosidic bond is formed by nucleophilic attack of aglyconic oxygen (b). We can also form the glycosidic linkage by nucleophilic attack of anomeric oxygen (a). This glycosylation procedure retains the anomeric oxygen (a) in the glycosidic bond. The glycosylation methods referred to in the title of this chapter are based on the latter concept. R. R. Schmidt and P. Kovac introduced the reverse concept to glycosylation methods and developed ‘anomeric O-alkylation’ and ‘glycosylation via locked anomeric configuration’, respectively. These methods, of which general procedures are shown in Scheme 2, use different approaches for stereocontrol of glycosidic bond formation. The ‘anomeric O-alkylation’ method utilizes an anomeric sodium or potassium alkoxide as a nucleophile. Stereoselectivity in the coupling reaction depends on the different reactivities of the anomeric c1 and anions. Factors controlling the reactivity, such as conformation of the nucleophile, are described in detail in Section 8.2. Review articles by Schmidt have described the ‘anomeric O-alkylation’ method [ 1-31. ‘Glycosylation via locked anomeric configuration’ employs a stannylene acetal at the 1,2-position of the hemiacetal. The stannylene acetal fixes the anomeric oxygen in the 1,2-cis configuration and leads to 1,2-ci.stype glycoside. Details are described in Section 8.3.
8.2 Anomeric O-Alkylation In addition to temperature, solvent, promoter, and additive, we must consider the configuration of the nucleophile (including roles of substituent groups) and the re-
178
8 Glycosylution Methods: Alkylations of Reducing Sugars
Electrophile (Donor)
Nncleophile (Acceptor)
L
_-___-__ . I
Scheme 1. Conventional and new glycosylation methods. L = leaving group; M = metal atom.
11
m* Electrophile
Nucleophile
I
"Anomeric0-alkylation"
Nucleophile
1
Glycoside
I
J
I "GlycosylationvM locked anomeric configuration" I Scheme 2. General procedures for 'anomeric 0-alkylation' and 'glycosylation via locked anomeric configuration'.
activity of the electrophile (primary compared with secondary triflates). This section describes the factors that lead to the stereoselective coupling reaction.
8.2.1 Anomeric 0-Alkylation of Ribofuranose with Primary Triflates: Effect of the Protecting Group at 0 - 5 of Ribofuranose Schmidt and Reichrath performed the anomeric 0-alkylation of ribofuranose with primary triflates with complete stereocontrol as shown in Scheme 3 [4]. Ribofur-
8.2 Anomeric 0-Alkylation
179
TBDMSCI 0 0
KOtBu ITHF
I
0'
.\c (2)
I (4) 89 %
(5) 86 %
I
.\c
\I*
Scheme 3. Stereoselective syntheses of Ribfu and p(1-5)Ribf and plausible complex formation. S = solvent molecule; TBDMS = t-butyldimethylsilyl.
anose 2,3-O-isopropylidene acetal (1) and the corresponding 5-0-TBDMS derivative (2) were selected as nucleophiles. Reaction of these nucleophiles with primary triflate (3) stereoselectively gave a- and P-linked disaccharides (4 and 5), respectively, in high yields. Promoter and solvent seem not to have a significant effect on the reaction, although the substituent group at 0 - 5 of ribofuranose might be important for stereocontrol. Plausible intermediates explaining this specific reaction are shown in Scheme 3, bearing in mind the steric influence of the protecting group. 8.2.2 Anomeric 0-Alkylation of Mannofuranose with Primary Triflates: The Crown Ether Effect
Schmidt et al. reported an effect of a crown ether on the glycosylation of mannofuranose. Scheme 4 shows an example of stereoselective coupling of mannofuranose 2,3 : 5,6-di-O-isopropylideneacetal (6)with primary triflate (7) in the presence of NaH [5, 61. The coupling reaction in the absence of the crown ether afforded only P-glycosides (9). Mannofuranose is different from ribofuranose in having two endo hydroxyl groups at the 2,3-positions. Thus mannofuranose could be a better ligand for complex formation. In particular, the p anion preferentially forms a complex
180
8 Glycosylution Methods: Alkylutions of Reducing Sugars
r
1
1
(10)
Scheme 4. Anomeric 0-alkylation of mannofuranose and effect of crown ether.
(11) as speculated in Scheme 4. Addition of 18-crown-6 afforded the a-glycoside (8) exclusively. It is thought that the crown ether inhibited endo-type complex formation and equilibrium moved to favor the a-anion (lo), yielding the a-glycoside. Addition of NaI resulted in an equimolar anomeric mixture of the glycosides. Perhaps partial complex formation of 18-crown-6with the sodium ion was suppressed. 8.2.3 Anomeric 0-Alkylation of Gluco- and Galactopyranoses with Primary Triflates: High fl-Selectivity as a Result of the Reactive Anomeric p-Anion Schmidt et al. examined the reactivities of electrophile and nucleophile in the anomeric 0-alkylation of 2,3,4,6-tetra-0-benzylglucose (12) [7]. Alkylation at room temperature gave decanoyl a- and P-glucosides in 7 :3 ratio, which reflected the anomer ratio of the hemiacetal in THF (2: 1). On the other hand, alkylation with less reactive methyl triflate at room temperature afforded a 2: 8 mixture of methyl a- and P-glycosides. When the reaction is performed with the less reactive electrophile, stereoselectivity reflects the reactivities of the nucleophiles instead of the original anomer ratio of hemiacetals. The effect of complex formation was also examined. Coupling of 12 and methyl triflate in THF or toluene at -50 "C gave the a/P mixture in the ratios 8 :2 and 1 :8, respectively. Addition of crown ether to the THF or toluene at the same temperature did not significantly alter the a/P ratio of
8.2 Anomeric O-Alkylution Primary triflate (3,15-19), t-BuOK or NaH OH
OBn
/ THF or dioxane
181
Bno&oR2
*
BnO OBn
(12) R' = Bn (13) R' = H (14) R ' = MTr
EL Bg% BnO
0'
I
BnO
(17) X= OBn (15) X= >CMe2 (16) X = > C H C ~ H I ~ (18) X=OMe
1) 17, NaH I THF B BnO n
O OBnGOH
(14)
2) H+
67 %
B BnOn
O
B
4
n
O
q
(20) BnO BnoOBn
Scheme 5. Anomeric O-alkylation of glucopyranoses and primary triflates. MTr = p-methoxytrityl
the products. In contrast with the two furanose examples described above, complex formation could not explain the stereoselectivity of the coupling of pyranoses with gluco and galacto configurations. Anomeric O-alkylation of benzylated glucopyranoses 12-14 with several primary triflates (3,15-19) gave p-glucosidesexclusively as shown in Scheme 5 [7,8].These pselectivities are a result of the greater reactivity of the anomeric p-anion. Although steric hindrance by the substituent group at 0-6 (MTr, Bn and H), solvent (THF and dioxane) and promoter (NaH and t-BuOK) did not affect the P-selectivity, there is one exception-glycosylation of 14, which has MTr at 0-6, with triflate (17) unexpectedly afforded the a-linked isomaltoside (20) only. Steric factors and the different reactivity on the triflates are primarily responsible for this behavior. The product from coupling of tribenzylglucopyranose (13) and triflate (19) was converted into lipoteichoic acid carrier fragment (21). Scheme 6 demonstrates the result and excellence of this anomeric O-alkylation method for the synthesis of a complex natural product (22) [2]. 8.2.4 Anomeric O-Alkylation of Acyl-Protected Nucleophiles with Primary Triflates
Despite of the presence of NaH, neither acyl migration nor orthoester formation occurred during the anomeric O-alkylation of alkaline labile acetyl-protected sugars (hemiacetal). Scheme 7 shows the results. Klotz and Schmidt reported preferential
182
8 Glycosylution Methods: Alkylutions of Reducing Sugars
Scheme 6. Synthesis of lipoteichoic acid fragment via anomeric 0-alkylation.
p-selectivities in the anomeric 0-alkylation of tetraacetylgluco- (23) and -galactopyranose (24), and of heptaacetyllactopyranose (25) with decyl triflate at room temperature [9]. This procedure is applicable to the synthesis of p-lactosyl ceramide. Klotz and Schmidt converted heptaacetyllactose (25)with the triflate and nonaflate(nonafluorobutane-sulfonate)of azidosphingosine (31 and 32) into plactosyl ceramide (33)in 49 and 42% yield, respectively (Scheme 8). In contrast, tetraacetylmannopyranose (26)was converted to decyl a-mannopyranoside (20) stereoselectivelyat -40 "C as shown in Scheme 7. This a-selectivity arises as a result of steric and electronic effects of the nucleophile and the reduced reactivity of the p-
(OAc
Scheme 7. Stereoselective anomeric 0-alkylation of acyl-protected sugars. DME = 1,2-dimethoxyethane.
8.2 Anomeric 0-Alkylution
183
y3
NaH I 1,2-diethoxyethane
OTBDMS
(31) R = Tf
Scheme 8. Synthesis of Iactosylceramide from acyl-protected lactose via anomeric 0-alkylation.
anion. When the temperature was reduced a-glycosides were also obtained from 23-25. This tendency is similar to that observed with alkyl-protected glucoses. Lubineau et al. reported an anomeric 0-alkylation (a-stereoselective Michael (GlcNAc) (34) on P-tosyloxaddition) of 3,4,6-tri-O-acetyl-N-acetylglucosamine yacrolein, as shown in Scheme 9 [lo]. The coupling product (35) was successfully converted into the corresponding glycoside with an a-linked spacer (36). The acetylprotected disaccharide nucleophile Galp( 1-3)GalNAc is also applicable to the above procedure. Very recently Lubineau et al. also accomplished stereoselective synthesis of aand P-glycosides [ 111. Allylation of acetylated GlcNAc (34) with ally bromide in the presence of NaH in CH2C12 gave the P-anomer (37b) exclusively. In contrast, addition of Bu4NI to the reaction mixture gave an a-glycoside (37a) only. This complete stereoselectivity is because the anomeric tetrabutylammonium alkoxide was
A AcO c O
L
,
,
NHAc
A AcO c
NaH, 18-crown-6 I THF
83%
O
G
AcHNo+%o (35)
1 ) Ph3P=CHCOzEt I THF 2) H2 I Pd 3) NaOEt
i
Scheme 9. Anomeric 0-alkylation of GlcNAc (a-selective Michael addition)
184
8 Glycosylation Methods: Alkylations of Reducing Sugars
A AcO c
a
,
NHAc
r
bco&ONa AcO
NHAc
AC& AcO
AcmONa
1 I
AllBr
/OAc A cAcO O-&$, AcHNOAll
Scheme 10. Stereoselective ally1 glycosylation of GlcNAc via anomeric 0-alkylation.
more reactive than its sodium counterpart and thus the alkylation occurred more rapidly than epimerization to the p-anomer (Scheme 10). 8.2.5 Anomeric 0-Alkylation of Mannopyranose with Primary Triflates: Possibility of Intramolecular Complexation of the Nucleophile The reactive p-anion of gluco- and galacto-type nucleophiles explains the pselectivities of anomeric 0-alkylations. Schmidt et al. examined this for the mannose-type nucleophile which is a C-2 epimer of glucose. Temperature-dependent stereoselectivity was observed in the coupling of tetrabenzyl- and tetraacetylmannopyranose with triflated solketal (15), behavior similar to that of the gluco and galacto series [6, 91. Alkylation of 2,3,4-tri-O-benzyl-6-O-p-methoxy-trityl mannopyranose (39) with 15 in THF, however, gave the P-mannopyranoside exclusively at temperatures in the range -30 to 0 "C [ 6 ] .Obviously, steric hindrance by the substituent group at 0 - 6 leads to P-selectivity. This result is different from that for glucopyranose. Tamura and Schmidt explained this selectivity by use of several mannopyranosyl nucleophiles with different types of protecting group [ 121. Stereoselectivities were found to be a result of the formation of more reactive intermediates derived from substituent groups on the nucleophiles. Variation of the substituent group at 0 - 6 evidently changes the yield and stereoselectivity. Coupling of triflate 15 and mannopyranosyl nucleophiles 39-41 with bulky substituent groups (R = MTr, Tr, and TBDPS) at 0 - 6 resulted in higher pselectivity [R = MTr; a : 15, f3 :74, R = Tr; a :3, p: 62, R = TBDPS; a : 14, p: 73 (%)I in THF than for the 6 - 0 unprotected nucleophile (38) [ a : 6 ,p:21 (%)I. The
8.2 Anomeric O-Alkylation
BnO
R 1-
f."
OH
I
185
(38)R=H (39) R = MTr (40)R = Tr (trityl) (41) R = TBDPS (t-butyldiphenylsilyl) rOTf
NaH
Ic) Scheme 11. Plausible complex formation (A-D) and coupling reaction of mannopyranose. Ln = solvent molecule as ligand.
equilibrium between the 1C (A) and C1 (B) conformations in Scheme 11 explained these results. In general, for thermodynamic reasons, mannopyranose with a bulky group at 0 - 6 tends to shift towards the C1 form (complex B) in the equilibrium. Thus, P-mannosides are afforded uia the reactive intermediate B. The 6-0unprotected mannopyranose, on the other hand, prefers 1C-type stable complex A formation, which leads to a lower yield in THF. MEM (methoxyethoxymethyl) at 0 - 2 certainly forms a highly stable crown ether-like complex C in THF, as depicted in Scheme 11. In addition, THF is an obligatory ligand in the formation of the above MEM ether complex. No coupling product was obtained in THF whereas alkylation in polar CH3CN afforded a- and p-mannopyranosides in yields of 23 and 48%, respectively. The combination of solvent and protecting group also affects yield and stereoselectivity. Anomeric O-alkylation of mannopyranose with isopropylidene acetal at in 0-2,3 afforded the corresponding mannopyranoside in high yield [a:9, P :61 ("A)] THF. The same reaction in CHZC12 resulted in a lower yield (27%) but exclusive pformation. Isopropylidene acetal at 0-2,3 contributed to the formation of a stable skew-boat-type complex D and CHZC12 acted as more suitable ligand than THF. 8.2.6 Anomeric O-Alkylation of KDO with Primary Triflates a-Glycosylation with KDO (2-keto-3-deoxyoctonate= 3-deoxy-~-manno-octulosonic acid) glycosyl halides, with the exception of fluorides, by the Koenigs-Knorr
186
8 Glycosylation Methods: Alkylations of Reducing Sugars
@ R' +R2 = (CH&, @=Solvent
Figure 1. Possible reaction intermediate leading to KDO glycoside.
method often gives unsatisfactory results because of elimination of the hydrogen halide and accompanying P-glycoside formation, whereas anomeric O-alkylation accomplishes a-selective KDO glycosylation [ 131. To increase the nucleophilicity of KDO it is better to convert it into the corresponding 4,5 :7,sdicyclohexylidenecarboxamide beforehand, because this compound can take a boat conformation and keep the 0 - 2 reactive quasi-equatorial configuration. Deprotonation of the carboxamide possibly stabilized the dianion species by complexation with metal ion as depicted in Figure 1. KDO glycosylation with some primary triflates gave the desired a-glycosides exclusively in 48-83% yields [ 141. Rembold and Schmidt accomplished the synthesis of lipid A with the [KDOa(2-6)GlcNP(26)GlcNI sequence which contains fatty acid and phosphate moieties [ 151. Anomeric O-alkylation can be a powerful method for KDO glycosylation. 8.2.7 Anomeric O-Alkylation of Partially Protected Aldoses with Primary Triflate Long-chain alkyl glycosides have become increasing important as surfactants because of their good physical performance and low toxicity. A shorter synthetic route for these compounds and use of simple materials are required by industry. Schmidt's group proved it was possible to reduce the substitution on the nucleophile in the anomeric O-alkylation. The use of 6-O-unprotected tribenzylglucopyranose (13) and P-selective coupling has been described in Section 8.2.3. Scheme 12 shows p-selective coupling of 2-O-unprotected tribenzylglucopyranose (42) with decyl triflate in CHzC12 to afford 43 in 60% yield [16]. These reactive positions (2- and 6OH) proved not to need protection. The amount of promoter was shown to control the generation of the regioisomer. The use of one equivalent of NaH in the alkyla(44)with triflate 15 afforded P-glycoside tion of 3,4,6-tri-O-benzylmannopyranose (45) exclusively, whereas the undesired 1,2-disubstituted isomer (46) was also obtained when two equivalents of NaH were used (Scheme 12) [ 121. TBDPS ether was employed at the primary position of glucose and lactose to increase solubility in common organic solvents [ 161. Scheme 13 shows the results of alkylation of these nucleophiles (47 and 50). Anomeric O-alkylation of the glucose (47) in toluene afforded a- and P-decylglycoside (48a, b) in 58% yield. The coupling of lactose (50) needed additional crown ether to yield the glycoside (51a, b). THF was not suitable because of formation of an undesired product (49a, b).
8.2 Anomeric 0-Alkylation ,OBn H O* ' " BnO
187
CioH21OTf NaHCH2CIZ (1.1 eq) OH
BnO& BnO o-
on
,/rb NaH ( 1 eq)
BnOB
X
o
BnO
H
(44)
I CHpClp
NaH (2 eq)
Scheme 12. Anomeric 0-alkylation of partially protected nucleophiles.
c1&210Tf, NaH
H HOo
g
o
-
OH
(48a,b)
HO
58 % (a$= 1:4)
OTBDPS
C,oH,,OTf, NaH
(47)
THF
- -&H
OH
(49a,b)
OTBDPS
(50)
OH
toluene 15-crown-5
55 %(a$ = 1:2)
- 0
OTBDPS
(51a,b)
OH
56 % (a$= 1:l)
Scheme 13. Anomeric 0-alkylation of less protected nucleophiles.
8.2.8 Anomeric 0-Alkylation of Unprotected Aldoses with Primary Triflate, Bromides, and Cyclic Sulfates We can also use 0-unprotected aldoses for anomeric 0-alkylation by employing an aprotic dipolar solvent, namely N,N'-dimethylhexahydropyrimidin-2-one (DMPU) [17]. DMPU is essential to accomplish the anomeric 0-decylation of 0-
188
8 Glycosylation Methods: Alkylations of Reducing Sugars
unprotected aldoses with didecyl sulfate in the presence of NaH. Glucose, mannose, N-benzoylglucosamine, and xylose are converted to the corresponding decylglycopyranosides in moderate yields as anomeric mixtures ( a :p = 2 : 1-1 : 10). Decyl agalactofuranoside and a/p-arabinofuranoside ( 1 : 3) were obtained exclusively from galactose and arabinose, respectively, under these reaction conditions. Benzyl and allyl glycosides of these aldoses can be obtained by this procedure with benzyl and allyl bromides. This is a much more effective method than conventional Fisher glycosylation for simple glycoside synthesis. Cyclic sulfates can be used as potent electrophiles [ 181. Cyclic sulfates with longer alkyl chains which are suitable for the lipophilic part of the surfactants were condensed with free glucose in the same manner as above in 65-72% yields (Scheme 14). Anomeric 0-alkylation of the unprotected sugar is a potent procedure for producing amphiphilic molecules in short steps and contributes to saving materials and energy.
Scheme 14. Anomeric 0-alkylation of free glucose with cyclic sulfates.
8.2.9 Anomeric 0-Alkylation with Secondary Triflates and Nonaflate Glycosidic linkage to secondary positions [e.g. Man( 1-4)GlcI can be accomplished via anomeric 0-alkylation with secondary triflate. This procedure is accompanied ~ by inversion of the stereochemistry of the electrophile as a result of the S N reaction. The use of promoters (15-crown-5and NaH) in toluene accomplished coupling of tetrabenzylglucose (12) with aliphatic triflates (isopropyl and 1-methylnonyl triflate) in 70% yield ( a :p = 1 :9) and quantitative yield respectively, whereas condensation of 12 and the triflate of 1,6-anhydr0-2,3-di-O-benzylgalactose (52) failed under these conditions. Schmidt’s group has recently employed aprotic dipolar solvent systems; DMF-HMPT and HMPT-THF to solve the problem (Scheme 15) [ 191. These new solvent systems gave the desired glycosides in high yields with a/S
(a),
(52) R = Tf (53) R = Nf
Scheme 15. Anomeric 0-alkylation procedure with secondary electrophiles. P = protecting group; HMPT = hexamethylphosphoric triamide.
8.3 Glycosylation via the Locked Anomeric Conjiguration
189
selectivities of 2 : 1-5 : 1. Exclusive a-selectivity was observed during formation of the sequence Mana( 1-4)Glc from tetrabenzylmannose and triflate 52. The corresponding nonaflate (53) is more effectivethan the triflate for this glycosylation [12].
8.3 Glycosylation via Locked Anomeric Configuration V. K. Srivastava and C. Schuerch have first introduced 1,2-0-dibutylstannylene acetal-mediated methyl and allyl P-glycosylation for mannopyranose in 1979 [20]. This has recently been successfully developed by G. Hodosi and P. Kovai: as a titled concept; Scheme 2 shows the general procedure used. Synthetically difficult 1,2-cis P-glycosides, especially p-mannopyranosides, can be obtained by this method. The possibilities and problems of the locked anomeric configuration method are discussed in this section.
8.3.1 Synthesis of Methyl, Allyl, and Benzyl Glycosides via Stannylene Acetals Stannylene-mediated glycosylation was first applied to alkyl mannosylations. Condensation of the stannylene acetal of 3,4,6-tri-O-benzylmannopyranose (44) with methyl iodide and allyl bromide in DMF afforded the desired methyl and allyl P-mannopyranosides, respectively, in almost quantitative yields [20]. Benzyl Smannopyranoside was obtained from the same stannylene acetal by use of BwNI in benzene [21].The electrophilic leaving group is responsible for the reactivity and stereoselectivity. Methylation with methyl tosylate and dimethyl sulfate gave methyl mannopyranosides as anomeric mixtures at temperatures above 75 "C [20]. Glycosylation of 3,4,6-tri-O-benzylglucopyranose (42) via stannylene acetal with methyl iodide resulted in the production of the 2-O-methyl ether (70%) and amethyl glycoside (30%) [20], presumably as a result of the formation of stannylene acetal on axial 0 - 1 and equatorial 0 - 2 . The equatorial 0 - 2 is more reactive during stannylene complexation, which leads the 2-O-methyl ether. Thus stannylenemediated alkylation of 6-O-tritylmannose and methyl a-mannopyranoside afforded 3-O-alkylated products because of the greater reactivity of the equatorial oxygen at the 3-position [20]. The reaction mechanism is discussed in detail in Section 8.3.4.
8.3.2 Epimerization at C-2 by Locked Anomeric Configuration Method Epimerization at C-2 sometimes occurs during dibutylstannylene complex formation. In particular, 1,2-cis P-per-O-acylation for formation of the stannylene acetals of free mannose, rhamnose, and lyxose is accompanied by epimerization [22]. Scheme 16 shows the epimerization process schematically. This unwanted phenomenon can be minimized by conducting the acetalation under milder conditions-brief reaction in dilute solution at low temperatures [23].
190
8 Glycosylution Methods: Alkylations of Reducing Sugars
OR I
Bu
Scheme 16. Epimerization of mannose by stannylene acetylation. P = protecting group.
We can, on the other hand, take advantage of this epimerization to obtain rare sugars. Hodosi and KovaC converted 6-0-tritylgalactose to talose in 70% yield by stannylene acetalation and C-2 epimerization [241.
8.3.3 Locked Anomeric Configuration Method for Rhamnosyl Stannylene Acetal
Locked anomeric configuration method results in complete p-selectivity in oligosaccharide synthesis [25, 261. The electrophile must be converted to an active triflate and inactive bromide, iodide and mesylate cannot be used. Addition of CsF or Bu4NF into the reaction effectively increases the solubility of the stannylene acetals. Accompanying formation of the formate derived from the primary triflate at room temperature can be suppressed by reducing the reaction temperature to -5°C. Table 1 shows results from coupling the 1,243-stannylene acetal of rhamnose (54) with primary and secondary triflates (57 and 58) in the presence of CsF.
8.3.4 Locked Anomeric Configuration Method for Mannosyl Stannylene Acetal: Isomerization of Acetal [25, 261
Anomeric oxygen on the analogous acetal of mannose is less reactive than that of rhamnose. Coupling of the stannylene acetal of mannose 55 with the primary triflate 57 in DMF at 0-25 "C afforded the desired p-linked disaccharide together with the (3-6) linked pseudo-disaccharide in 40 and 23% yield, respectively. Changing the solvent to CH3CN and addition of BQNF increased the yield and regioselectivity. Because of the low solubility and reactivity of the stannylene acetal in CH3CN, other aprotic polar solvents (e.g. DMF, abbreviated in Table 1. DMA, and DMSO) were employed for the coupling of secondary triflate 58, as shown in
8.3 Glycosylution via the Locked Anomeric Conjiqurution Table 1. Results from glycosylation via the locked anomeric configuration. DMA = N , N-dimethylacetamide. I I
191
I
Manp( 14)Glc
RhafJ( 1-4)Glc CsF I DMF 25 "C, 78 %
B z0
Tf+
Bzo OMe (58)
DMF DMA DMSO
40% 52% 59% 25 "C
Mang( 1-4)Glc CsF I DMF 25 "C, 67 %
Table 1. The secondary triflate did not react with 3-0 of the nucleophile; the anomeric oxygen was more reactive. Isomerization of the stannylene acetal from 1,2-0 to 2,3-0 and the resultant equatorial 3-0 activation rationalize the formation of the pseudo-disaccharide as shown in Scheme 17. The epimerization of the stannylene acetal seems much faster than alkylation. The use of the analogous 3-O-benzyl ether effectively prevented the unfavorable coupling. Thus, generation of the etheric by-product was suppressed in the reaction with the same primary triflate (57).
8.3.5 Locked Anomeric Configuration Method for Stannylene Acetal with the Glucose Configuration 125,261 Scheme 18 shows results obtained from use of the locked anomeric configuration method for coupling of gluco-type stannylene acetals with triflates. Reaction of the stannylene acetal of maltose (59) and the secondary triflate (58) resulted in generation of an ether (2-4) linked pseudo-trisaccharide similar to the result with glucose. It is worthy of note that the coupling of the stannylene acetal of 4,6-O-benzylideneglucose (61) with the primary triflate (57) predominantly afforded the p( 1-6) disaccharide (62). The stereochemistry of the main product is inconceivable, however; it is plausible that the anomer oxygen favored a configuration close to equatorial orientation as the most stable 1,2-0-stannylene complex conformation.
192
8 Glycosylation Methods: Alkylations of Reducing Sugars
Scheme 17. Isomerization between 1,242- and 2,3-O-stannylene acetals of mannose.
HO
H$%o&
1) BuzSnO / MeOH (59) HO
Ho
OH
HO
-$ .*
2) 58, B u g F / DMF OHO + L -
(60) 75 %
BzO
OMe
Scheme 18. Coupling reaction via locked anomeric configuration for maltose and glucose moieties.
8.4 Conclusion The advantages of the ‘anomeric 0-alkylation’ and ‘glycosylation via locked anomeric configuration’ methods are:
1) primarily we can use unprotected sugars as nucleophiles; 2) we can also perform glycosylation reactions under non-acidic conditions; 3) we can also use polar solvents such as DMF, DMA, and DMSO, and even CH3CN; and 4) inversion of configuration occurs at the 0-glycosidically linked carbon of the electrophile. These points reduced the limits of glycosylation reactions and often result in increased coupling yields, compared with conventional glycosylation. With regard to point 4,we can avoid the use of poorly reactive hydroxyl groups, which reduce coupling yields in Koenigs-Knorr-type glycosylation. ‘Anomeric 0-alkylation’ and ‘locked anomeric configuration method’ can be alternatives to traditional KoenigsKnorr-type glycosylation.
References
193
References I . R. R. Schmidt, Angew. Chem. Int. Ed. Engl., 1986,25, 212-235 (197 references). 2. R. R. Schmidt, Pure Appl. Chem., 1989, 61, 1257-1270 (69 references). 3. R. R. Schmidt, Modern Methods in Carbohydrate Synthesis, Hanvood Academic publishers, 1996, pp. 20-54 (57 references). 4. R. R. Schmidt and M. Reichrath, Angew. Chem. Int. Ed. Engl., 1979, 18, 466-467. 5. R. R. Schmidt, M. Reichrath, and U. Moering, Tetrahedron Lett., 1980,21, 3561-3564. 6. R. R. Schmidt, U. Moering, and M. Reichrath, Chem. Ber., 1982, 115, 39-49. 7. R. R. Schmidt, M. Reichrath, and U. Moering, J. Carbohydr. Chem., 1984,3, 67-84. 8. R. R. Schmidt, U. Moering, and M. Reichrath, Tetrahedron Lett., 1980, 21, 3565-3568. 9. W. Klotz and R. R. Schmidt, J. Curbohydr. Chem., 1994, 13, 1093-1101. 10. A. Lubineau, H. Bienayme, and J. Le Gallic, J. Chem. Soc., Chem. Commun., 1989, 19181919. 1 1. A. Lubineau, S. Escher, J. Alais, and D. Bonnaffk, Tetrahedron Lett., 1997, 38, 4087-4090. 12. J. Tamura and R. R. Schmidt, J. Curbohydr. Chem., 1995,14, 895-911. 13. R. R. Schmidt and A. EDwein, Angew. Chem. Int. Ed. Engl., 1988,27, 1178-1180. 14. A. EBwein, H. Rembold, and R. R. Schmidt, Curbohydr. Rex, 1990,200, 287-305. 15. H. Rembold and R. R. Schmidt, Carbohydr. Res., 1993,246, 137-159. 16. R. R. Schmidt and W. Klotz, Synlett, 1991,3, 168-170. 17. W. Klotz and R. R. Schmidt, Liebigs Ann. Chem., 1993, 683-690. 18. W. Klotz and R. R. Schmidt, Synthesis, 1996, 687-689. 19. Y. E. Tsvetkov, W. Klotz, and R. R. Schmidt, Liebigs Ann. Chem., 1992, 371-375. 20. V. K. Srivastava and C. Schuerch, Tetrahedron Lett., 1979,35, 3269-3272. 21. A. Dessinges, A. Olesker, and G. Lukacs, Carbohydr. Res., 1984, 126, c6-c8. 22. G. Hodosi and P. KovaE, XVIII Int. Carbohydr. Symposium Abstr., Milano, 1996, pp. 394. 23. G. Hodosi and P. KovaE, Carbohydr. Rex, 1997,303, 239-243. 24. G. Hodosi and P. KovaE, J. Carbohydr. Chem., 1998,17, 557-565. 25. G. HodosiandP. KovaE, J. Am. Chem. SOC.,1997,119,2335-2336. 26. G. Hodosi and P. KovaE, Curbohydr. Res., 1998, 308, 63-75.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
9 Other Methods of Glycosylation Luigi Panza and Luigi Lay
Introduction and Summary The biological relevance of oligosaccharides in molecular recognition phenomena stimulated the development of many synthetic approaches for the formation of glycosidic linkage, which is the main problem in oligosaccharides chemistry. Besides the most popular methods, which will be discussed in other chapters of the book, many other protocols have been explored.
X = "exotic"leaving group
New methods should, in general, have advantages towards the existing protocols (e.g. to be more efficient, simpler, to use cheaper reagents, to allow better selectivity, to be environmental friendly etc). This chapter will also deal with common methods not covered by other chapters, but more attention will be given to those approaches which fulfil, at least in part, the above advantages. Highlights
1. Use of a protecting group at the anomeric position easily transformed into a good leaving group.
196
9 Other Methods of Glycosylution
Example: an anomeric 2-butenyl glycoside can be isomerized into a propenyl ether which has been demonstrated to be an efficient leaving group. Introduction of the concept of latent-active glycosyl donor
OBn 78%, a/p 1:20 TMSOTf CH$N
BnO
G . J. Boons, S. Isles, Tetrahedron Lett., 1994,35, 3593.
BnO
Scheme B.
Advantages: a protecting group can be easily converted into an efficient leaving group. Disadvantages: use of mercury salts for the preparation of 2-butenyl glycosides.
2. Use of an heterogeneous acid catalyst. Example: an heteropoly acid act both as promoter and dehydrating agent in the glycosylation of an anomeric hydroxy sugar donor
Go&$ 82%, pia 1090
BnO BnO
N3
K. Toshima, H. Nagai, S. Matsunnua, Synlett, 1999, 1420
Scheme C.
Advantages: the procedure is simple, the promoter is reusable and environmental friendly. Disadvantages: the method seems to work well only with reactive donors; only a few examples available so far.
3. New achievement with old methods. Example: use of ortho esters as intermediates in regio- and stereoselective glycosylations.
9.1 En01 Ethers
197
TMSOTf CH2C12
W. Wang, F. Kong, J. 0%.Chem., 1998,63,5744.
Scheme D.
Advantages: good regioselectivity with partially protected acceptors, multiple glycosidic bond formation. Disadvantages: multistep procedure; requires AgOTf for the ortho esters formation.
N. B. Because of the vast scope of this chapter, it is almost impossible to include all the pertinent literature. The authors have made an attempt to organise in a logical manner most of the multifarious approaches for the formation of the glycosidic linkage not included in other chapters. They apologize for any omission they can have made unintentionally.
9.1 Enol Ethers This section will examine the preparation of glycosides and oligosaccharides starting from sugar derivatives which contain a double bond substituted with an ethereal or an acetalic oxygen atom. The term enol ether is therefore used in this context in a quite broad sense. The following general scheme summarizes the approaches which have been explored for synthesis of glycosides from enol ethers. The different approaches can be classified into two main groups: a) strategies in which the starting compound is an hydroxy enol ether already containing the aglycone moiety and from which the glycoside is obtained by cyclization with an electrophile (from endo-en01 ethers, retrosynthetic pathway d and exo-enol ethers retrosynthetic pathway e, Scheme 1); b) approaches in which a properly activated donor, which can be derived from or still contain an en01 ether function, reacts with a glycosyl acceptor (a sugar alcohol) in a more classical way (from exo-glycals, retrosynthetic pathway a, endo-glycals, retrosynthetic pathway b and vinyl glycosides, retrosynthetic pathway c, Scheme 1).
198
9 Other Methods of Glycosylation
&'
from exoenol ethers
I
H'R' endo-enol ethers
Y
PO
S
O
R
Scheme 1. Retrosynthetic pathways for the synthesis of glycosides from enol ethers.
Furthermore, in some of these approaches the diverse structure of the starting compound may be used to obtain different kinds of glycosides, namely aldosides or ketosides (endo-en01 ethers and endo-glycals), whereas other starting structures allow only the formation of ketosides (exo-enol ethers and exo-glycals). The following examples illustrate some applications of the different approaches shown above.
9.1.1 Endo-Enol Ethers The first example of the use of sugar hydroxy enol ethers was originally proposed by Suzuki and Mukaiyama [ 11 in 1982 for the synthesis of 2-deoxyglycopyranosides (Scheme 2). The enol ether precursors 3 were obtained by a Horner-Wittig reaction 1 with an alkoxymethyldiphenylphosphine of 2,3,5-tri-O-benzyl-~-arabinofuranose oxide 2, as an E/Z-mixture separated by careful chromatography. Treatment of each diastereoisomer with various electrophiles (Hg(I1) salts, PhSeX, NIS) induced a 6-endo cyclization. The substituent at position 2 was then reductively removed to give the desired 2-deoxydisaccharides 4 and 5. It is noteworthy that, in such an approach, the factors which control the stereochemistry of the process are completely different from those of a classical glycosylation and will be discussed later. In this case the stereochemistry at the anomeric position was dependent on the stereochemistry of the double bond and on the electrophile used.
Scheme 2. Reagents: i) LDA, THF, -78 to 0°C; ii) KH, THF, 40"C, 75% Z/E 2: 1; iii) Hg(I1) salts, PhSeCl or NIS; iv) NaBH4, Bu3SnH or LiAlH4 respectively.
9.1 En01 Ethers
199
BnO ?Me COOMe
BnO v
f- l o R =
CHO
i,ii BnO
COOMe
9
yo OMe 6
I
i,iii BnO
BnO
BnO
16
BnO
COOMe
I
14
BnO
COOMe
17 R = HgCl 19R=H
IZOR=H
Scheme 3. Reagents: i) THF, NaH, O T , 90%, Z/E 2: 1 from 7, 85%, Z/E 2 : 3 from 14; ii) CF3COOH/H20 9 : 1, 0 "C, 80%; iii) MeONa in MeOH, 95%; iv) Hg(OCOCF3)2,THF, 0 "C then KCI, 80%; v) Ph3SnH, anhydrous AcONa, toluene, 90%.
Sinay and Paquet cleverly applied this approach to the synthesis of sialic acid [2] and Kdo [3] glycosides (Scheme 3). The enol ether precursors were prepared in good yield by a Horner-Emmons reaction between the properly protected aldehydo sugar derivative 7 or 14 and the sugar containing alkoxyphosphono acetate 8, and the E/ Z mixtures obtained were easily separated by chromatography. With respect to the previous procedure, the double bond in the starting enol ether was trisubstituted. In the case of sialic acid disaccharides, the mercuriocyclization of the E diastereisomer
200
9 Other Methods of Glycosylation
9 gave the a-linked mercurioderivative 11 with complete stereo- and regioselectivity. Likewise, cyclization of the Z isomer 8 afforded exclusively the p-linked mercurioderivative 10. The remarkable stereoselectivity of the reaction has been rationalized in terms of the attack of the electrophile from the less hindered side of the double bond controlled by the adjacent stereocenter and by the chair conformation in the cyclization transition state. Treatment of the mercurioderivatives with triphenyltin hydride and sodium acetate gave the p- and a-sialic acid disaccharides 12 and 13. When the procedure was applied to the Kdo enol ether precursors 15 and 16, the cyclization was again highly stereoselective affording, after demercuriation, the a-linked Kdo disaccharide 19 from the Z isomer and the p-linked Kdo disaccharide 20 from the E isomer. The reaction was also applied to a phosphonoacetate linked to the 3 position of a properly protected N-acetylgalactosamine. In this case the Horner-Emmons reaction was completely stereoselective giving exclusively the E isomer, from which the P-linked Kdo disaccharide was easily obtained in good yield. The above procedures allow access to 2-deoxyglycosidesand oligosaccharides. In an analogous way such an approach could be used for the synthesis of 2-hydroxy glycosides provided that the enol ether is allowed to react with an electrophilic oxygen. Russo et al. developed this idea [4]through the epoxidation of hydroxy enol ethers followed by ring closure of the intermediate epoxides to give the desired 2-hydroxy glycosides. It is worthy of mention that these compounds, having a single free hydroxyl group, allow further manipulation at position 2. The starting enol ethers E- and 2-3were prepared according to Mukaiyama [ 11, through a Horner-Wittig reaction of alkoxymethylphosphine oxides with 2,3,5-tri0-benzyl-D-arabinose (Scheme 4) and ribose. After separation of the E/Z mixture, epoxidation with MCPBA of the of the hydroxy enol ethers, either in Z or E configuration of the double bond, gave directly the cyclized products 21-24 owing to the high reactivity of the intermediate enol ether epoxides, with a 6-endo-tet ring closure. As mentioned above, the stereochemical course of this process is controlled by factors which are completely different from those of a “classical” glycosylation reaction. In this case two new stereocenters are introduced, at the anomeric position and at C-2, and the stereochemical outcome of the process depends on the config-
OR
BnO
-
Bno&OR BnO
E-3
BBnOn
O 2 3
+
21 OH
e OR
B,,O
OR 23
Scheme 4. Reagents: i) MCPBA, CH2C12, Na2HP04.
+
BBnOn
I
22
OR
O
3
24 HoOR
9.1 En01 Ethers
201
uration of the double bond, on the stereoselectivity of the epoxidation reaction, and on the stereospecificity of the ring closure. While the epoxidation was not highly stereoselective, the ring closure was stereospecific in all cases studied except for the formation of mannosides with oxygenated aglycons. The same procedure was also applied to the synthesis of ketofuranosides [5] such as fructosides and psicosides, where the starting enol ethers contained an extra hydroxymethyl group linked to the double bond. This structural feature allowed the use, besides the MCPBA epoxidation, of the Sharpless epoxidation. Although the epoxidation reaction gave, in general, better results than in the former case, it is noteworthy that the stereoselectivity of the Sharpless epoxidation was somewhat unpredictable. This is probably due to the presence of a second hydroxyl group which can coordinate the titanium and, in some cases, counteract the normal course of the Sharpless epoxidation. 9.1.2 Exo-Enol Ethers
The synthesis of glycosides from exo-enol ethers through a iodoetherification reaction was initially proposed by Barrett [6].It is obvious that from this method only glycosides of ketosugars can be obtained. In this approach a hydroxy group of a sugar either at the anomeric carbon or at another position is initially converted into an ester with a series of carboxylic acids containing a silyloxy function. Different procedures were used for the esterification step at the anomeric position in order to obtain a- or p-anomeric esters with good stereoselectivity. The esters were methylenated using the Tebbe reagent, desilylated and iodocyclized to give reducing and nonreducing disaccharides in fair to good yield. It is noteworthy that, in the case of iodocyclization of anomeric vinyl ethers, the anomeric C-0 bond is not cleaved at the intermediate iodonium ion stage. The stereochemical outcome of the cyclization reaction is strongly dependent on the structure of the vinyl ether, and the stereoselectivity varies from modest to excellent. Barrett applied this procedure to the synthesis of sucrose (Scheme 5) [7]. 2,3,4,6Tetra- 0-benzyl-D-glucopyranose was converted into the a-glucopyranosyl ester 25 with excellent selectivity. In this case the methylenation using Tebbe reagent was unsuccessful. However, the Nozaki-Takai protocol gave the desired vinyl ether. After desilylation, the cyclization reaction gave only the (3-fructofuranosyl derivative 26 when the reaction was performed in the presence of silica. The sN2 displacement of the iodide proved to be difficult, as expected for a neopentylic position. The problem was solved using a radical-mediated substitution with TEMPO, and final deprotection afforded sucrose 29. A similar approach was used by Sinay (Scheme 6) for a new synthesis of Kdo glycosides and disaccharides [8]. The mannose derived hydroxyheptenitol 30 was converted into the exo-enol ether 31 through a multistep synthesis. Cyclization was achieved using potassium t-butoxide and iodine at low temperature to give the disaccharidic derivative 32 as the only product of the reaction. The transformation of the iodomethyl appendage into the carboxylic group required a difficult nucleo-
202
BnO B n
9 Other Methods of Glycosylation
OBn QSiPh;Bu
Bn007/yvoB -
O
25
q
0
...
i,u
OBn
111
26
Bnd
27
: \OBn
28
Bn6
Acb
bAc
OBn
HV
OH
Scheme 5. Reagents: i) TiC14, TMEDA, Zn, CH2Br2, THF (Nozaki-Takai protocol), 68%; ii) TBAF on silica, THF, then tBuOK, 12, THF, 25 "C, 65%; iii) TEMPO, Bu3SnH, benzene, hv, 80%; iv) Na, NH3, THF then Ac20, pyridine, 90%; v) MeONa, MeOH, 96%.
philic displacement with cesium acetate in HMPA at 140 "C followed by a standard sequence of deprotection, oxidation and esterification with diazomethane to afford the final disaccharide 33. 9.1.3 Endo-Glycals Glycals have been extensively used for the synthesis of 2-deoxyglycosides and oligosaccharides since Lemieux introduced their reaction with simple alcohols in the presence of iodine and silver salts [9]. Interesting results were obtained using elec-
COOMe
30
BnO BnO 31
BnoOMe
Scheme 6. Reagents: i) PivC1, pyridine, DMAP, 98%; ii) 9-BBN, THF, then H202, NaOH, 71%; iii) RuC13/NaI04 CC14, CH3CN, H20, 92%; iv) Methyl 2,3,4-tri-O-benzyl-a-~-glucopyranoside, DCC, DMAP, 78%; v) Tebbe reagent, 81%; vi) LiAlH4, THF, O"C, 93%; vii) tBuOK, then 12, THF, 92%; viii) CsOAc, HMPA, 130 "C, 68%; ix) MeONa in MeOH, rt, 98%; x) (COC1)2/DMSO then Et3N; NaC102, H202, CHjCN; xi) CHzN2 Et20, 68%.
9.1 En01 Ethers
BnO BnO
203
Bz0 34
35 31 39 R=H
Scheme 7. Reagents: i) IDCP, CH2C12,4A ms, 58% of 36, 79% of 38; ii) Ph-iSnH, AIBN, benzene, 94%.
trophiles such as IDCP, again described by Lemieux, and more recently exploited by Danishefsky [lo], who applied the concept of armed-disarmed glycosylation (Scheme 7). In this manner it was possible to use the benzyl protected glycal34 as a donor, and the benzoyl protected glycal35 as an acceptor, in the initial glycosylation reaction. The product still contains a glycal function which can be submitted for a second glycosylation reaction to finally give compound 39. Other groups have proposed the use of NIS and NBS as electrophiles. For example, Tatsuta introduced [ l l ] and applied the NBS mediated glycosylation to the synthesis of natural 2deoxyglycoside containing compounds [ 121. Thiem [ 131 developed the NIS-mediated glycosylation, which was applied by many groups to the synthesis of various natural products containing oligosaccharidic chains [ 141. Thiem has improved the method for the synthesis of disaccharide side chains of antracyclines by the transformation of the alcohol acceptor into the corresponding tin-alkoxide to enhance its reactivity, as in the glycosylation of 41 (Scheme 8) 1151. All these 2-deoxy-2-haloderivatives can be reductively dehalogenated to give the desired 2-deoxyglycosides. Sinay introduced the use of phenylselenyl chloride as an electrophile [ 161, which was exploited by Barrett in the avermectin a-disaccharide synthesis [ 171. Ogawa
HOIt..
/ 0
i ____)
40
42 R=I
41 43 R=H
AcO
Scheme 8. Reagents: i) (Bu3Sn)z0,benzene then NIS, CH3CN 65%; ii) BqSnH, AIBN, 76%.
204
9 Other Methods o j Glycosylation (OBn
BnO& BllO
t
34
ratio
Scheme 9. Reagents: i) (PhS)2, PhSC1, SbC15, CH2Cl2 -6O"C, 70°h
described [ 181 the addition of phenylsulfenate esters to glycals in the presence of TMSOTf while Franck performed the reaction using phenylbis (pheny1thio)sulfonium salts, in which the alcohol acceptor was again presented as the stannyl ether (see e.g. the glycosylation of 34 with 44, Scheme 9) [19]. The addition product was reduced to the 2-deoxyglycosides by using either Raney-Ni or a tin hydride. In these glycosylation reactions the use of NIS, NBS or phenylselenyl chloride afforded mainly the a-glycosides, whereas other electrophiles gave the pglycosides as the main products with moderate selectivity. Franck studied quite extensively the face-selectivity of his reaction and found that it depends on the arylthio substituents, on the nucleophile and on the glycal structure. Such an approach was also applied to the synthesis of glycofuranosides starting from furanoid glycals. Some examples of activation of glycals using protons as electrophiles are also described. PTSA [20], CSA [21], triphenylphosphine hydrobromide [22], and AG50 WX2 resin [23] were used as promoters allowing the direct formation of 2-deoxyglycosides with predominant a-anomeric configuration. For example, Tatsuta effected the glycosylation of a glycal in the presence of CSA for the synthesis of the antibiotic elaiophylin [21]. The main drawback of this procedure is the possible occurrence of a competitive Ferrier rearrangement. Glycals were also transformed into the corresponding epoxides and used as glycosy1 donors, as will be described in another chapter. 9.1.4 Exo-Glycals
The NIS-mediated glycosylation introduced by Thiem, was applied not only to endo-glycals, but also to exo-glycals for the preparation of glycosides of ketosugars. It was initially studied [24] on the exo-glycal of leucrose 48 (Scheme 10) which was obtained in a classical way by reductive elimination on the glycosyl bromide 47 with zinc dust. Treatment of 48 with NIS in the presence of the sugar acceptor 49 gave the trisaccharide 50 in about 50% yield as a single anomer. Similarly, van Boom obtained [25] a iodomethyl disaccharide by treatment of the exo-glycal derived from tetra- 0-benzybglucopyranose with IDCP in the presence of a protected mannose acceptor.
205
9.1 End Ethers
i
OBz 41
BzO
OAc
Br 48
49
50
OAc
Scheme 10. Reagents: i) Zn, CH3COONa, CH3COOH/H20 1 : I, -10"C, 65%; ii) NIS; 3 A ms, CH3CN, rt, 47%.
Attempts to apply an analogous approach to the synthesis of Kdo disaccharides were made by Sinay by reaction of an exo-glycal obtained from 2,3,5,6-di-O-isopropylidene-D-mannose with iodine and potassium t-butoxide, but after a positive result with methanol, sugar acceptors did not give the expected disaccharides [8]. Russo [26] developed the use of exo-glycals such as 51 and 54 for the synthesis of glycosides of ketosugars by epoxidation with dimethyldioxirane followed by a Lewis acid-mediated glycosylation (Scheme 11). In this way glycosides and disaccharides of fructose 56 and gluco-heptulose 53 were obtained. The stereoselectivity of the reaction was dependent on the diastereomeric ratio of the epoxides in the case of simple alcohol acceptors, while less reactive sugar alcohols gave moderate selectivity for fructose glycosides and good to excellent a-selectivity in the case of pyranosyl derivatives. The method was further developed for the synthesis of Kdo disaccharides (Scheme 12) [27]. The exo-glycal57, previously synthesized by Sinay [8], was epoxidized with dimethyldioxirane and glycosylated with different alcohol and sugar acceptors to give the hydroxymethyl derivatives such as 59. The advantage of this approach with respect to the NIS mediated glycosylation is the direct introduction of the oxygenated function at C-1 of the Kdo moiety thus avoiding the cumbersome substi-
OBn
9
ABn
55 a/!3 11:
Scheme 11. Reagents: i) DMD, CH2C12, 0°C; ii) ZnCl2 'OEt2, CH2C12, -78"C, (55-90%)
206
xF--
9 Other Methods of Glycosylation
OH
11
*
5 8 "
57
Scheme 12. Reagents: i) DMD, CH2C12, -78 "C; ii) ZnCl2 . OEt,, CHzC12, -78 "C, 82%).
tution of the iodide. The reaction occurs with excellent a-selectivity and its stereochemical outcome has been rationalized by means of semiempirical calculations. 9.1.5 Vinyl Glycosides This method may be seen as a more classical approach as vinyl ethers can act as leaving groups in glycosylation reactions (Scheme 13a). The method was again introduced by Sinay [28] and derived from the observation that mercury(I1) chloride induces cyclization of 1-propenyl glycosides of 2-acetamido sugars to give oxazoline derivatives. Some initial positive results using 1-propenyl glycosides promoted the
a)
BBnO n 0
G o BnO
T
+
BBnO n 0
i
BnoOCH3
60
b,
Ho% BnO
BBnO n O BnO G o Brio $
62
61
G
o
H
-
+
N3
63
BBnO
n N3
65
64
OBn
Brioo
Pia 5:1
BnoOCH3
~ p/a 6.611 o BnoOCH3
OBn
BnO
66
61
Scheme 13. Reagents: i) BF3. OEt,, CH3CN, -25°C; ii) TMSOTf, CH3CN, -25"C, 75% of 76 and 92% of 79.
~
9.1 En01 Ethers
207
development of isopropenyl glycosides as glycosyl donors. Thus, treatment of the anomeric acetates of tetra-0-benzylglucopyranose with the Tebbe reagent gave the desired isopropenyl glucoside 60. The donor was activated both with TMSOTf and BF3 . OEt2. The glycosylation reactions were performed both in acetonitrile and in dichloromethane. The first solvent gave the better yield and p-selectivity, as expected, owing to the formation of transient a-nitrilium species. The rationalization of the process in terms of an intermediate mixed acetal formation led to the hypothesis that the glycosylation should also occur by reverting the reacting partners, namely using a vinyl derivative of the acceptor with a 1-hydroxy sugar. This reversed approach was successfully applied to the glycosylation of 2-azido derivative 63, for which the isopropenyl glycoside itself is not available as the Tebbe reagent is not compatible with the azido group (Scheme 13b). Finally the same paper describes the use, as donors, of isopropenyl glycosyl carbonates, easily obtained from hemiacetal derivatives with the commercially available isopropenyl chloroformate (Scheme 13c). Strictly speaking these donors are anomeric carbonates but are included here as their efficiency as glycosyl donors derives from the presence of the double bond. The glycosylation reaction of donors such as 66 with various acceptors occurred smoothly in the presence of TMSOTf to give disaccharides in good to excellent yield. Isopropenyl glycosides were also studied by Chenault [29] with particular attention to mechanistic aspects (Scheme 14). The stereoselective synthesis of pivaloyl protected isopropenyl glycosides was achieved either using Tebbe reagent or by reaction of glycosyl halides with bis(acetony1)mercury. The glycosylation reaction (e.g. of 69 with 37) was promoted with various electrophiles the best results being obtained with Tf20, TMSOTf and NIS/TfOH. The use of IDCP gave electrophilic addition to the double bond with subsequent formation of acetals such as 71. Chenault found that there are interesting influences of the reaction conditions on the reactivity of the isopropenyl glycosides towards transglycosylation or electrophilic addition on the double bond. (OPiv
(OPiv
69
Scheme 14. Reagents:
NIS, TfOH, CH3CN, 70'31, ii) IDCP, CH3CN or CH2C12, 53%
208
9 Other Methods of Glycosylation
AcO
Br 12
-Ai:030n i
BnO BnO+
AcO
iv
13
74
Bno$&oF+ OBn
B;:oGonv
BnO active
BnO
6'
latent
BnO
BnO
l5
op-
latent
& $-on.
alp 1:20
BnO BnO BnO 71
Scheme 15. Reagents: i) 3-Buten-2-01, Hg(CN)Z,HgBrz, CH3CN, 70%; ii) MeONa in MeOH, 90%, iii) NaH, BnBr, DMF, 820/0; iv) (Ph3P)3RhCl/nBuLi,THF, 92%; v) TMSOTf, CH3CN, 78%.
Boons developed the use of vinyl glycosides as donors by modifying the leaving group and exploiting the concept of latent-active glycosylation strategy (Scheme 15) [30]. In his approach a sugar was protected at the anomeric position as 3-buten-2-yl glycoside (72+ 74). The isomerization of the double bond with Wilkinson's catalyst in the presence of a base afforded 2-butene-2-yl glycosides such as 75, which can undergo a Lewis acid catalyzed glycosylation reaction. The activation of the butenyl group with TMSOTf promoted the glycosylation reaction giving disaccharides in good yield. Such interesting procedures exploit the easy transformation of a protecting group at the anomeric position into an efficient leaving group. The use of an allyl glycoside (e.g. 76) as acceptor allows the reiteration of the procedure. Moreover, the stability of the allyl glycoside allows quite a simple and flexible protecting group strategy. This latent-active glycosylation strategy was also applied by Boons to the synthesis of libraries of both linear [31] and branched trisaccharides [32]. The main drawback of the procedure is the preparation of the starting glycosides [ 301: These are obtained from acetobromosugars and 3-buten-2-01 using HgBr2/ HgCN2 as promoters, a procedure quite laborious and problematic from a health and environmental point of view. Boons recently tried to improve the preparation of substituted allyl glycosides by enzymatic means, using almond 0-glycosidase [33]. Another type of vinyl glycoside was used by Schmidt for glycosylation reactions [34].The glycosyl donor was obtained by treatment of a sugar hemiacetal with ethyl phenyl propiolate and sodium hydride. Glycosylation reactions were performed using TMSOTf as a promoter and demonstrated that such vinyl ethers at the anomeric position are not very good leaving groups. Surprisingly, Takeda et al. proposed very recently the same method [35] without mentioning Schmidt's paper. The only difference is the procedure used for the preparation of the vinyl glycosides which are obtained by addition of tetra-0-benzylD-glucopyranose to some alkynyl esters and ketones in the presence of tributylphosphine. The glycosylation reaction is carried out in the presence of molecular sieves and requires 10 equivalents of TMSOTf, thus confirming the relatively low reactivity of such donors.
9.2 I-Hydroxy Sugars
...
111
209
B n O & o q BnO
Me0
MeoOCH3
81
82
Scheme 16. Reagents: i) Nysted reagent, THF, CH2C12, 75%); ii) TfOH, CHZC12, -2O”C, 67%; iii) Raney-Ni, yield very much batch-dependent (50-90%).
Finally, some examples of the use of bicyclic systems in which the anomeric vinyl ether is partly or completely contained in a ring are present in the literature [36]. Franck described the use of cycloaddition products obtained from glycals as donors (e.g. 78, Scheme 16). Compound 78 exhibited low reactivity, so it was modified by introduction of a second double bond using the Nysted reagent. The vinyl glycoside 79 so obtained revealed excellent glycosyl donor properties when activated with Lewis or protic acids, the best results being obtained with TfOH, to give fair to good yields of P-linked products. Raney-Ni desulfurization afforded the 2-deoxy-Pglycosides. The difference in reactivity between 78 and 79 introduces the possibility of using these compounds as latent-active donors [ 3 7 ] . Hetch et al. used cyclic ketene acetals for glycosylation reactions [38]. The donor is obtained from a glycosyl bromide by treatment with silver oxide and Hunig’s base and is activated with CSA in the presence of the acceptor to give an orthoester. TMSOTf catalyzed rearrangement of such an ortoesther gave the desired disaccharide.
9.2 l-Hydroxy Sugars The use of l-hydroxy sugars for the formation of glycosidic bonds would be one of the most fascinating glycosylation methods, owing to the simplicity and the stability of the “donor”. Of course, the OH group itself is not a good leaving group and needs to be activated. The simplest way for the activation of the OH group is the use of an acidic promoter in order to have a water molecule as leaving group. In this case, better results are obtained in the presence of a water scavenger. Other methods involve the transformation of the anomeric hydroxy group into an intermediate glycosyl donor, which is used without isolation in glycosylation reactions.
210
9 Other Methods of Glycosylation
\ OTBDPS
+ +
i
Ho H AllO O%
OTBDPS
OH 83
,O 84
AllO H O &
*'
Scheme 17. Reagents: i) pyridinium p-toluenesulfonate, CH2C12, azeotropic dehydration, 73%.
Finally, the activation at the anomeric position through the Mitsunobu reaction will be considered. Besides the 1-hydroxy sugars, the l-O-silylated glycosides used as glycosyl donors will also be included in this section. The classification into the following sub-sections is in some senses questionable, but may help the reader to find a selected topic more easily. 9.2.1 Acidic Activation Apart from the obvious problems given by the water formed during the reaction, the direct transformation of l-hydroxy sugars into glycosides suffers of two other main problems: Their poor reactivity as donors and the strong tendency to selfcoupling to give trehalose like structures. Such problems are easily overcome in the formation of simple glycosides when the alcohol acceptors can be used in large excess (i.e. they are sufficiently volatile and cheap). After Fischer proposed the acid catalyzed formation of simple glycosides [39], some other methods appeared in literature [40]. Wessel [41] used the triflic acid for the formation of simple glycosides using the alcohol as solvent. The method was improved by Fraser-Reid [42] for the preparation of pentenyl glycosides, easily obtained using DMSO as solvent, TfOH and only 3 equivalents of alcohol. Glycosylation with sugar acceptors was performed on the Kdo precursor 83 as donor (Scheme 17), using pyridinium p-toluenesulfonate as acid catalyst and removing water either with 4 A ms or by azeotropic dehydration [43]. Glucuronic acid glycosides have been obtained by treatment of the methyl 2,3,4tri-O-acetyl-D-glucuronate with alcohols and phenols in the presence of TMSOTf [44]. According to the reactivity of the alcohol, the reaction is believed to proceed via a SN2 like reaction on a silylated hydroxy group to give a-glycosides, or by formation of an oxonium ion to produce the P-anomers owing to the participation of the acetate. The reactivity of N-acetyl-D-glucosamine and galactosamine in hydrogen fluoride solutions was also studied [45]. More recently Mukaiyama developed a method for the glycosylation of 2,3,5-triO-benzyl-D-ribose using a catalytic amount of trityl salts as promoters, in the pres-
9.2 I-Hydroxy Sugars
BnO&o, BnO
f
B
$
o
G
Brio OMe 87
86
2 11
pla ,6,84
BnBnO O&%O+ Brio OMe
88
Scheme 18. Reagents: i) 5 mol%,TrB(CsF5)4, benzene/toluene 10: 1, Drierite, O T , 83%.
ence of Drierite as a dehydrating agent [46]. Without any additive the p-anomer was formed preferentially, while the presence of lithium salts reversed the stereoselectivity leading to a large predominance of the a-anomer. The same procedure was also applied to the synthesis of 2-deoxy-~-glucopyranosides88 (Scheme 18)
PI.
A very interesting possibility has arisen in recent years, namely the use of solid acidic catalysts both as a promoter and as a dehydrating agent. Such an approach, when applicable, has many advantages: The procedure is simple and environmental friendly, the catalysts are usually quite cheap and reusable. The main limitation to be noted from literature data, is that these catalysts are efficient in glycosylation of donors which form the oxonium ion quite easily, such as deoxysugars. Montmorillonite K-10 was used for glycosylation reactions with simple alcohols and sugar acceptors [48]. In particular, olivosides were obtained using this Lewis acid solid catalyst. Another recent example of dehydrative glycosylation of 1-hydroxy sugars was performed [49] using an heteropoly acid, H4SiW12040 (Scheme 19). Different glycosyl donors and acceptors were used, with the following results: The glycosylation of deoxy sugars requires small amounts of promoter, whereas a larger amount is needed for benzylated hexoses; the formation of simple glycosides is described for all the donors, while the preparation of disaccharides, such as 90, is performed only on deoxy sugars; the glycosylation reaction is usually quite stereoselective giving mainly the a-anomer. Although at an initial stage, such promoters deserve attention because they can also be used for the activation of less reactive glycosyl donors. Acidic Activation With Additional Reagents
Several other attempts to activate 1-hydroxy sugars with cationic activators or Lewis acid catalysts have appeared in literature, in which a second active species is generated in situ by additional reagents. Inanaga et al. used catalytic amounts of Yb(OTf)3 and methoxyacetic acid as promoters to obtain glycosides of perbenzylated glucose 91 and ribose in excellent
&
BnO BnO
OH
86
+
8'
N3
HO
89 N3
p/a 10:90
90
Scheme 19. Reagents: i) heteropoly acid (H4SiW12040) 10 Wt%, CH3CN, 2 5 T , 82%.
212
9 Other Methods of Glycosylation
BnO%oH BnO
BBnOn OBn
91
O
q
-
67 Brio OMe
i
BBnO n o &OBn o ~ o M ~
2515
68
Scheme 20. Reagents: i) Yb(OTf)s, CH30CH2COOH 10 mol% each, CH2C12, ms 4A, reflux, 99%.
yield (Scheme 20) [50]. In the same conditions it is also possible to obtain thioglycosides. Although the role of methoxyacetic acid is not clear, no reaction took place when it was absent. The use of trimethylsilyl derivatives in the presence of various Lewis acids is also described. Susaki has published [51] a glycosylation reaction promoted with TMSCl and Zn(0Tf)Z. In a similar way, Mukaiyama described [ 521 successful catalytic glycosylations using (TMS)20 in combination with four different Lewis acids, again studying the effect of LiC104 on the stereoselectivity of the reaction. In a further development the same group studied the use of Sn(0Tf)z with various trimethylsilyl derivatives [ 531. 9.2.2 Dehydrative Glycosylation
Starting from observations from different fields of organic synthesis, Mukaiyama introduced the use of dehydrating reagents for the activation of the anomeric position of 1-hydroxy sugars. In a first example [54]a diphosphonium salt, generated by reaction of a trialkyl- or a triarylphosphine oxide and triflic anhydride allowed the synthesis of 1,2-cis-ribofuranosides when used in combination with a base (Hunig's base),1!4 ms and an appropriate acceptor. Similar results were obtained when the phosphine oxide was substituted with an oxotitanium derivative [ 551 in conditions similar to those previously described. A third paper [56] introduced the use of a different metal derivative in order to improve the already good yields and selectivities on the glycosylation of 2,3,5-tri-O-benzylribose92. Excellent results were obtained with diphenyltin sulfide and triflic anhydride in the presence of LiC104, with CsF as an acid scavenger, using alcohols or trimethylsilyl ethers as acceptors (Scheme 21). It is worthy of note that most of the tin reagent is recovered at the end of the reaction.
Scheme 21. Reagents: i) diphenyltin sulfide, Tf20, CsF, CH2C12, 0°C; ii) -23"C, 99%.
1% 1
6-
RO RO
OH
1
OR 91R=Bn 94R=Bz
- OTf
RO
OR
9.2 I-Hydroxy Sugars
RROO
&
o GBnO OR
213
Brio OMe
96a R = Bn, 85%, alp 6040 %b R = Bz, 72%, p only
Scheme 22. Reagents: i) diphenylsulfoxide, Tf20, 2-chloropyridine, toluene/CHzClz 3 : 1, -78123 "C.
More recently, Gin et a]. developed [57] the glycosylation with 1-hydroxy sugars using diphenyl sulfoxide and triflic anhydride to give glycosides and disaccharides in good yields. Benzyl or benzoyl protected glycosyl donors (91 and 94, Scheme 22) reacted equally well, and the procedure even allowed the N-glycosylation of an amide. In the Presence of the Acceptor From the Beginning
In spite of their efficiency, the above procedures involve the prior activation of the anomeric position of the donor, followed by the addition of the acceptor. The selective activation of the donor in the presence of the acceptor would allow the presence of both reagents from the beginning of the reaction. Koto et al. introduced [ 5 8 ] a glycosylation reaction in which a mixture of both the donor and the acceptor, together with CoBr2, is treated with methanesulfonic acid to give the glycoside or disaccharide through the in situ formation of a glycosyl bromide. The procedure was further modified, in order to obtain a better control of the stereoselectivity, with Et4NC104 [59] or using TMSBr instead of methanesulfonic acid [60]. The same group also introduced the activation of the anomeric position of 1-hydroxy sugars with p-nitrobenzenesulfonyl chloride, silver triflate and Et3N, probably through the formation of an anomeric sulfonate intermediate [61]. From this procedure glycosides and disaccharides were obtained with predominant pconfiguration. The addition of N,N-dimethylacetamide reverted stereoselection to predominant formation of the a-anomer of 68 (Scheme 23) [62]. A simplified procedure, described by Szeja [63], involves in situ formation of glycosyl sulfonates under phase-transfer conditions followed by alcoholysis to give mainly a-glycosides. Recently Koto et al. proposed [64] another dehydrative glycosylation in which DAST is used to activate the donor in the presence of the acceptor, Sn(OTf)Z,
BBnOn O g o H OBn 91
+
BBnOn 61
O q Brio OMe
alp 13:2?
Scheme 23. Reagents: i) NsCl, AgOTf, dimethylacetamide, Et3N, CHzCIz, -40 "C, 88%.
214
9 Other Methods of Glycosylation
B n o S O H BnO
1
95
"Me,
B n*;BnO C -) Brio OMe ma
alp 82:18
Scheme 24. Reagents: i) (CCl3CO)20, TMSC104, ms 5A,EtzO, rt, 91%.
Bu4NC104, and Et3N. The reaction probably proceeds through the formation of a glycosyl fluoride, which is further converted into an anomeric triflate and eventually to the glycoside. Finally, an even more recent paper [65] described a novel dehydrative glycosidation using trifluoro- or trichloroacetic anhydride in the presence of TMSC104 and 5 A ms with preferential formation of the a-anomer of 96a (Scheme 24). The anhydride is believed to form the anomeric ester with the essential contribution of the 5 A ms as acid scavengers. The ester is then activated by the TMSC104 to give glycosides such as 96. 9.2.3 Mitsunobu Glycosylation
A particular method for the anomeric activation of 1-hydroxy sugars is the Mitsunobu reaction which has mainly found application for the synthesis of aryl glycosides. Following from an earlier publication [66], Garegg described [67] the use of this reaction for synthesis of aryl 8-D-mannopyranosides. Lubineau studied [68] the influence of the pK, of the acid or phenol in the formation of aryl and acylglycosides derived from three pesticides. Mitsunobu reaction was employed by Ogawa in the synthesis of the antibiotic hygromycin A [69]. A more extensive study was made by Roush in a project devoted to the synthesis of analogs of aureolic acid family [70], which contain oligosaccharidic chains constitutes of 2,6-dideoxypyranosides. It was observed that the glycosylation reaction proceeds through an SN2 mechanism, as the stereochemistry of the products reflect the anomeric configuration of the starting hemiacetal. Moreover, the reaction was also performed using 2-thiophenyl derivatives as donors (Scheme 25). The preferential formation of 8-glycosides, such as 99, seems to be due to the effect of 2-thiophenyl substituent on the anomeric ratio of 97 and not to a neighboring group-participation. In a recent paper, Dondoni et al. obtained [711
Scheme 25. Reagents: i) 2-naphthol, Ph3P, DEAD, toluene, O T , 74%.
9.2 1-Hydroxy Sugars
AcO 100
OSiMe,
OMe
BzO
OAc
101
i h -
215
AcO
OBz
OAc lo'
BzO
OMe OBz
Scheme 26. Reagents: i) TMSOTf, CH2C12: -50&-30"C, 76%.
glycosylated calix[4]arenes under Mitsunobu conditions. Mitsunobu reaction was also performed between glucuronic acid and phenolic chromium tricarbonyl complexes [72]. The phenol complexation is used in order to increase its acidity. Glycals ' of were shown to react under Mitsunobu conditions through an S N ~displacement the allylic hydroxy group [73]. Anomeric 0-acylation was exploited by Amos Smith within the synthesis [74] of phyllanthoside, an antineoplastic glycoside containing a (3-glycosyl ester linkage. Mitsunobu reaction has rarely been used for the synthesis of alkyl glycosides owing to the lower acidity of an alcoholic hydroxy group. An old example describes the formation of alkyl glycosides and disaccharides using, besides triphenylphosphine and DEAD, a mercuric salt [75].Descotes et al. studied the reactivity of 1,3,4,6-tetra-O-benzoyl-~-fructofuranose in Mitsunobu conditions with different nucleophiles including alcohols [ 761. Finally, Nicolaou exploited [77] the Mitsunobu reaction for the formation of an 0-glycosyl hydroxylamine in the synthesis of calicheamicin oligosaccharides. 9.2.4 1-0-Silyl Glycosides
The use of 1-0-silylglycosides as glycosyl donor was introduced by Tietze for the synthesis of aryl glycosides [78].Reaction of trimethylsilyl glycosides, protected as ester or benzyl ethers, with phenyltrimethylsilyl ethers in the presence of TMSOTf gave good yields of aryl glycosides. The method was developed by Glaudemans (Scheme 26) [79],in a version in which the hydroxy group of the acceptor was protected as a t-butyldiphenylsilyl ether (e.g. 101) and the 1-0-TMS derivative or galactopyranose 100 used as a donor, in the of 2,3,4,6-tetra-O-acetyl-~-glucopresence of TMSOTf. In this way it was possible to avoid the prior deprotection of the acceptor. Simple glycosides were obtained from the reaction between 1-0-TMS glycosides and alcohols using BF3 . OEt2 as promoter [go]. Kolar et al. used silylated glycosyl donors derived from 2,6-dideoxysugars for the synthesis of anthracycline derivatives [811. 2-Deoxy- and 2,6-dideoxy silylglycosides were also studied by Priebe et al. [82], particularly for their conversion into other synthetically useful anomeric derivatives. Starting from the observation of a transient anomeric silyl derivative in a previously studied glycosylation reaction on 1-hydroxy sugars [56], Mukaiyama applied a similar procedure (a catalytic amount of a Lewis acid, diphenyltin sulfide) to 1-0-silyl derivatives of furanoses and pyranoses. Thus 1,2-
2 16
9 Other Methods of Glycosylution
trans- or 1,243 ribofuranosides, and 1,2-& glucopyranosides were obtained using the appropriate reaction conditions [83]. Finally, a very recent work described the synthesis of spacer-linked neodisaccharides of L-daunosamine [ 841. In a glycosylation reaction on a monosilylated 1,4-butanediol the main product obtained was the monoglycosylated unprotected diol. This may suggest either a desilylation due to the acidic conditions or a higher reactivity of the silylated oxygen of the acceptor.
9.3 Esters and Related Derivatives 9.3.1 Esters
Among the various glycosyl derivatives, glycosyl esters are the most readily accessible and frequently serve as useful precursors for other glycosyl donors such as glycosyl halides. Despite the impressive development of new and efficient glycosylation methods, described in other chapters of this book, glycosyl esters still offer a good alternative as glycosyl donors, especially when more classical procedures fail. In this section, we will focus mainly on the 1-0-acyl donors introduced in the last decade, even if some older methods are mentioned. For a more detailed description of the most classical methods we refer to ref. [86] and references cited therein. The first example of 1-0-acyl sugars as glycosyl donors dates back to 1933, when Helferich described the stereoselective syntheses of both a and fl glucosides using zinc chloride or p-toluenesulfonic acid as a catalyst and 1,2,3,4,6-penta-O-acetyl-BD-glucose as a donor [85]. Since then, several applications of 1-0-acyl donors, namely 1-0-acetyl, l-O-(2’-substituted)-acetyland 1-0-benzoyl sugars, have appeared in the literature [86]. Furthermore, many different Lewis acids have been used as effective promoters in the glycosylation reactions, including tin tetrachloride [87], ferric chloride [88], BF3 . OEtz [89] and TMSOTf [90]. Mukaiyama made an extensive contribution to this area. First, he showed that 2,3,4,6-tetra-0-benzyl-l-O-bromoacetyl-fl-~-glucopyranose could be stereoselectively converted into a-glucosides with primary and secondary alcohols using trityl perchlorate as an activator [91]. This method was further developed using different promoters and applied to the synthesis of 0-glycosides and disaccharides from 1-0acyl donors and 0-trimethylsilylated nucleophiles (Scheme 27).
cat.
PO
PO
X=H, halogen, -OR
Scheme 27. Glycosylations from 1 -0-acyl donors and 0-trimethylsilylated acceptors.
9.3 Esters and Related Derivatives
r (OBn
103
1
I
L
I
,OBn B BnOn
B BnO n o S O A c BiO
217
O
G
BnO 105
ClO,
J
TMSOR
(OBn
TMSOAc
TMSCIO,
Scheme 28. General mechanism proposed for glycosylation reactions promoted by MCI,-AgC104 systems.
Thus, D-ribofuranosides with high a-stereoselectivity were obtained with the combined use of a catalytic amount of tin tetrachloride and tin(I1) triflate and a stoichiometric amount of lithium perchlorate using 1-0-acetyl-2,3,5-tri-O-benzylp-D-ribofuranose as a glycosyl donor and 0-trimethylsilylated alcohols or monosaccharides as acceptors [92]. Moreover, highly stereoselective syntheses of 1,2-cis glucosides from 1-0-acetyl glucose having a non participating group have been achieved using a combination of a Lewis acid and silver perchlorate; several Lewis acids corresponding to the general formula MCl, have been screened, tin tetrachloride and gallium trichloride ~ being the most effective [93]. The reaction was assumed to proceed via the S N type mechanism and the species such as 104 were postulated to be the active catalyst, generated from MCl, and silver perchlorate (Scheme 28). The activation of the anomeric acetoxy group affords the intermediate oxonium ion 105 stabilised by the perchlorate ion. This large counter anion blocks the p-face of the anomeric centre; silyl alkoxide attack from the a-face predominantly produces the a-glucoside along with trimethylsilyl perchlorate and MCln-lOAc, from which the catalyst is regenerated. Further, the effect of the leaving group on the stereoselectivity of this glycosylation reactions was explored by screening various l -0-acyl-2,3,4,6-tetra0-benzyl-D-glucopyranoses with tin tetrachloride and silver perchlorate as the catalytic system. The 2-(2-methoxyethoxy)acetoxygroup gave the best results. Using this glycosyl donor, either c1 or p glucosides were obtained with high stereoselectivity [94], depending on the catalytic system. Mainly a-glucosides were formed employing tin tetrachloride and silver perchlorate in diethyl ether at -5 "C (a/p ratios ranging from 96/4 to 98/2), whereas P-glucosides were achieved with tetra-
218
9 Other Methods of Glycosylution
chlorosilane-silver perchlorate in acetonitrile at - 10 "C (a/p ratios ranging from 9/91 to 1/99). It is worthy of note that the stereoselectivities of the above glycosylations were demonstrated to be independent on the a/p ratio of the starting substrate, confirming the formation of the oxocarbenium cation intermediate and therefore the S N type ~ mechanism. Mukaiyama's protocol was successfully applied to the synthesis of other pglycopyranosides and a or p furanosides. 1-0-Acetyl-2,3,4,6-tetra-0-pivaloyl-~-~-glucopyranose afforded in high yields 0glucosides and disaccharides using methyltrichlorosilane and silver perchlorate as the catalytic system [95].2-Amino-2-deoxy-~-~-glucoand galactopyranosides, which are still challenging tasks in carbohydrate chemistry, have been prepared in high yields from the corresponding 1-0-acetyl donors either using trichlorotin triflate or tin(I1) triflate as a catalyst, the latter being more advantageous for practical reasons [96]. The p configuration of the newly formed glycosidic linkages was ensured by exploiting the participation of N-2,2,2-trichloroethoxycarbonyl(Troc) as a neighbouring group. 2,3,5-Tri-O-benzyl-l-O-iodoacetyl-~-ribofuranose (alp = 1/5) gave the best results as a glycosyl donor in the synthesis of both a and p ribofuranosides: the combined use of silver perchlorate and lithium perchlorate afforded ribofuranosides with high a stereoselectivity. On the other hand, P-ribosides were obtained with different catalytic systems, such as the combination of silver perchlorate and diphenyltin sulfide. The milder tin(I1) chloride also gave high yields of p-ribosides, when used in combination with 2 mol% of tetrachlorosilane [97]. More recently, Reynolds described the syntheses of D-arabinofuranosides, Dgalactofuranosides and L-rhamnopyranosides viu tin tetrachloride mediated glycosylation of the corresponding l -0-acetyl donors [98]. Finally, the combination of dimethyl dichlorosilane and silver perchlorate was proved to be efficient in the a-sialylation of the 6-OH of a P-D-galactopyranosyl acceptor, using a 2-0-acetylated sialyl donor: the disaccharide product, a useful building block for the synthesis of sialyloligosaccharides, was obtained in 76% yield in propionitrile at -28 "C (alp = 83/17) [99]. As stated above, the 1-0-acetyl donors may still be an effective alternative to classical methods, as recently witnessed by the synthesis of a kedarcidin chromophore analogue (Scheme 29). In a key step of the synthesis, the alcohol 106 was
Kedarosamine
106
Scheme 29. Reagents: i) BF3 . OEtz, toluene, 0 "C, 80%.
107
9.3 Esters and Related Derivatives
219
AcO OAc
OAc 108
109
110
Scheme 30. Reagents: i) Lewis acid, CH2C12, -1O"C, 76-950/0; ii) Bu3SnH, AIBN, toluene, 1 lO"C, 80-95%.
glycosylated with the N-protected 1-0-acetyl kedarosamine and afforded the uglycoside 107 in 80% yield. It is worthy of note that the use of either the glycosyl imidate or the phenyl thioglycoside of the kedarosamine led to lower yields of the product [ 1001. An efficient synthesis of P-glycosides of 2,6-dideoxy sugars, occurring in many biologically relevant antibiotics such as digitoxin, venturicidins and olivomycin, was achieved using acetyl 3,4-di-O-acetyl-2,6-anhydro-2-thio-~-~-altropyranoside 108 as a glycosyl donor (Scheme 30). Several Lewis acids promoters and solvents were examined. The best results, in terms of yields and stereocontrol, were obtained when the glycosylations were carried out in dichloromethane in the presence of TMSOTf (1.1 eq.) at -10°C. The 2,6-anhydro-2-thio bridge of the obtained p-glycosides can be easily converted into the corresponding 2,6-dideoxy derivatives either by standard hydrogenolysis using Raney-Ni as a catalyst or by the treatment with tributyltin hydride and AIBN in toluene at 110 "C [loll. Lanthanoid(II1) triflates catalyzed glycosylations with 1-O-methoxyacetyl sugars as donors have been described. After a preliminary screening of many lanthanoid triflates, ytterbium triflate, Yb(OTf)3, was selected as the catalyst and acetonitrile as the reaction solvent. By this method, a variety of glycosides including disaccharides and thioglycosides were prepared in good to excellent yields. The stereoselectivities are sometimes modest, but they could be improved by increasing the reaction temperature to 53 "C. Under these conditions, the thermodynamically more stable p-isomer was obtained as the major product [102]. The Nicholas reaction [lo31 has been successfully exploited for a novel glycosylation reaction based on the internal delivery concept (Scheme 3 la). The suitably O-protected glycopyranoside derivative 111 bearing the cobalt-complexed 4-alkoxy6-phenyl-5-hexynoate residue at the anomeric position affords the corresponding propynyl cation 112 under Lewis acid catalysis. The intermediate 112 collapses spontaneously to form the oxonium ion delivering the cobalt-complexed y-lactone 113. The oxocarbenium cation is then intramolecularly captured by its counter alkoxy anion leading to glycoside 114. The rather laborious preparation of the starting substrates for the investigation of this glycosylation reaction is described in Scheme 3 1b. Various glycosyl "donors" of the gluco, galacto and manno series were synthesized, either O-benzylated or 67 was O-benzoylated, whereas only methyl 2,3,4-tri-O-benzyl-cr-~-glucopyranoside used as nucleophile.
220
9 Other Methods of Glycosylation
Ph b)
TBDPSO
BnO
115 61
117
(OBn
pp. (CO)co
k$co)3
0
118
alp 9191
113
Scheme 31. Reagents: i) CO~(CO)S, Et20, rt; ii) 67,BF3 . OEt2, CH2C12, 0°C; iii) CAN, MeOH, rt, overall yield 74%; iv) TBAF, THF, rt, 92%; v) PDC, DMF, rt; vi) 91, DCC, DMAP, HOBt, Et3N, CH2C12, rt; vii) Co2(CO)8, Et20, rt; viii) TMSOTf, EtCN, -65 "C, 740/0.
As an illustrative example, compound 117 was synthesized from 115 and 116. The treatment of 117 with Coz(C0)~gave the corresponding cobalt complexes, which afforded the disaccharide 118 with high p-stereoselectivity. The y-lactone 113 was also recovered in high yields [ 104). The p-nitrobenzoate was successfully employed as a leaving group in the synthesis of the biologically important disaccharide H, 2-O-(a-~-fucopyranosyl)-~-galactopyranose. Although the p-nitrobenzoate is not an excellent leaving group, in the case of the highly reactive 2,3,4-tri-O-benzyl-t-fucosyl derivative (a/p = 5/1) it was efficiently activated using BF3 . OEt2 in the presence of 1,3,4,6-tetra-O-acetyl-a-~galactopyranose as a glycosyl acceptor. The disaccharide H was obtained in yields
9.3 Esters and Related Derivatives
221
OBn-p-CI p-CBnO p-CBnO p-ClBnO 119
HO 0
BnO OMe
87
p-CBnO p-CBnO 120
alp 6194
Scheme 32. Reagents: i) TrB(C6F5)d 10 mol%, BuCN-CH2C12 2/1, Drierite, -23 "C, 97's.
ranging from 70Y0to 79%, and with a a/p ratio of 11 : 1, from which crystallization gave pure a anomer in 40-50% yield [ 1051. 9.3.2 Sugar Carbonates and Derivatives Glycosyl phenyl carbonates as glycosyl donors have been reported by Mukaiyama et al.: after activation in the presence of an acceptor, the leaving group decomposes into carbon dioxide and phenol. First, efficient a-sialylation of a 6-O-unprotected galactosyl acceptor was achieved with a 2-O-phenyl carbonate derivative of Nacetylneuraminic acid, under dimethyl dichlorosilane-silver perchlorate catalysis [99]. Glucosyl phenyl carbonate 119 was activated with a catalytic amount of trityl tetrakis(pentafluoropheny1)borate to provide (1 +6), (1'3) and (1+4)-linked disaccharides in very good yields and with excellent p-stereoselectivity (Scheme 32). Interestingly, the reaction can be performed on alkyl thioglycoside as acceptors, giving access to one-pot sequential syntheses of trisaccharides upon the activation of the alkyl thio group of the disaccharide product [ 1061. An attractive strategy for the formation of glycosidic bonds is based on bridging donor and acceptor. The decarboxylative glycosylation method, developed by Ikegami and co-workers [107], is an example. Donor and acceptor are connected through a carbonate bridge and the glycosidic bond is obtained by internal removal of carbon dioxide promoted by a Lewis acid (Scheme 33a). The mixed carbonates were prepared as a mixture of anomers by l-O-acylation of pyranoses (glucose and galactose) with activated alkyl carbonates of alcohols or monosaccharides (glucose and galactose). The decarboxylative glycosylations were best promoted by stoichiometric amounts of trialkylsilyl triflates (TMSOTf or TBDMSOTf) and the choice of the solvent was crucial. The highest yields were obtained using toluene and mesitylene. The stereoselectivities were only modest when a non participating group was present, furthermore, the anomeric ratio of the starting materials was not reflected in the products, as deduced starting from anomerically pure carbonates. Although an intramolecular mechanism was initially claimed, Schmidt et al. [ 1081 demonstrated with competition experiments that the acid promoted decarboxylative glycosylation follows an intermolecular course. Good a-stereoselectivities were obtained applying a catalytic version of the decarboxylative glycosylation, using the combinations of tin tetrachloride-silver perchlorate or CpzHfCl&ver perchlorate as promoters and EtzO as a solvent [ 1091. On the other hand, very high p-stereoselectivities were reported in the decarboxylative glycosylations of O-acyl protected
222
9 Other Methods of Glycosylation
BnO
123 121
Scheme 33. Reagents: i) K2C03, toluene, 88%; ii) TMSOTf, CH2C12, rt, 89%, only 0.
sugar carbonates. Using 0-acyl protected donors (i.e. 94), selective synthesis of mixed P-carbonates (122) was achieved from 0-succinimidyl-activated carbonates of various acceptors, mainly in the presence of K2CO3 (Scheme 33b) [ 1lo]. Ley and co-workers reported that various carbohydrate derivatives react readily via the C-1 hydroxy group with carbonyldiimidazole or thiocarbonyldiimidazole in ether (EtlO or THF) to give the 1-imidazolylcarbonyl-or thiocarbonyl- glycosides 124 and 125, respectively (Scheme 34). These compounds were reacted with alcohols and glycosyl acceptors in the presence of zinc bromide or silver perchlorate affording the corresponding glycosides in good yields. When the reactions were carried out in diethyl ether as a solvent, a glycosides were obtained with good stereoselectivity, whereas in dichloromethane the quantity of the anomers was increased, suggesting that a SN1 type process is operating [ 1111.
124
0
Scheme 34. 1 -1midazolylcarbonyl- (1 24), 1 -imidazolylthiocarbonyl- (125) and 2-pyridincarboxyl(126) glycosides.
9.3 Esters and Related Derivatives
223
The authors suggest that the displacement is facilitated by chelation of the metal atom to the carbonyl oxygen (or thiocarbonyl sulfur) and the N-3 of the imidazolyl group. This can be related to the remote activation concept originally introduced by Hanessian [ 1121. The same strategy, i.e. the introduction of a bidentate ligand as a leaving group, was exploited using glycosyl2-pyridinecarboxylates 126 as glycosyl donors (Scheme 34). These compounds were easily prepared by esterification of the anomeric hydroxy group of the sugar with picolinoyl chloride in the presence of triethylamine. Mild Lewis acids, such as tin(I1) triflate and copper(I1) triflate, were found to be more effective than other reagents, such as BF3 . OEtz and TMSOTf, in promoting the activation of the 2-pyridinecarboxy group. Interestingly, in preliminary experiments carried out both with alcohols and monosaccharides as nucleophiles, the stereochemical outcome of the glycosylation reactions was observed to be dependent on the choice of the metal salt and the solvent [ 1 131. This procedure was further applied to more glycosyl donors, namely mannopyranose, 2-azido-2-deoxy-glucopyranose and 2-deoxy sugar derivatives [ 1141, demonstrating that the stereochemistry of the glycosylation reactions was influenced not only by the promoter and the solvent, but also by the structure of the donor. Glycosyl 2-thiopyridylcarbonates have been described as effective glycosyl donors by Hanessian et al. However, this leaving group has been used mainly for the formation of N-glycosidic linkages in the synthesis of 1,2-cis and 1,2-trans nucleosides [115]. A new glycosylation method based on the mild activation of 0-glycosyl N-ally1 carbamates with soft electrophiles has been described by Kunz and co-workers [ 1161. This activation is similar to that of 0-glycosyl4-pentenoyl esters reported by Fraser-Reid [ 1171, with formation of an oxazolidinone instead of a lactone as the leaving group at the anomeric center. Anomeric N-ally1 carbamates were readily obtained from anomerically unprotected carbohydrates by reaction with ally1 isocyanate. Among the electrophiles used for the activation, the methyl bis-methylthiosulfonium hexachloroantimonate (TMTSB) was found to be the most effective in promoting the glycosylations. Various alcohols and O-unprotected sugars were used as acceptors, and the corresponding glycosides were obtained in good yields but with low stereoselectivity, except in cases where neighbouring-group participation occurred. The authors applied this procedure to the synthesis of the mucine core trisaccharide 131 showing that many different protecting groups, such as alkyl and silyl ethers, esters and acetals, remain unaffected under the mild conditions required for the TMTSB-promoted activation of 0-glycosyl N-ally1 carbamates (Scheme 35) [ 1 181. 9.3.3 Orthoesters and Oxazolines The first appearance of 1,2-0-orthoesters as glycosyl donors in glycosylation reactions was in 1971. Kochetkov et al. reported a teut-butyl orthoester as a donor upon activation with 2,6-dimethylpyridinium perchlorate [ 1 191 and extensively
224
9 Other Methods of Glycosylation Ph
I
127
A c AcO
C
Y
128
&
oms+ yo
&
AcO
N3
129
NPht
130
129
... AcO
0
oms AcO AcO 131
Scheme 35. Reagents: i) TMTSB, CH2C12, -15°C + -5"C, 67%; ii) HS(CH&$H, CH2C12, 83%; iii) TMTSB, CH2C12, -29°C + -1O"C, 48%.
PTSA,
explored this methodology introducing the 1,2-0-(1-cyanoethylidene) group, whose activation occurred under milder conditions. Other orthoester derivatives, such as 1,2-0-[1-(p-methylpheny1)thiolethylideneand 1,2-0-[I-"-( 1-phenylethylidene)amino]oxyl]-2,2-dimethylpropylidene, were also described [861. A recent useful application of the 1,2-0-(1-cyanoethylidene) glycosyl donors was found in the highly regioselective glycosylations of primary-secondary ditrityl ethers acceptors. Quite surprisingly, secondary trityl ethers were essentially more reactive than the primary ones, using cyanoethylidene derivatives of mannose and galactose as donors. All glycosylations gave the 6-0-trityl derivatives of 1-2 and 1-4 linked disaccharides in good yields and very high regioselectivity, even in the case of such a poorly reactive acceptor as the 0-4 of galactose. This unusual regioselectivity was attributed to the formation of an early transition state, where electronic factors, i. e. electron density at oxygen, rather than steric factors will become determinant [ 1201. There are two main advantages in using 1,2-0-orthoesters as glycosyl donors: -
-
the orthoester ring directs the nucleophilic attack to the back side, leading to 1,2trans glycosides; the nucleophilic opening of the orthoester ring (for example, with a simple alcohol) allows the contemporary and orthogonal protection of the C-1 and C-2 functionalities of the sugar.
During the last decade sugar orthoesters have been widely employed. However, their use has been more as protecting groups rather than as glycosyl donors in oligosaccharide synthesis. The new achievements concerning glycosylation reactions with sugar orthoesters exploit their chemistry in a quite different way. Recently, Kong and co-workers reported a new methodology for highly regio-
225
9.3 Esters and Related Derivatives
(OAC
/OH
(OAc AcO AcO Br 72
+
AcO
Ho%
BnO
AcO
-
Me (OH
OMe
y:o%
132
OMe
OMe
ii
7
E
133: R = H 1 3 4 R = Ac
AcO
TMSOTf
OMe 135:R=H 136R=Ac
Scheme 36. Reagents: i) AgOTf, 2,4-lutidine, CH2C12, rt, 95%; ii) AczO, pyridine, qu.; iii) TMSOTf, CH2C12, O"C, 78%.
and stereoselective synthesis of oligosaccharides. The procedure is based on the preparation of sugar-sugar orthoesters by coupling acetobromosugars with unprotected or partially protected sugar acceptors in high yields and excellent selectivity. Subsequent rearrangement in the presence of catalytic quantities of TMSOTf yields the oligosaccharide [121]. In a typical example, acceptor 132 was regioselectively converted into orthoester 133 from acetobromoglucose 72 using silver triflate in the presence of 2,4-lutidine (Scheme 36). Rearrangement of 133 with a catalytic amount of TMSOTf selectively afforded disaccharide 135 in 78% yield. The same compound was obtained in 80% yield when 4-0-acetylation of 133 ( 4 134) was performed prior to the rearrangement. These results are mainly due to steric factors (Scheme 36). The less hindered hydroxy group attacks the acyloxonium carbon of A generating orthoester 133 which, during the following rearrangement, is fragmented again into A and the trimethylsilylated acceptor: finally, a regio- and stereoselective backside attack on the C-1 of A gives disaccharide 135. Many other examples reported by the authors demonstrate the excellent regioselectivity in the orthoester formation. This new method was widely explored and applied to the synthesis of complex branched oligosaccharides. First, a variety of acetobromosugars and glycosyl acceptors were examined in terms of their ability to afford regioselective orthoester formation. It was found that both the regio and the stereoselectivity were dependent on the acceptor rather than the donor structure, according to the mechanism depicted in Scheme 36. Indeed, acetobromo derivatives of glucose, galactose,
226
9 Other Methods of Glycosylation
mannose, rhamnose, lactose and maltose invariably led to highly regioselective formation of the orthoesters with various acceptors [121], [122], [123]. On the other hand, the behaviour of the glycosyl acceptors under the orthoester formation conditions can be summarized as follows: n,6-unprotected acceptors (n = 2-4) always afford 1+6 linked oligosaccharides; selective 3-0-glycosylations were achieved with 2,3- and 3,4-unprotected sugars (glucose, mannose); - the rhamnose behaviour is an exception: Very high 3-regioselectivity was observed both with 3,4- and 2,3,4-unprotected sugars, whereas the 2,3-diol gave good 2-regioselectivity, further demonstrating that orthoester formation is very sensitive to steric factors [ 1221. -
The mannose-type acceptors gave particularly interesting results: The 3,4,6unprotected derivatives can be directly converted into the corresponding 3,6diorthoester with very high regioselectivity. Thus, using acetobromomannose 137 as a donor, the biologically important 3,6-branched mannotriose 141 was obtained in 88% yield by rearrangement of the diorthoester 140 (Scheme 37). When acetobromolactose was employed as a donor, a 3,6-branched pentasaccharide diorthoester was formed (88%)) the rearrangement of which afforded the 3,6-branched pentasaccharide in 740/0yield [124], [125]. Likewise, both 2,3,4- and 3,4-unprotected mannosyl acceptors gave 3-0-glycosylation regioselectively, by rearrangement of the corresponding orthoesters, leading to various very important mannose-based building blocks [ 1251. The versatility of this methodology was demonstrated by its application to the synthesis of biologically relevant branched oligosaccharides, such as the phytoalexin elicitor hexasaccharide [ 1241, [ 1261, [ 1271, a trisaccharide part of the Escherichia coli K26 antigen [ 1221 and the trisaccharide repeating unit of the 0 2 antigen occurring in the lipopolisaccharide of Stenotrophomonas maltophilia [ 1281. A related approach to the stereoselective synthesis of glycosidic linkages is based on the reductive cleavage of cyclic orthoesters generated by two sugar moieties. A C O y OAC ACOAAcOc
OAC O
q
+
Br 137
138
ii
139: R = H
141: R = Ac
140: R = Ac
Scheme 37. Reagents: i) AgOTf, 2,4-lutidine, CH2C12, rt, 91%; ii) Ac20, pyridine, qu.; iii) TMSOTf, CH2C12, -30 "C, 88%.
9.3 Esters and Reluted Derivatives
227
i
OMe
R, = H, R4= OBn R2=OBn, R, =OBn, % = H
142: R, = OBn, R, = H,
143: R, = H ,
OMe 144: 82% 145 74%
Scheme 38. Reagents: i) LiAlH4, AlC13, CH>ClZ/Et20, rt.
Such cyclic orthoesters are available from the corresponding sugar lactones through reaction with sugar diols in the presence of methoxy trimethylsilane and TMSOTf [ 1291. Using this procedure, 4,6-O-glucopyranosylidene, galactopyranosylidene and mannopyranosylidene acetals were synthesized. For the cleavage of the orthoesters, the combination LiAlH4-AlC13 was found to be the most effective reducing agent. The treatment of glycopyranosylidene acetals afforded the p-(1-+4)-linkeddisaccharides, with high regio- and stereoselectivity [ 1301. It is worthy of note that only the most sterically congested p-(1-4) glycosides were obtained, and not one of the other possible isomers, i.e. the p-(1-6) glycosides andfor a-glycosides, were detected in the reaction mixture, even in the case of the mannose-type donor. This high selectivity was attributed to the predominant coordination of the less hindered oxygen atom followed by the axial attack of a hydride. The procedure was further extended to the synthesis of glycosyl-galactosides (Scheme 38). The corresponding glycopyranosylidenes 142 and 143 were reductively cleaved with LiAlH4-AlC13 giving the highly congested p-( 1-4)-glycosyl-galactosides 144 and 145 with excellent regio- and stereoselectivity [131]. The mannose 1,2,6-orthoester 147 was recently reported as a potential glycosylating agent; after protection of the remaining hydroxy groups, the ring opening of the orthoester with nucleophiles could be achieved with 2-OH and 6-OH groups being orthogonally protected (Scheme 39). Compound 147 was prepared by mannose 1,2-0rthoester 146 using tert-butyldiphenylsilyl chloride and imidazole in DMF or, better, pyridinium triflate in pyridine. O-Acetylation or O-benzylation of 147 gave orthoesters 148a,b. The glycosylating properties of 148b were then examined: BF3 . OEtz was found to be the best promoter, and glucoside 149 was chosen as an exemplificative acceptor (Scheme 39). Disaccharide 150 was obtained in good yields. Interestingly, when an excess of orthoester was used, the partial formation of the trisaccharide due to the glycosylation of the 6’-hydroxy group took place [ 1321. Sugar tricyclic orthoesters, such as glucose 1,2,4-0rthoester derivatives, were used as monomers in polymerization reactions to afford cellooligosaccharides with a degree of polymerization up to 20. The 1,2,4-0rthopivalate was found to be the best monomer, both in terms of regio- and stereoregularity of the polymer produced, and it was successfully employed in the first chemical synthesis of cellulose [133].
228
9 Other Methods of Glycosylation
HO
HO HO
RO
146
147
148a:R = Ac 148b:R = En
-
+ En0
BnO 149
yo Bn0-&&/ooct BnO
148b
En0 150
Scheme 39. Reagents: i) Py . TfOH, pyridine, 89%; ii) AczO, pyridine, qu. or BnCI, raH, DMF, qu.; iii) TMSOTf, ClCH2CHzC1, 0 "C, 68%.
The electronic and steric effects of diverse orthoester groups (orthopropionate, orthoacetate and orthobenzoate) on the ring-opening polymerization of glucose 1,2,4-0rthoester were then explored. Although these compounds gave stereoregular polysaccharides, having only P-glucosidic bonds, the polysaccharides were not regioregular, consisting of a mixture of P-(1+4) and P-(1+2) units. These results were largely attributed to steric rather than electronic effects [ 1341. The 1,2-O-orthoesters derivatives of D-mannose and D-galactose were used as glycosyl donors to incorporate a-mannoside and P-galactoside branches at C-6 of chitin and its N-deacetylated form chitosan [ 1351 employing a N-phthaloyl 6-0-trimethylsilylated chitosan derivative as an acceptor. Sugar oxazolines are obtained from 2-acylammido-2-deoxy glycosyl donors through deprotonation of the oxazolinium ion intermediate 151 (Scheme 40). They are quite stable and can be used as glycosyl donors in 1,2-truns glycosylation reactions. In acidic media, the oxazoline 152 generates the oxazolinium ion, which can react with a suitable acceptor to provide the corresponding P-glycoside 153.
151
R
Y 153
Scheme 40. Glycosylation via oxazolinium ion intermediate.
9.3 Esters and Related Derivatives
229
Due to the strongly acidic conditions usually required for their activation, the oxazoline method has not found broad application in the field of oligosaccharide synthesis. They are incompatible with acid-sensitive protecting groups and with poorly nucleophilic acceptors. Their employment seems to be restricted to reactive acceptors. Despite these severe limitations, some important examples of the application of the oxazoline method have been reported [ 1361. Recently, Martin et al. described the synthesis of a- and P-u-glucosamine and galactosamine 1-phosphates using the more reactive trifluoromethyl oxazolines and dibenzyl phosphate [ 1371. was used as a monomer The methyl oxazoline of 3,6-di-O-benzyl-~-glucosamine for the synthesis of a dibenzylchitin-type polysaccharide. The polymerization under 1O-camphorsulfonic acid catalysis provided the polyaminosaccharide [ 1381. was used as a glycosyl The methyl oxazoline of 3,4,6-tri-O-acetyl-~-glucosamine donor in the glycosylation of ally1 6-O-pivaloyl-~-~-galactopyranoside: the reaction took 3-4 days in the presence of BF3 . OEtl and afforded the (3-(1+3)-disaccharide as the only regioisomer in 45-500/0 yield [ 1391. Finally, the unprotected methyl oxazoline of D-glucosamine was used as a substrate for the glycosidase chitinase in the synthesis of the N,N’-diacetylchitobiose, the repeating unit of chitin, via an enzymatic glycosylation [ 1401. 9.3.4 Phosphorus and Sulfur Derivatives Various glycosyl donors incorporating a variety of phosphorus-containing leaving groups have been developed and used for the stereoselective construction of a- and p- glycosidic linkages, with or without a neighbouring participating group [ 861. Besides the classical glycosyl phosphates and phosphites, the use of glycosyl diphenylphosphineimidates [141] and glycosyl phosphorodiamidimidothioates [142] as glycosyl donors have been described. New achievements in this area include both pentavalent and trivalent phosphorous-containing leaving groups. Glycosyl phosphoroamidates, phosphorimidates and phosphoramidimidates come into the first category. prepared from Shelf-stable glycosyl N,N,N’,N’-tetramethylphosphoroamidates, both O-benzyl and O-benzoyl-protected D-glucopyranose and D-galactopyranose, were coupled with a variety of primary and secondary glycoside alcohols in propionitrile in the presence of TMSOTf at -78 “C. In all cases, the chemical yields were very high as well as the p-stereoselectivities, demonstrating the considerable versatility of this glycosylation method [ 1431. Recent reports detail how this methodology has been extended. The high stability of glycosyl N,N,N’,N’-tetramethylphosphoroamidates allowed the reactivity to be modulated either by varying the nature of the protecting groups (benzyls or benzoyls) or the glycosylation conditions (Lewis acid and reaction temperature). This feature was exploited in a novel application of the “armed-disarmed” principle introduced by Fraser-Reid [ 1441. Thus, the “armed” donor 154 was coupled with the “disarmed” acceptor 155 under the aforementioned conditions to provide disaccharide 156 in 86% yield ( a :(3 = 5:95). Switching the protective groups from
230
9 Other Methods of Glycosylation
alp 5/95
BnO RO 154
155
156: R = Bz
ii
(OBn
158
0
oP@Me2)
157: R = Bn
BzO 0PWez)z
Scheme 41. Reagents: i) TMSOTf, EtCN, -78”C, 86%; ii) NaOMe, MeOH, then BnBr, NaH, DMF, 89% overall yield; iii) 155, TMSOTf, EtCN, -78 “C, 83%.
benzoyls to benzyls, the “armed” donor 157 was obtained, the glycosylation of which with acceptor 155 gave trisaccharide 158 in 83% yield (a: = 13:87, Scheme 41) [145]. On the other hand, the “armed-disarmed’’ concept was applied to glycosylation reactions between glycosyl N,N,N’,N‘-tetramethylphosphoroamidates and glycosides bearing other phosphorous-containing leaving groups, such as glycosyl diphenylphosphinimidates, phosphites and phosphorodiamidimidothioates, on each occasion taking advantage of their well documented reactivity differences [ 1451. An illustrative example is showed in Scheme 42. Fully benzylated glycosyl phosphites, such as 159, are known to be readily activated with BF3 . OEt, even at -78 “C giving very high p-selectivities, whereas the corresponding tetramethylphosphoroamidates were unreactive at below - 10 “C in the same conditions. The “armed” donor 159 was therefore coupled with
BnO
alp 3197
BnO
160
BnO
Bn 157 159
161
OMe
67
Scheme 42. Reagents: i) BF3 OEtl, CH2C12, -7O”C, 66%, only -78”C, 88%.
p; ii) 67, TMSOTf, EtCN,
9.3 Esters and Related Derioatives
-T&
T&OP(OEtJ2
-,
NF’h
II
0 OEt P-OEt
23 1
promoter ROH
(OMe
alp 1/30 0-P-OEt Me0
Me0 162
I
OEt
+
BnO I63
BnO BnO
164
Scheme 43. Reagents: i) PhN3, benzene, 50°C; ii) TMSOTf, EtCN, -78”C, 7 5 %
the “disarmed” acceptor 160 at -70°C giving disaccharide 157 in 66%1 yield (only p-anomer). This was subsequently activated with TMSOTf at -78°C in the presence of the alcohol 67 to afford the trisaccharide 161 (88%) a : p = 3 :97). Glycosyl N-phenyl diethylphosphorimidates were prepared by the treatment of the corresponding glycosyl phosphites with phenyl azide and used without further purification in glycosylation reactions with primary and secondary glycosyl acceptors. The chemical yields were generally good, whereas the stereoselectivities ranged from fair to only moderate. Scheme 43 shows an overview of the procedure and a selected example [ 1461. Interestingly, the authors observed a general a-selectivity using lutidinium ptoluensulfonate/tetrabutylammonium iodide as promoters and P-selectivity in the presence of TMSOTf. Moreover, the highest selectivities (a or p ) were obtained for the primary acceptor alcohols, whereas the ratios drop with secondary acceptors. The treatment of glycopyranoses with ethoxy bis(diisopropy1amino)phosphine in the presence of diisopropylammonium tetrazolide afforded the corresponding glycosy1 phosphoramidites 166 which, by reaction with phenyl azide, gave the glycosyl phosphoramidimidates 167. When they were used as glycosyl donors with various acceptors, the corresponding disaccharides were obtained, but only with modest pstereoselectivities. The best results were achieved with primary alcohols in propionitrile, in the presence of TMSOTf or BF3 . OEt2 (Scheme 44)[ 1471. The glycosyl phosphoramidites themselves were explored as glycosyl donors. First, 2-deoxyglycosidic linkages were synthesized. Excellent a-stereoselectivities were achieved using 2-deoxyglycosyl N,N-diisopropylphosphoramiditesas glycosyl donors, even in the case of hindered alcohol acceptors [148]. Compounds 169 were then coupled with selected primary and secondary glycosyl acceptors to give disaccharides with high p : a ratios, either using TMSOTf or BF3 . OEt2 as promoters. Propionitrile was found to be the best solvent for achieving high selectivities as well as good chemical yields (Scheme 45) [149]. Glycosyl diphenylphosphinyl donors were recently explored for the synthesis of D-ribofuranosides: the couplings were conducted with 1.5 eq. of TMSOTf at low temperature using simple alcohols or a-aminoacids as acceptors. Although only
232
9 Other Methods of Glycosylation
Me0 Me0
\
Me0
-
NPf, 165
(OMe
0-P-OEt
I
Me0
NPr',
166
...
167
ci/p 1/20
111
.___)
/OH
67
BnO
OMe
168
Scheme 44. Reagents: i) EtOP(NPr2')2, diisopropylammonium tetrazolide, CH2C12, rt; ii) PhN3, benzene, 45 "C; iii) 67,TMSOTf, EtCN, -78 "C, 73%.
modest stereoselectivities were observed, the glycosides were obtained in excellent yields. However, when a glucosyl acceptor was tested, the formation of a complex mixture of products was observed [ 1501. Glycosyl phosphorodithioates 171 (Scheme 46), prepared by action of 0,Odialkylphosphorodithioic acid on the corresponding glycals, were employed as glycosy1 donors in the synthesis of 2-deoxydisaccharides. The glycosylations of primary and secondary acceptors in the presence of silver fluoride proceeded sluggishly (from 7 to 12 days) and the a-stereoselectivities were somewhat low, except in a few cases [151]. This procedure was extended by the preparation of the related glycosyl phosphorothioates 172 and phosphoroselenoates 173 (Scheme 46). They were used as donors in the glycosylation of Diclofenac, a non-steroidal agent with potent antiinflammatory, analgesic and antipyretic activity, in order to produce new Diclofenacderived prodrugs. The corresponding glycosyl esters were obtained in good yields (72-84%) and with excellent P-selectivity, using silver carbonate as a promoter [ 1521. Among the sulfur containing leaving groups, glycosyl sulfonates have been described 11.531, but they were not further developed in recent years, although glycosyl triflates have been claimed as reactive intermediates in many glycosylation reaction 11541. Only one example of the use of glycosyl sulfates as glycosyl donors has been reported [ 1551. They were easily prepared from gluco- and mannopyranoses and em-
0-P
BnO BnO 169
BnO
9/91
BnO 95
Scheme 45. Reagents: i) BF3 . OEtz, EtCN, -78 "C, 92%.
170
References
171
172
233
173
Scheme 46. Glycosyl phosphorodithioates (171), glycosyl phosphorothioates (172) and glycosyl phosphoroselenoates (173).
ployed in the glycosylation of simple alcohols as well as primary and secondary sugar acceptors, providing the corresponding glycosides in fair to good yields, but with low stereoselectivities. Finally, sugar 1,2-cyclic sulfites have been investigated as glycosyl donors. Preliminary studies exploited these compounds for the introduction of reactive nucleophiles, such as azide or benzoate, on the anomeric position [156]. Effective b-0glycosylations with sugar cyclic sulfites were then reported, under catalysis of lanthanide(II1) triflates. However, the reactions required high temperatures (from 80 to 100 "C),limiting their application only to simple alcohols [ 1571. References I. a) K. Suzuki, T. Mukaiyama, Chem. Lett., 1982, 683-686; b) K. Suzuki, T. Mukaiyama, Chem. Lett., 1982, 1525-1528. 2. a) J. M. Beau, R. Schauer, J. Haverkamp, L. Dorland, J. F. G. Vliegenthart, P. Sinay, Curbohydr. Rex, 1980, 82, 125-129; b) F. Paquet, P. Sinay, Tetrahedron Lett., 1984, 25, 30713074. 3. F. Paquet, P. Sinay, J. Am. Chem. Soc., 1984, 106, 8313-8315. 4. a) F. Nicotra, L. Panza, F. Ronchetti, G. Russo, L. Toma, Tetrahedron Lett., 1985, 26, 807808; b) F. Nicotra, L. Panza, F. Ronchetti, G. Russo, L. Toma, J. Chem. SOC.,Perkin Trans. 1987, I , 1319-1324. 5. A. Boschetti, L. Panzd, F. Ronchetti, G. Russo, J. Chem. Soc., Perkin Trans. 1988, 1 , 33533357. 6. A. G. M. Barrett, B. C. B. Bezuidenhoudt, A. F. Gasiecki, A. R. Howell, M. A. Russell, J. Am. Chem. Soc., 1989, I l l , 1392-1396. 7. A. G. M. Barrett, B. C. B. Bezuidenhoudt, L. M. Melcher, J. Org. Chem., 1990; 55, 51965197. 8. A. Haudrechy, P. Sinay J. Org. Chem., 1992, 57, 4142-4151. 9. R. U. Lemieux, S. Levine, Can. J. Chem., 1%4,42, 1473-1480. 10. R. W. Friesen, S. J. Danishefsky, J. Am. Chem. SOC.,1989, 111, 6656-6660. 1 1 . K. Tatsutd, K. Fujimoto, M. Kinoshita, S. Umezawa, Carbohydr. Rex, 1977, 54, 85-104. 12. a) K. Tatsuta, T. Yamauchi, M. Kinoshita, Bull. Chem. Soc. Jpn. 1978, 51, 3035-3038; b) K. Tatsuta, Y . Amemiya, Y. Kanemura, H. Takahashi, M. Kinoshita, Tetrahedron Lett., 1982, 23, 3375-3378; c) K. Toshima, K. Tatsuta, M. Kinoshita, Tetrahedron Lett., 1986, 27, 47414741. 13. a) J. Thiem, H. Karl, J. Schwenter, Synthesis, 1978, 696-699; b) J. Thiem, H. Karl, Tetrahedron Lett., 1978, 4999-5002. 14. a) J. Thiem, S. Kopper, Tetrahedron, 1990, 46, 113-138; b) D. Horton, W. Priebe, M. Snaitzman, Carbohydr. Res., 1989, 187, 149-153; c) B. Abbaci, J. C. Florent, C. Monneret, J. Chem. Soc., Chem. Commun., 1989, 1896-1897; d) S. J. Danishefsky, H. G. Selnick, D. M. Annistead, F. E. Wincott, J. Am. Chem. Soc., 1987, 109, 8119-8120.
234 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
9 Other Methods of Glycosylation
J. Thiem, W. Klafie, J. Org. Chem., 1989, 54, 2006-2007. G. Jaurand, J. M. Beau, P. Sinay, J. Chem. Soc., Chem. Commun., 1981, 572-573. A. G. M. Barrett, T. A. Miller, Tetrahedron Lett., 1988,29, 1873-1874. Y. Ito, T. Ogawa, Tetrahedron Lett., 1987, 28, 2723-2726. G. Grewal, N. Kaila, R. W. Franck, J. Org. Chem., 1992, 57, 2084-2092. C. J. Tu, D. Lednicer, J. Org. Chem., 1987, 52, 5624-5627. See e.g. K. Toshima, K. Tatsuta, M. Kinoshita, Bull. Chem. Soc. Jpn., 1988, 61, 2369-2381. V. Bolitt, C. Mioskowski, S. G. Lee, J. R. Falck, J. Org. Chem., 1990, 55, 5812-5813. S. Sabesan, S. Neira, J. Org. Chem., 1991, 56, 5468-5472. J. Thiem, M. Kleeberg, Carbohydr. Rex, 1990, 205, 333-345. D. Noort, G. H. Veeneman, G. J. P. H. Boons, G. A. van der Marel, G. J. Mulder, J. H. van Boom, Synlett, 1990,205-207. 26. F. Nicotra, L. Panza, G. Russo, Tetrahedron Lett., 1991, 32, 4035-4038. 27. L. Lay, F. Nicotra, L. Panza, G. Russo, G. Sello, J. Carbohydr. Chem., 1998,17, 1269-1281. 28. A. Marra, J. Esnault, A. Veyrieres, P. Sinay, J. Am. Chem. Soc., 1992, 114, 6354-6360. 29. a) H. K. Chenault, A. Castro, L. F. Chafin, J. Yang, J. Org. Chem., 1996, 61, 5024-5031; b) H. K. Chenault, L. F. Chafin, J. Org. Chem., 1998,63, 833-840. 30. a) G. J. Boons, S. Isles, Tetrahedron Lett., 1994, 35, 3593-3596; b) G. J. Boons, S. Isles, J. Org. Chem., 1996, 61, 4262-427 1. 31. G. J. Boons, B. Heskamp, F. Hout, Angew. Chem. Int. Ed. Engl., 1996,35, 2845-2847. 32. M. Johnson, C. Arles, G. J. Boons, Tetrahedron Lett., 1998, 39, 9801-9804. Perkin Trans. 1997, I , 3357-3360. 33. R. R. Gibson, R. P. Dickinson, G. J. Boons, J. Chem. SOC., 34. Y. D. Vankar, P. S. Vankar, M. Behrendt, R. R. Schmidt, Tetrahedron, 1991,47, 9985-9992. 35. Y. Osa, K. Takeda, T. Sato, E. Kaji, Y. Mizuno, H. Takayanagi, Tetrahedron Lett., 1999, 40, I53 1-1 534. 36. C. H. Marzabadi, R. W. Franck, J. Chem. Soc., Chem. Commun., 1996, 2651L2652. 37. R. W. Franck, C. H. Marzabadi, J. Org. Chem., 1998,63, 2197-2208. 38. M. L. Sznaidman, S. C. Johnson, C. Crasto, S. M. Hetch, J. Org. Chem., 1995, 60, 39423943. See also the preparation of disaccharides by cycloaddition to sugar cyclic ketene acetals: S. C. Johnson, C. Crasto, S. M. Hetch, J. Chem. Soc., Chem. Commun., 1998, 1019-1020. 39. E. Fischer, Chem. Ber., 1893, 2400-2412. 40. See e.g.: a) A. A. Pavia, J. M. Rocheville, S. N. Ung-Chhun, Carbohydr. Res., 1980, 79, 7989; b) A. A. Pavia, S. N. Ung-Chhun, Can. J. Chem., 1981, 59, 482-489 (synthesis of trehaloses); c) A. Lubineau, J. C. Fischer, Synth. Commun., 1991,21, 815-818 (glycoside synthesis). 41. H. P. Wessel, J. Carbohydr. Chem., 1988, 7, 263-269. 42. P. Konraddson, C. Roberts, B. Fraser-Reid, Reel. Trav. Chim. Pays-Bas, 1991, 110, 23-24. 43. S. Horito, M. Tada, H. Hashimoto, Chem. Lett., 1991, 117-120. 44. B. Fischer, A. Nudelman, M. Ruse, J. Herzig, H. E. Gottlieb, J. Org. Chem., 1984, 49, 49884993. 45. J. Defaye, A. Gadelle, C. Pedersen, Carbohydr. Rex, 1989, 186, 177-188. 46. a) H. Uchiro, T. Mukaiyama, Chem. Lett., 1996, 79-80; b) H. Uchiro, T. Mukaiyama, Chem. Lett., 1996, 271-272 47. K. Takeuchi, S. Higuchi, T. Mukaiyama, Chem. Lett., 1997, 969-970. 48. a) K. Tomooka, Y. Nakamura, T. Nakdi, Synlett, 1995, 321-322; b) T. Jyojima, N. Miyamoto, Y. Ogawa, S. Matsumura, K. Toshima, Tetrahedron Lett., 1999, 40, 5023-5026. 49. K. Toshima, H. Nagai, S. Matsumura, Synlett, 1999, 1420-1422. 50. J. Inanaga, Y. Yokoyama, T. Hanamoto, J. Chem. Soc., Chem. Commun., 1993, 1090-1091. 51. H. Susaki, Chem. Pharm. Bull., 1994, 42, 1917-1918. 52. T. Mukaiyama, K. Matsubara, M. Hora, Synthesis, 1994, 1368-1373. 53. H. Uchiro, K. Miyazaki, T. Mukaiyama, Chem. Lett., 1997, 403-404. 54. T. Mukaiyama, S. Suda, Chem. Lett., 1990, 1143-1146. 55. S. Suda, T. Mukaiyama, Chem. Lett., 1991, 431-434. 56. T. Mukaiyama, K. Matsubara, S. Suda, Chem. Lett., 1991, 981-984. 51. B. A. Garcia, J. L. Poole, D. Y. Gin, J. Am. Chem. Soc., 1997,119, 7597-7598. 58. S. Koto, N. Morishima, S. Zen, Chem. Lett., 1976, 1109-1110. 59. S. Koto, N. Morishima, S. Zen, Bull. Chem. Soc. Jpn., 1982, 55, 1543-1547.
References
235
60. S. Koto, N. Morishima, C. Kusuhara, S. Sekido, T. Yoshida, S. Zen, Bull. Chem. Soc. Jpn., 1982,55,2995-2999. 61. S. Koto, T. Sato, N. Morishima, S. Zen, Bull. Chem. Soc. Jpn., 1980, 53, 1761-1762. 62. a) N. Morishima, S. Koto, S. Zen, Chem. Lett., 1982, 1039-1040; b) S. Koto, N. Morishima, M. Owa, S. Zen, Carhohydr. Res., 1984, 130, 73-83. 63. W. Szeja, Synthesis, 1988, 223-224. 64. M. Hirooka, S. Koto, Bull. Chem. Soc. Jpn., 1998, 71, 2893-2902. 65. M. Wakao, Y. Nakai, K. Fukase, S. Kusumoto, Chem. Lett., 1999, 27-28. 66. G. Grynkiewicz, Carbohydr. Rex, 1977, 53, CI 1-C12. 67. a) P. J. Garegg, T. Iversen, T. Norberg, Carbohydr. Res., 1979, 73, 313-314; b) K. Akerfeldt, P. J. Garegg, T. Iversen, Acta Chem. Scand. B, 1979, 33, 467-468. 68. A. Lubineau, E. Meyer, P. Place, Carbohydr. Rex, 1992, 228, 191-203. 69. a) N. Chida, M. Ohtsuka, S. Ogawa, Chem. Lett., 1988, 969-972. 70. a) W. R. Roush, X. F. Lin, J. Org. Chem., 1991,56,5740-5742; b) W. R. Roush, X. F. Lin, J. Am. Chem. Soc, 1995,117, 2236--2250. 71. A. Dondoni, A. Marra, M. C. Scherrmann, A. Casnati, F. Sansone, R. Ungaro, Chem. Eur. J., 1997, 3, 1774-1782. 72. G. T. Badmdn, D. V. S. Green, M. Voyle, J. Oryanomet. Chem., 1990, 388, 117-121. 73. A. Sobti, G. A. Sulikowski, Tetrahedron Lett.: 1994, 35, 3661-3664. 74. a) A. B. Smith, 111, K. J. Hale, R. A. Rivero, Tetrahedron Lett., 1986, 27, 5813-5816; A. B. Smith, 111, R. A. Rivero, K. J. Hale, H. A. Vaccaro, J. Am. Chem. Soc, 1991, 113, 2092-2112. 75. W. A. Szarek, H. C. Jarpell, J. K. N. Jones, Curhohydr. Res., 1977, 57, C13-CI6. 76. A. Bouali, G. Descotes, D. F. Ewing, A. Grouiller, J. Lefkidou, A. D. Lespinasse, G. Mackenzie, J. Curbohydr. Chem., 1992, 11, 159-169. 77. K. C. Nicolaou, R. D. Groneberg, J. Am. Chem. Soc, 1990, 112, 4085-4086. 78. L. F. Tietze, R. Fischer, H. J. Guder, Tetrahedron Lett., 1982, 23, 4661L4664. 79. E. M. Nashed, C. P. J. Glaudemans, J. Org. Chem., 1989, 54, 6116-6118. 80. D. X. Qiu, F. Y. Wang, M. S. Cai, Syntli. Coinmun., 1989, 19, 3453-3456. 81. a) C. Kolar, G. Kneissel, Angew. Chern. Int. Ed. Engl., 1990, 29, 809-810; b) C. Kolar, G. Kneissel, U. Knodler, K. Dehmel, Curhohydr. Rex, 1991, 209, 89-100. 82. W. Priebe, G . Grynkiewicz, N. Neamati, Tetruhrdron Lett., 1991, 32, 2079-2082. 83. T. Mukaiyama, K. Matsubara, Chem. Lett., 1992, 1041- 1044. 84. A. Kirschning, G. Chen, Tetrahedron Lett., 1999, 40, 4665~-4668. 85. B. Helferich, E. Shimitz-Hillebrecht, Chem. Ber., 1933, 66, 378--383. 86. K. Toshima, K. Tatsuta, Chem. Rev.,1993, 93, 1503-1531 87. a) R. U. Lemieux, W. P. Shyluk, Can. J. Chem., 1953, 31, 528-535; b) S. Hanessian, Y. Banoub, Curhohydr. Res., 1977, 59, 261-267. 88. a) M. Kiso, L. Anderson, Carhohydr. Res., 1979, 72, C15-CI7; b) L. M. Lerner, Carhohydr. Rex 1990,207, 138-141. 89. J. Dahmen, T. Frejd, G. Magnusson, G. Noori, Carbohydr. Res., 1983, 114, 328-330. 90. T. Ogawa, K. Beppu, S. Nakabayashi, Carbohydr. Res., 1981, 93, C6--C9. 91. T. Mukaiyama, S. Kobayashi, S. Shoda, Chem. Lett., 1984, 907-910. 92. T. Mukaiyama, T. Shinipuku, T. Takashima, S. Kobayashi, Chem. Lett., 1989, 145-148. 93. a) T. Mukaiyama, T. Takashima, M. Katsurada, H. Aizawa, Chem. Lett., 1991, 533-536; b) T. Mukaiyama, M. Katsurada, T. Takashima, Chem. Lett., 1991, 985-988. 94. K. Matsubara, T. Sasaki, T. Mukaiyama, Chem. Lett., 1993, 1373-1376. 95. T. Mukaiyama, M. Katsurada, Y. Iida, Chem. Lett., 1992, 2105-2108. 96. T. Mukaiyama, K. Matsubara, Chem. Lett., 1992, 1755-1758. 97. a) T. Mukaiyama, N. Shimomura, Chem. Lett., 1993, 781-784; b) N. Shimomura, M. Saitoh, T. Mukaiyama, Chem. Lett., 1994, 433-436. 98. A. K. Pathak, Y. A. El-Kattan, N. Bansal, J. A. Maddry, R. C. Reynolds, Tetrahedron Lett., 1998,39, 1497-1500. 99. T. Mukaiyama, T. Sasaki, E. Iwashita, K. Matsubara, Clzem. Lett., 1995, 455--456. 100. T. Vuljanic, J. Kihlberg, P. Somfai, J. Org. Chem., 1998, 63, 279--286. 101. K. Toshima, Y . Nozaki, S. Mukaiyama, K. Tatsuta, Tetrahedron Lett., 1992, 33, 1491-1494. 102. Y. Inanaga, Y. Yokoyama, T. Hanamoto, Tetrahedron Lett., 1993,34, 2791-2794.
236 103. 104. 105. 106.
9 Other Methods of Glycosylation
K. M. Nicholas, Acc. Chem. Res., 1987, 20, 207-214. C. Mukai, T. Itoh, M. Hanaoka, Tetrahedron Lett., 1997, 38, 4595-4598. F. Nicotra, L. Panza, A. Romano, G. Russo, J. Curbohydr. Chem., 1992, 11, 397-399. a) T. Mukaiyama, K. Miyazaki, H. Uchiro, Chem. Lett., 1998, 635-636; b) T. Mukaiyama, Y. Wakiyama, K. Miyazaki, K. Takeuchi, Chem. Lett., 1999, 933-934. 107. T. Iimori, T. Shibazaki, S. Ikegami, Tetruhedron Lett., 1996, 37, 2267-2270. 108. G. Scheffler,R. R. Schmidt, Tetrahedron Lett., 1997,38, 2943-2946. 109. T. Iimori, I. Azumaya, T. Shibazaki, S. Ikegami, Heterocycles, 1997, 46, 221-224. 110. I. Azumaya, T. Niwa, M. Kotani, T. Iimori, S. Ikegami, Tetrahedron Lett., 1999, 40, 46834686. 111. a) M. J. Ford, S. V. Ley, Synlett, 1990, 255-256; b) M. J. Ford, J. G. Knight, S. V. Ley, S. Vile, Synlett, 1990, 331-332. 112. S. Hanessian, C. Bacquet, N. Lehong, Curbohydr. Res., 1980, 80, C17-C22. 113. K. Koide, M. Ohno, S. Kobayashi, Tetrahedron Lett., 1991, 32, 7065-7068. 114. H. Furukawa, K. Koide, K. Takao, S. Kobayashi, Chem. Pharm. Bull., 1998,46, 1244-1247. 115. a) S. Hanessian, J. J. Conde, B. Lou, Tetrahedron Lett., 1995, 36, 5865-5868; b) S. Hanessian, J. J. Conde, H. H. Khai, B. Lou, Tetrahedron, 1996, 52, 10827-10834. 116. H. Kunz, J. Zimmer, Tetrahedron Lett., 1993, 34, 2907-2910. 117. J. C. Lopez, B. Fraser-Reid, J. Chem. Soc., Chem. Comrnun., 1991, 159-161. 118. H. Herzner, J. Eberling, M. Schultz, J. Zimmer, H. Kunz, J . Carbohydr. Chem., 1998, 17, 759-776. 119. N. K. Kochetkov, A. F. Bochkov, T. A. Sokolovskaya, V. J. Snyatkova, Curbohydr. Rex, 1971,16, 17-27. 120. Y. E. Tsvetkov, P. I. Kitov, L. V. Backinowsky, N. K. Kochetkov, Tetrahedron Lett., 1993, 34, 7977-7980. 121. W. Wang, F. Kong, J. Org. Chem., 1998,63, 5744-5745. 122. Y. Du, F. Kong, J. Carbohydr. Chem., 1999,18,655-666. 123. W. Wang, F. Kong, J. Curbohydr. Chem., 1999, 18, 451-460. 124. W. Wang, F. Kong, J. Org. Chem., 1999,64, 5091-5095. 125. W. Wang. F. Kong, Angew. Chem. Int. Ed. Engl., 1999,38, 1247-1250. 126. W. Wang, F. Kong, Tetrahedron Lett., 1998, 39, 1937-1940. 127. W. Wang, F. Kong, Curbohydr. Rex, 1999, 315, 117-127. 128. W. Wang, F. Kong, J. Curboydr. Chem., 1999,18, 263-273. 129. T. Iimori, H. Ohtake, S. Ikegami, Tetrahedron Lett., 1997,38, 3413-3414. 130. T. Iimori, H. Ohtake, S. Ikegami, Tetrahedron Lett., 1997,38, 3415-3418. 131. H. Ohtake, T. Iimori, S. Ikegami, Synlett, 1998, 1420-1422. 132. S. Hiranuma, 0. Kanie, C.-H. Wong, Tetrahedron Lett., 1999, 40, 6423-6426. 133. F. Nakatsubo, H. Kamitakahara, M. Hori, J. Am. Chem. Soc., 1996,118, 1677-1681. 134. M. Hori, H. Kamitakahara, F. Nakatsubo, Macromolecules, 1997, 30, 2891-2896. 135. a) K. Kurita, M. Kobayashi, T. Munakata, S. Ishii, S. Nishimura, Chem. Lett., 1994, 20632066; b) K. Kurita, H. Akao, M. Kobayashi, T. Mori, Y. Nishiyama, Polymer Bull., 1997,39, 543-549; c) K. Kurita, K. Shimada, Y. Nishiyama, M. Shimojoh, S. Nishimura, Mucromolecules, 1998, 31, 4764-4769. 136. a) L. Lay, L. Panza, G. Russo, D. Colombo, F. Ronchetti, E. Adobati, S. Canevari, Helu. Chim. Actu, 1995, 78, 533-538; b) D. Colombo, L. Panza, F. Ronchetti, Curbohydr. Rex, 1995,276, 437-441; c) J. Banoub, P. Boullanger, D. Lafont, Chem. Rev., 1992, 92, 1167-1 195 and references cited therein. 137. P. Brusca, 0. R. Martin, Tetrahedron Lett., 1998, 39, 8101-8104. 138. a) J. Kadokawa, Y. Watanabe, M. Karasu, H. Tagaya, K. Chiba, Mucromol. Rapid Commun., 1996,17, 367-372; b) J. Kadokawa, S. Kasai, Y. Watanabe, M. Karasu, H. Tagaya, K. Chiba, Macromolecules, 1997, 30, 8212-8217. 139. J. Xia, C. F. Piskorz, R. D. Locke, E. V. Chandrasekaran, J. L. Alderfer, K. L. Matta, Bioorg. Med. Chem. Lett., 1999, 9, 2941L2946. 140. S. Kobayashi, T. Kiyosada, S. Shoda, Tetrahedron Lett., 1997, 38, 21 11-21 12. 141. S. Hashimoto, T. Honda, S. Ikegami, Tetruhedron Lett., 1991, 32, 1653-1654. 142. S. Hashimoto, T. Honda, S. Ikegami, Tetrahedron Lett., 1990, 31, 4769-4772.
References
237
143. S. Hashimoto, Y. Yanagiya, T. Honda, H. Harada, S. Ikegami, Tetrahedron Lett., 1992, 33, 3523-3526. 144. D. R. Mootoo, P. Konradsson, U. Udodong, B. Fraser- Reid, J. Am. Chem. Soc., 1988, 110, 5583-5584. 145. S. Hashimoto, H. Sakamoto, T. Honda, S. Ikegami, Tetrahedron Lett., 1997,38, 5181-5184. 146. S. Pan, H. Li, F. Hong, B. Yu, K. Zhao, Tetrahedron Lett., 1997, 38, 6139-6142. 147. M.-J. Chen, K. Ravindran, D. Landry, K. Zhao, Heterocycles, 1997, 45, 1247- 1250. 148. H. Li, M.-J. Chen, K. Zhao, Tetrahedron Lett., 1997, 38, 6143-6144. 149. D. Niu, M.-J. Chen, H. Li, K. Zhao, Heterocycles, 1998, 48, 21-24. 150. G. Singh, I. Tranoy, Carbohydr. Lett., 1998, 3, 79 -84. 151. H. Bielawska, M. Michalska, J. Carbohydr. Chem., 1991, 10, 107-112. 152. J. Borowiecka, Liebigs Ann., 1997, 2147-21 50. 153. R. Eby, C. Schuerch, Carbohydr. ReJ., 1982, 102, 131-138. 154. D. Crich, W. Cai, J. Ory. Chem., 1999, 64, 4926-4930. 155. L. Cipolla, L. Lay, F. Nicotra, L. Panza, G. Russo, Tetrahedron Lett., 1994,35, 8669-8670. 156. A. Meslouti, D. Beaupere, G. Demailly, R. Uzan, Tetrahedron Lett., 1994, 35, 3913-3916. 157. W. J. Sanders, L. L. Kiessling, Tetrahedron Lett., 1994, 35, 7335-7338.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
10 Polymer-Supported Synthesis of Oligosaccharides Jiri J. Krepinsky and Stephen P. Douglas
10.1 Introduction Among the variety of biological activities of oligosaccharides, antiinfective effects are the reason some oligosaccharides are close to a practical use [ 11, provided that the oligosaccharides can be cleanly made on large scale. Glycosylations by enzymic (cf. Part 3 of this Volume) and chemical methods compete to meet this challenge. Classical chemical methods of oligosaccharide synthesis invariably require extensive purification of the desired reaction products, mostly by chromatography, which makes chemical syntheses of oligosaccharides very laborious and expensive. Similar problems encountered in the syntheses of oligopeptides and oligonucleotides were dealt with successfully using polymer-supported synthetic and purification techniques. A polymer support facilitates quick and efficient purification of products assuming that these reactions proceed to completion. Although some indications suggest that polymer supports may influence the glycosylation reaction itself [2], the primary purpose of the polymer support is rapid purification after every step in the reaction sequence leading to the formation of an oligosaccharide [ 3 ] . Therefore, polymer supports will not make the chemistry of oligosaccharide synthesis any simpler which is unfortunate since this chemistry is complex and many details important for the synthesis remain unknown despite the rapid progress in this area during recent years documented, for instance, in this Volume. However, because of the significantly simpler purification, synthetic sequences become enormously faster. Furthermore, for syntheses of combinatorial oligosaccharide libraries, polymer support is essential since it allows purifications of the desired intermediates or products as groups, which is difficult to accomplish otherwise. Four factors combine to make oligosaccharide synthesis challenging: i) several hydroxyl groups of similar reactivity are present in monosaccharides, from which oligosaccharides are generally constructed and which almost invariably require laborious protection-deprotection schemes; ii) difficulty to form anomerically pure glycosidic linkages; iii) acid lability of the glycosidic linkages (hemi-acetals); iv)
240
10 Polymer-Supported Synthesis of Oligosaccharides
incompletely known intramolecular interactions. The most common glycosylation technique utilizes a nucleophilic substitution at the anomeric carbon with a hydroxyl group (“acceptor”) of the aglycon. In order to prevent other hydroxyls from taking part in this reaction, they must be protected [4] (and subsequently deprotected) by methods compatible with the labile glycosidic linkages [ 5 ] . As is common for acetal formation reactions, glycosylations are often both incomplete and accompanied by numerous side-reactions, so that chromatography is usually needed to clean desired products after each synthetic step. Therefore, the synthesis of an oligosaccharide in solution is extremely time consuming, costly, and environmentally unfriendly in part because of the large volumes of solvents used. Despite the ability of this methodology (c.f. this Volume) to provide almost any oligosacaccharide [6], if the chromatographic purification is the bottleneck step in the chemical oligosaccharide syntheses, removal of the requirement for chromatography may make chemical synthesis of oligosaccharides significantly more practical. Methods employing enzymes have been developed to overcome these difficulties (cf. Part 3 of this Volume). Glycosyltransferases [7] and glycosidases working in reverse [S], or combined enzyme and chemical strategies [9, 101 have been examined. Syntheses employing glycosyl transferases using soluble polymer supports, such as water-soluble polyvinyl alcohol [ 1I] and polyacrylamide [ 121, to which a monosaccharide was bound via a photolabile (O-nitrobenzyl) linker, also established that a spacer between the polymer support and the oligosaccharide must be of certain length so that the reactions can occur [13]. Despite considerable advantages, such as not needing a hydroxyl protection-deprotection scheme, enzymic syntheses have their own problems. One problem is the requirement of glycosyl transferases to employ relatively unstable and difficult to prepare intermediates. Furthermore, transferases cannot as yet be used to synthesize oligosaccharides unknown in Nature.
10.2 General Reflections Polymer supported designs of oligosaccharide synthesis began to be explored thirty years ago encouraged by the solution of analogous problems in the syntheses of oligonucleotides and oligopeptides by polymer-supported strategies. The spectacular success of solid-state syntheses of oligopeptides and oligonucleotides on polystyrene resins and controlled-pore glass is due not only to the polymer support but also to specifically designed chemical procedures to be performed on solid state support. The main benefit of procedures involving solid state reactants, i.e. the purification of desired intermediates from by-products, excess reagents and products of their decomposition by simple washing, is combined with prompt quenching of the reaction and fast work-up, eliminating overexposure of reaction products to excess reagents and by-products. The first polymer-supported oligosaccharide synthesis by Schuerch’s group employed the Koenigs-Knorr reaction and its variants, using an immobilized glycosyl acceptor on an organic solid polymer (Scheme 1) [ 141. It was followed by inves-
10.2 General Reflections
+
J
t
P
& O Q ! ! j
P
24 1
242
10 Polymer-Supported Synthesis of Oligosaccharides
tigations of an inorganic support, namely porous glass, zirconized and silanized [ 151. The results were not satisfactory; among the problems were decreased reaction rate and lower yields compared to solution strategies, incomplete coupling, and poor anomeric specificity. These problems seemed to be attributed to the solid state rather than its incompatibility with certain aspects of Koenigs-Knorr chemistry. Unfortunately, these difficulties stymied further progress in polymer-supported glycosylations, although the method was not entirely abandoned by van Boom [16, 171 and others [ 181, providing various oligosaccharides using Merrifield cross-linked polystyrene and controlled-pore glass. Although it was suggested that the use of a soluble polymer could improve the results [19], it took another twenty years until the first report in 1991 from Krepinsky’s group on successful1 applications of polyethyleneglycol as soluble support was published (Scheme 1) [20]. Since both Koenigs-Knorr (promoted by silver triflate) and trichloroacetimidate chemistry gave comparable results on polyethylene glycol, it seemed to confirm the belief that it was the solid state support, i.e. two phase condition, which failed glycosylations [20]. That this conclusion was erroneous was demonstrated when the glycal method of glycosylation developed by Danishefsky’s group 1211, was exploited by that group for the synthesis of oligosaccharides using immobilized glycosyl donors on polystyrene support 1221. This scenario is reported to provide excellent anomeric specificity although an exception was noticed [23, 241, which was subsequently remedied by a modification of the procedure 1251. Oligosaccharide chains can be extended (i) from the reducing end to the nonreducing terminus (using polymer-supported acceptors), as well as (ii) from the nonreducing end to the reducing terminus (using polymer-supported donors) (Scheme 2). Both strategies have serious problems due to incomplete glycosylations and/or donor decomposition. Assuming that a small portion (realistically perhaps 3-5%) of polymer-supported monosaccharide M1 acceptor fails to be glycosylated to a disaccharide D1, the disaccharide D1 in the subsequent step is extended to a trisaccharide T1, but a portion of the previously unglycosylated monosaccharide M 1 gives disaccharide D2. These second round glycosylations are again incomplete. After the third glycosylation the following compounds will be polymer-bound in addition to the desired tetrasaccharide: the monosaccharide M 1, disaccharides D 1, D2, and D3, and trisaccharides T1 and T2. After each additional glycosylation the mixture becomes even more complicated, yet all impurities originating from the donors are absent. Thus the desired product must be separated from the truncated sequences (= failures), after detachment from the polymer support. To alleviate the situation, excess glycosylation reactants are recommended to be applied in portions (Scheme 2), as is practiced to obtain “difficult sequences” in polymer-supported oligopeptide syntheses. Incomplete anomeric specificity causes further complications. Starting the synthesis from the nonreducing terminus, i.e. using a polymer-bound donor presents the same problem of truncated sequences. Since donors are usually quite reactive, a portion of a donor may be transformed into molecules which do not react anymore with acceptors and since they are fastened to the polymer, they are carried through the synthetic sequence (Scheme 2) and must be removed by chromatography at the end of the synthesis. This will remain problematic unless
10.2 General Reflections
k
\
4
I
4
0
\
243
244
10 Polymer-Supported Synthesis of Oligosaccharides
Glycal Assembly
0
2 Self-policingFailure
ZnClDHF Due to their relatively higher polarity the "failures" are easily separated after the synthesis by simple Chromatography.
Scheme 3. Two-phase polymer supported oligosaccharide synthesis using galactal donor attached
to an insoluble polymer.
stable donors are employed, which react specifically with acceptors. One solution has been developed by Danishefsky's group (cf. Schemes 3 and 4) [26]. Failures are self-policing in that they are easily removed at the end of the synthesis by simple chromatography owing to their highly polar nature [26]. An ingenious solution of this problem was developed by Ogawa's group (Scheme 5 ) [27]. The monosaccharide added last to the sequence is equipped with a hydrophobic group, such as 2-(trimethylsilyl)ethyl, which after detachment from the polymer permits isolation of the sequence containing the hydrophobic group by reversephase chromatography (investigated by Hindsgaul's group) [28], thus separating it from most failure sequences. As already mentioned, solid-state oligopeptide and oligonucleotide syntheses required the development of synthetic methods compatible with the solid state processes. This should be equally or more indispensable for oligosaccharide synthesis since syntheses of oligosaccharides are an even more daunting task than the syntheses of oligonucleotides or oligopeptides. This is actually happening, since in spite of this complexity, the progress in designing glycosylation methods is steadily accelerating.
10.2 General Reflections
245
Glycal Assembly
B
> opiv
0BnO 1
MeOTUDTBP
OPiv
BnO
OBn
I
=Bn*
--
BnO
,o& BnO
OH
opiv
Scheme 4. Two-phase polymer supported oligosaccharide synthesis using on-support transformation of glucal into thioglycoside donor attached to insoluble polymer.
+
E
" L & Y
Repeat
-
r
Y
Non-tagged impurities easily removed by chromatography Scheme 5. Polymer supported oligosaccharide synthesis utilizing hydrophobic handle attached in the final stage of the synthetic sequence. The handle allows to separate most of failure sequences accumulated during the synthesis.
246
10 Polymer-Supported Synthesis of Oligosuccharides
10.3 Polymer Supports Automated two phase polymer-supported syntheses of oligopeptides and oligonucleotides provide their products in micromolar to millimolar quantities because of the combination of loading capacity (the loading capacity is a number of anchoring sites in millimoles per gram of a support) of solid polymers (cross-linked polystyrene, controlled-pore glass, Tentagel, Argogel) and the limit of the support volume (mass) practically usable. Aside from mechanical stability of the support, the most important consideration is the chemical stability of both the product and the supporting system (i.e. the polymer and the tether). Since prolonged contacts with the reagents may induce decomposition of the polymer-bound product and the supporting system itself, the reactions should be as fast as possible. Given a small scale of the synthesis, the reagents can be used in a huge excess (e.g. hundredfold) to ensure rapid reactions. However, to synthesize larger quantities of shorter sequences it became necessary to employ polymeric supports which can be practicably handled in substantially larger volumes than the solid polymers, such as polyethyleneglycol o-monomethylether (MPEG) of m.w. = 5,000-6,000 [29, 301, albeit the loading capacity of MPEG at approximately 0.2 mmol/g is not much different from loading capacities of polystyrene-based supports. MPEG is soluble in most solvents including water and thus the polymer-bound synthon can be dissolved under many reaction conditions. After completion of the reaction an ether such as methyl t-butyl ether is poured into the reaction mixture and the polymer-bound product precipitates because MPEG is insoluble in ethers. The remaining components of the reaction mixture can be removed from the desired MPEG-bound product by simple filtration (Scheme 1). One of the original arguments for using a soluble polymer support for oligosaccharide syntheses was based on the necessity to obtain oligosaccharides in larger quantities because biological effects are only observed in higher concentrations. Furthermore, it was assumed that glycosylations on soluble supports are more easily controlled than those immobilized on insoluble matrices, and that all reactants in solution would allow reaction kinetics and anomericity control similar to those observed in solution chemistry [19, 20, 311. Fortunately these arguments did not prevent solid supports from being investigated. Thus it was shown in quick succession that almost any solid support used previously for oligopeptide and oligonucleotide syntheses could be employed for oligosaccharide syntheses (Table 1): polystyrene crosslinked with divinylbenzene (1-2%) [21] usually derived from “Merrifield and Wang resins” [32] or Rink resin [33], polystyrene with grafted polyethylene glycol (TentaGel [34], Argogel [35]), and controlled-pore glass (CPG) [36], as well as novel supports such as polystyrene crown [37], in combination with a suitable chemistry and a tether between the polymer and oligosaccharide chain. Now it appears that there are no significant differences between glycosylations performed in solution without a polymer support, with a soluble polymer support, or using a solid polymer support [38], although some variations were observed 12). It should be noted, of course, that insoluble catalysts are much more likely to be
10.4 One-Phase Systems (Syntheses in Solution)
247
Table 1. Polymer supports showing the functionality to which linkers are attached. Supports for one-phase (solution) syntheses are marked with an asterisk (*); the remainder are supports for twophase syntheses. Except more heavily crosslinked macroporous polystyrene, all supports for two phase syntheses are 1-2% cross-linked polystyrene-divinylbenzene.The first four resins (Merrifield, Wang, Rink, aminomethyl) have loading capacity 0.1-0.3 mmol/g, and swell in solvents such as dichloromethane, methanol, dimethylformamide, and pyridine. Macroporous polystyrene has loading capacity 0.3-0.6 mmol/g, and maintains constant pore size in most solvents, including water. In both Tentagel and Argogel, swelling in organic solvents, polyethylene glycol is grafted on 1-2% cross-linked polystyrene-divinylbenzene with loading capacity 0.3 mmol/g. Argogel is claimed to be more chemically stable. Soluble Polvstvrene* Aminornew
Polvethvlene Glvcol*
Merrfield
\, TentaGelTM
X= OH, NHz, others
effective in association with a soluble support, which can be considered a protecting group, as exemplified by the removal of boronate diester from 4,6-positions of galactose by Amberlite IRA-743 [39].
10.4 One-Phase Systems (Syntheses in Solution) In the synthesis of oligosaccharides, a soluble polymer is bound to the first member of the oligosaccharide chain by a linker permitting removal of the polymer from the
248
I 0 Polymer-Supported Synthesis of Oligosaccharides
completed oligosaccharide, after the completion of the synthesis. Most physical properties of such synthons, are dictated by the polymer and they are not much different from those of the polymer, unless oligosaccharides on the polymer become very large. The polymer can be linked to either the acceptor or the donor. Polyethyleneglycol o-monomethylether (MPEG) and linear polystyrene have been investigated, although the latter has been used only infrequently (Table 1). Polyethyleneglycol = ,-monomethylether (MPEG)
MPEG of different average molecular weights is commercially available [40]. MPEG of average m.w. = 5,000 are most frequently used, but m.w. = 2,000 and m.w. = 12,000 were used as well. Depending on size, MPEG forms rigid rod-like structures stabilized by intramolecular interactions [41]. It is soluble in most apolar solvents (benzene, toluene, methylene chloride, chloroform, acetonitrile, acetone) as well as in water, and insoluble in hexane, diethylether and t-butylmethyl ether, and cold ethanol. It can be dissolved in hot tetrahydrofurane and ethanol, therefore it is commonly purified by reprecipitation from ethanol. Otherwise it is purified by precipitation with an ether from its solutions in the organic solvents cited above. Although MPEG is strongly hygroscopic, the adsorbed water can be almost completely removed by azeotropic distillation, e.g. with toluene or benzene; wet MPEG precipitates with ether only reluctantly. t-Butylmethyl ether is safer to use for the precipitation than diethylether since its boiling and flash points are higher [42]. Recovery of MPEG derivatives is usually no less than 95%. This recovery is substantially lower when MPEG is complexed with metal cations (e.g. mercury, chromium, cerium). Therefore many common metal-containing reagents cannot be used with substrates containing MPEG. MPEG also precipitates less easily and is more difficult to filter if it carries higher molecular weight synthons. Precipitations with alcohols (e.g. methanol) give similar results. To improve precipitation properties of MPEG when the synthesized oligosaccharide extends beyond five monosaccharides and its protected form approaches m.w. 5,000, a suitable remedy is to use MPEG of average mw = 12,000 [43]. Also, shorter MPEG of average mw = 2,000 was used in some cases, and instead of the precipitation a filtration through a silica gel column was used for purification [44]. Although MPEG is stable enough under many reaction conditions that it can be considered a protecting group, some conditions cause its partial degradation [29]; for instance pyridinium salts peel off one or more ethylene glycol units [45]. Furthermore, MPEG cannot be located directly on the anomeric carbon. Treatment of an MPEGyl glycoside, e.g. 2,3,4,6-tetra-O-acetyI-~-D-galactoside with a glycosylating agent such as acetobromomannose causes a glycosyl exchange to take place is formed, and soluble glycolyl and MPEGyl 2,3,4,6-tetra-O-acetyl-a-D-mannoside or (bis)glycolyl 2,3,4,6-tetra-O-acetyl-p-D-galactoside can be isolated from the mother liquors [46] after the MPEG-bound saccharide is precipitated (Scheme 6). The solubility of MPEG in organic solvents allows monitoring of the progress of the synthesis by high field 'HNMR spectroscopy using the signal of its single OCH3 group (6 = 3.380 ppm) as internal standard. Since the glycol peak of MPEG at
10.4 One-Phase Systems (Syntheses in Solution)
249
AcO-7
Ace% AcO
OAc
OCH~C€IZ(OCH~CH~),,OCH~+
AcoA%AcO X
1 AcoA* AcO
Promoter
+
Scheme 6. An example of instability of polyethylene glycol directly attached to the anomeric carbon.
6 = 3.640 ppm is very strong, it must be suppressed by irradiation for 3.0 s to increase the dynamic range and obtain a good quality spectrum. Despite this, sufficient structural information to identify the product can be usually obtained from its 'HNMR spectrum. Linear Polystyrene As with MPEG, linear polystyrene is soluble in benzene, toluene, chloroform, and insoluble in alcohols (methanol) and ethers (e.g. 2-methoxyethyl ether). The first monosaccharide was linked via thioglucoside to chloromethylated polystyrene (prepared from the polymer using chloromethyl methyl ether in the presence of stannic chloride) in benzene solution, and subsequent glycosylations were also performed in benzene. Polymer bound disacharides were isolated by precipitation using 2-methoxyethyl ether or methanol. Although the original report seemed unpromising [ 18b], an application of recent methods of oligosaccharide synthesis is desirable since linear polystyrene should be stable under conditions incompatible with the use of MPEG. Linear polystyrene to which a protected monosaccharide was bound directly was also prepared by copolymerization of styrene with four different derivatives of protected glucose substituted on C-6 with 4-vinylbenzoate, and some reactions have been investigated [ 19a].
250
10 Polymer-Supported Synthesis of Oligosaccharides
10.4.1 Linkers First members of growing oligosaccharide chains usually are not bound to polymeric supports directly but through a linker. Two major reasons for this arrangement are that a too short span between the carbohydrate and the support may create interactions detrimental to the course of the synthesis (however, some reactions may be improved [2]). Thus the tether maintains a safe distance between the support and the growing oligosaccharide chain. The major reason for inserting a tether is that it allows for an easy release of the carbohydrate from the polymer support. Usually the direct bond between the saccharide and the support would be stronger than the glycosidic bonds between two monosaccharides making impossible the removal of the oligosaccharides from the supports. Should the bond between a saccharide and the polymer involve the anomeric carbon, this bond often would be too labile to tolerate all reactions required by the synthetic sequence without a partial decomposition of this bond. Several linkers were examined for one phase systems (Table 2). The choice of a linker is determined by the reactions employed in the reaction sequence and on the form of the final oligosaccharide, which further depends on the eventual use of the oligosaccharide and the possible utilization of the linker or a part of it. As in any organic chemical synthetic scheme, reagents and protecting groups must act in harmony; soluble polymers-linkers can be regarded as special protecting groups. The following examples will illustrate such criteria. Succinoyl Diester The extensive use of succinoyl diester in polymer-supported solution syntheses of oligopeptides and oligonucleotides invited its application in polymer-supported synthesis of oligosaccharides. Since it is essential that both the protective groups and the linker are stable during the synthetic sequence, it is obvious that the baselability of ester linkages prohibits temporary protection with ester groups to be removed before the completion of the synthesis. Particularly unstable are ester groups located on anomeric carbons. Moreover, suitably located succinoyl-MPEG groups are prone to migrations in acidic environment, for instance 2-O-SuMPEG+ 3-0-SuMPEG in acceptor P-mannosides with OH-3 unprotected [47]. Despite these restrictions the acceptor-bound succinoyl-MPEG [48] was found to permit glycosylations with glycosyl trichloroacetimidates promoted by boron trifluoride, triflic anhydride, and trimethylsilyl or triethylsilyl triflates, with glycosyl halogenates promoted by silver triflate, and with thioglycosides promoted by the iodonium ion. It is compatible with ester long-term protection, with allylic, benzylic type (including benzylidene-type acetals), and certain silicon-based protecting groups, and deprotection with hydrogenolysis. To synthesize an oligosaccharide, MPEG is bound to a hydroxyl of the first monosaccharide through the succinoyl diester linker (Scheme 1). Next, another hydroxyl of this monosaccharide is deprotected, glycosylated, temporary protection on the second monosaccharide is removed and the above process is repeated until the required oligosaccharide is synthesized. Although incomplete glycosylation can
0
-S-Polystyrene
-0CHz \ /-CHzO-PEG
CH&BnOH
3) Acetolytic PEG Removal with Sc(OTO3/AczO
Removal
2) Hydrogenolytic PEG-DOX
1) Reductive PEG Removal
'Abbreviations. Donors: A= Trichloroacetimidate; B= Bromide; C= Sulfoxide; D= ThioalkyVaryl; E= Fluoride
7) Thio
6 ) DOX
Removal
!) Hydrogenolysisfor Complete
Removal
18b
55
54
54
27
0
) NaOMdMeOH for PEG
5 ) Benzylphenol
53
TEA/DCM
44
Dioxirane oxidation then NaOMmeOH
4) Fluorene
2,209 49, 51,52,91
Reference
Base-catalysed (DBU; NaOMe)
Removal
44
Donor(s) Used'
Hydrogenolysis
-0CHZ \ 1-O(CH&COO-PEG
-OCO(CHz)zCO-PEG
Structure
3) Phenylacetamide
2) Thioethanol
1) Succinoyl
Linker
~
Table 2. Major linkers used in one-phase (solution) oligosaccharide synthesis showing chemical structure of the linker, glycosylating agent. and removal methods used.
252
10 Polymer-Supported Synthesis of Oligosaccharides
be minimized by using glycosylating agents in excess, it is probably more efficient to add an excess of the glycosylation agent in several portions. The synthesis of the methyl heptaglucoside 1 is an excellent example of a regioselective glycosylation of the primary hydroxyl in 4,6-diol using succinoylated MPEG and iodonium promoted glycosylations with thioglycosides [49]. After the glycosylation, the unreacted OH-4 was capped by acetylation. A comparison with a classical solution synthesis [50]confirmed, as expected, that the MPEG-supported synthesis was significantly faster. Another example of a synthesis using MPEG-Subound acceptor are syntheses of heparan sulphate-like oligomers up to a dodecamer [51]. MPEGSu- can be also linked to a donor as shown in the synthesis of an oligomer of N-acetylneuraminic acid [521. Succinoyl diester has been further modified by linking to 9-hydroxymethylfluorene (Table 2) and using amino MPEG (i.e. MPEG terminating with amino group). Increasing sensitivity of the linker towards bases, this modification made it possible to distinguish between base-labile long-term protecting groups such as pivaloyl on the oligosaccharide while removing it from the polymer-linker with a mild base [53]. Dioxyxylyl Diether (DOX) The base-lability of succinoyl diester linker severely limits the selection of protecting groups available for an oligosaccharide synthesis, so a more versatile tether was required. A diether bond of a,a'-DiOxyXylyl diether, - O C H Z C ~ H ~ C H ~(DOX) O-, [54] is not limited by restriction of the succinoyl linker: (i) bound via a hydroxyl or as an 0-glycoside, it is stable under many reaction conditions including glycosylation; (ii) it is easily removable from a finished oligosaccharide by hydrogenolysis under mild acidic condition; (iii) using suitable hydrogenolytic conditions MPEG only is removed and DOX is converted into 4-methylbenzyl protecting group; (iv) ,using scandiumtictriflate/aceticanhydride[551 DOX can be converted into 4(acetoxymethy1)benzyl protecting group by removal of MPEG only (Scheme 7); (v) it is easily accessible from a,a'-dichloro-p-xylene. The MPEG-DOX grouping is readily synthesized from MPEG-OH and c1,a'dichloro-p-xylene by Williamson ether synthesis yielding MPEG-DOX-CI, and, after hydrolysis, the alcohol MPEG-DOX-OH [ 541. MPEG-DOX-OH can be
10.4 One-Phase Systems (Syntheses in Solutiorz)
Carbohydrate Portion
253
QiQxy Xylyl Portion u MetbylPEG Portion
Carbohydrate-OCH
HJH+
AczO/DCM Carbohydrale-OH
I?$?
CH,C,
,CCH,
Carbohydrate-OCH,
Scheme 7. An example of a versatile linker that can be transformed during or at the end of a synthetic sequence into other synthetically useful forms.
glycosylated to attach a saccharide at the anomeric position (an impossible connection through succinoyl tether [20, 461) to create an MPEG-supported acceptor. In a practical example (Scheme l), the MPEG-DOX-OH is glycosylated by 2-0acety1-3,4,6-tri-O-benzyl-a-D-mannopyranosyl trichloroacetimidate. The 2-0-acetyl group as a short-term protecting group can be easily deacetylated to a hydroxyl acceptor for the next glycosylation; it also serves for steric control of glycosylation via neighbouring group participation. Repetition of the glycosylation and hydrolytic steps gave the expected protected MPEG-DOX-bound pentasaccharide. Standard deprotection including the removal from the polymer support yielded the desired pentamannopyranoside [Manp(al-2)]4Manp(2). Target oligosaccharides are often contaminated with impurities originating in deprotection steps which usually can be removed by chromatography of peracetylated oligosaccharides. Peracety-
254
10 Polymer-Supported Synthesis of Oligosaccharides
lated oligosaccharides are commonly more suitable for final purification by chromatography than deprotected compounds; after deacetylation pure oligosaccharides are obtained readily [56]. The attachment of MPEG-DOX to a carbohydrate hydroxyl other than the anomeric one is achieved by the reaction of such hydroxyl with MPEG-DOX-Cl by Williamson ether synthesis [541. Syntheses of oligosaccharides on MPEG, tethered with dioxyxylyl (DOX) and related 4-oxybenzyloxy diethers, can be performed even more speedily using orthogonal glycosylation [27] and multicomponent carbohydrate [ 571 coupling strategies, which are described in detail elsewhere in this Volume. 10.4.2 Chemistry Investigations In addition to its use as polymer support for oligosaccharide syntheses, MPEGDOX-OH established itself as a useful aide for investigations of problems associated with glycosylations. This is possible because electrophiles competing for nucleophilic acceptors with glycosylating agents can be readily identified after precipitating products of reaction between such electrophiles and MPEG-DOX-OH. These competing reactions complicate results of glycosylations including those polymer supported; therefore it is urgent to suppress them as much as possible. Better understanding of the competing reactions makes this objective realizable. Thus it was established that the trans (p) anomeric specificity of some 2-acetamido glycosylating agents does not require oxazoline intermediacy, and that formation of oxazolines represents a side reaction. It was further found that oxazoline cannot be an obligatory intermediate since MPEG decomposes under the conditions required
10.5 Two-Phase Systems (Syntheses on Solid Supports)
I
BnTBnO
OBnOBn
O
A
’h3q
TBSO
TBSO
SMe
Ph-
255
BnO SMe
OBnOBn
OH
Scheme 8. “Reverse” use of a polymer support for the synthesis of P-mannoside linkage. The synthetic helper arm (MPEG) is removed from the desired product by simple precipitation.
by oxazoline intermediacy [58]. The origins of acyl transfer from 2-acyloxy glycosylating agents to acceptors [59a] have been studied in combination with calculations based on density functional theory [59b] in order to eliminate this very common side reaction. Novel reactants in glycosylation reactions can be efficiently evaluated using MPEG-DOX-OH as illustrated by the investigation of dimethylboron triflate as a promoter [60]. The formation of the P-mannoside linkage by intramolecular aglycon delivery [61] was modified to be performed on MPEG support: in the last step of the synthesis the j3-mannoside remains in solution after precipitation of MPEG-bound byproducts (Scheme 8) [62].
10.5 Two-Phase Systems (Syntheses on Solid Supports) Polymers used as a support for oligosaccharide synthesis should be commercially available or easily modifiable, for instance by introduction of spacers and linkers. The supports should be both mechanically and chemically stable, and have reasonable loading capacity. Three main types of solid support for oligosaccharide syntheses were essentially “borrowed” from oligonucleotide and oligopeptide solid state syntheses (cf. Table 1): (i) functionalized controlled pore glass (CPG); (ii) functionalized polystyrene cross-linked with divinylbenzene (e.g.: Merrifield, Wang, Rink resins); and (iii) crosslinked polystyrene resin with grafted functionalized polyethylene chains (TentaGel, Argogel). The last two types of supports (ii and iii) were compared as to their reaction kinetics. It was concluded that, contrary to the
256
I0 Polymer-Supported Synthesis of Oligosaccharides
common belief, reactions on PEG-containing beads do not proceed faster and that the kinetics depends on the reaction rather than on the support [63]. Compared to one-phase syntheses, monitoring progress of the synthesis of twophase syntheses presents a problem because of the insolubility of the support. Usually the product is detached from a portion of the polymer-bound material and analyzed by convential techniques, such as TLC, NMR spectroscopy and mass spectrometry. Monitoring of the progress of the synthesis without the loss of material by high resolution magic angle spinning NMR spectroscopy of solids is an important development [641. Controlled Pore Glass This has become the support of choice for oligonucleotide syntheses. It is mechanically quite sturdy, does not change its volume in different solvents, and it withstands fairly strong chemical conditions to which it is exposed for a short time (for instance, in an oligonucleotide synthesizer. However, it becomes unstable on prolonged exposure to bases and acids [65] and has a very low loading capacity. Cross-Linked Polystyrene This support was well tested for oligopeptide syntheses, and this led to its adoption for solid state synthesis of oligosaccharides. It has to be preswollen before the synthesis, and it also changes its volume during the synthesis as the synthesized chain becomes more voluminous. It is acid and base resistant, and can be used with linkers cleavable by photolysis, oxidation, fluorolysis, and several other conditions (Table 3). However, it does not swell in some solvents, for instance diethylether. Macroporous highly cross-linked poly(styrene-co-divinylbenzene) resins [661, such as Argopore [67],maintain a constant volume in organic solvents, including protic solvents, and even water. Polyethyleneglycol Grafts on Cross-Linked Polystyrene While the above mentioned supports are rigid, functionalized polyethyleneglycol chains on polystyrene in TentaGel [68] and Argogel [69] are highly mobile and can be considered to be in “solution” [70]. TentaGel (Table 1) is used extensively for oligonucleotide and oligopeptide syntheses; because of the polyethyleneglycol nature of the support, somewhat diminished stability towards acids would be expected. Because of the polystyrene matrix, Tentage1 should be swollen before use; swelling of its beads was determined to be higher or comparable to 2% cross-linked polystyrene-divinylbenzene beads [71]. It has been observed that the purity of oligonucleotides synthesized on TentaGel is significantly better than after synthesis on Teflon or CPG supports [65]. It is not known if this applies also to the synthesis of oligosaccharides. Mechanically TentaGel is more delicate than crosslinked polystyrene supports. Properties of Argogel are similar to those of TentaGel but it is claimed that its chemical stability is significantly improved (cf. Table 1 for its chemical structure), and also because of its double loading capacity.
~~
Linker
*
-
0 1 0 CPG 0,
O2N
= TG = PS(C)
,0(CH&O-PS
-NHCO(CH~)SCONH-AP
-0-CH2-p
CH3
-0(CHZ)~-S~-(CH~)&ONHCH~-PS
I I
CH3
-So-OCH2CONH-TG
A
-OCO(CH2)2CONHCH2COO-TG
-O-CO(CH2)2CO-NH-PS
-O-CO(CH2)2CO-NH-TG
-0-CO(CH2)2CONH-CPG
\
-S(CH2)3Si-
/
/
Structure
D. H. Rich, S. K. Gurwa, J. Am Chem. SOC.,1975,97,1575.
F
b. Rich's Linker
9) a. Pentenyl
I) Nitrobenzyl
r) p-Acylaminobenzyl
butylcarbonamide
i ) Ethoxydimethylsilyl-
acetamide
); QMercaptophenol-
I)CMercaptophenol
3) Sucanoyl-glycine
1) Succinoyl
1) Mercaptopropyl
~
Donor(s) Used
34,36b
32% Aq. Ammonia
NIA
hv /THF
DDQ/DCM, H20
NIA
Hg(OCOCF3)2 DCM, H 2 0
NaOMe/MeOH
37
32€
67
32j
S4,86
32a
82
36b
36b, 77
32%Aq. Ammonia
32%Aq. Ammonia
36a
~
Reference
NBS in THF/MeOH with DTBP
Removal
Table 3. Major linkers used in two-phase (solid state) oligosaccharide synthesis showing chemical structure of the linker, glycosylating agent, and removal methods used.
-S(CH&O-PS(M)
A
32b, 32i
3%
DMTSB-DIPEA DCM/MeOH NBSlDTBP
32e
32e
21
TrBFflCM
NaOMelMeOH
TBAFl HOAc
24,25b
22
32d
1)hvmHF for Partial Removal 2) PhSSiMeflnI2 for Conversion to Thioglycoside TBAF/ HOAc TBAM HOAc
32b
73
Reference
NaI, then NaOAc or N d
AcOH/THF/HzO
Removal
'Abbreviations. Donors: A= Trichloroacetimidate; B=Thioalkyl/aryl; C= Sulfoxide; D= n-Pentenyl; EFluoride; F= Glycal Polymers: CPG= Controlled-Pore Glass; TG= TentaGelR; PS(M)= Merrifield Resin; PS(C)= Polystyrene-Graft Crown (Cbiron Mimotopes, San Diego, CA, 92121); AP= ArgoPoreR
b. Mercaptopropanol
A
1s) a. Mercaptothiopropanol
A
F
F
F
Donor(s) Used
A
-S(CH&-S-PS(M)
R= Ph, 'Pr
R=pb R= 'fi
OZN
-TPS R
I
R
-S02(CH&-PS(M)
Et
I
-Si-(CH2)4-PS
I
Et
Structure
b. p-Oxybenzyl
14) a. p-Alkoxybenzyl
13) Diaryl(a1kyl)silyl
11) Sulfonyl
10) Diethylsilyl
Linker
Table 3 (continued)
10.5 Two-Phase Systems (Syntheses on Solid Supports)
259
10.5.1 Linkers
As in one-phase polymer supported synthesis, linkers must not interfere with the synthetic procedure, must be completely compatible with all synthetic steps, and must facilitate easy removability under conditions which harm neither the oligosaccharide nor the support. Many linkers have been adopted from other branches of solid state chemistry, others have been designed de now, and many more will be designed according to the requirements of a particular synthesis. It should be noted, of course, that a more general practical adoption of polymer-supported synthetic methodologies depends to a certain extent also on the general employability of a linker. Linkers utilized in the two-phase synthesis of oligosaccharides are surnmarized in Table 3. Dialkyl- or Diaryl-Silyl In some cases the solid support, i.e. polystyrene, has to be functionalized first, and the novel functional group represents the linker. The case of silicon-based linkers is a fitting example since it is easily removable by fluoride anions (e.g. tetrabutylammonium fluoride) and yet stable under most conditions utilized in oligosaccharide synthetic strategies. Cross-linked polystyrene-divinylbenzeneis first randomly lithiated [72],followed by reaction with diphenylsilyldichloride to create polystyrene-bound silyl chloride [24,271 which can be linked to a carbohydrate hydroxyl. The relative instability of the diphenylsilyl group to acidic conditions led to the replacement of the phenyl group with the isopropyl group [2Sb], using diisopropylsilyldichloride. Diethylsilyl functionality indirectly linked to the polymer [ 731 was investigated as a linker in a series of glycosylations under different conditions. Another promising silicon-based linker is S-(2-oxyethyldimethylsilyl)valeroyl[32JI which, in analogy with the cleavage of (trimethylsily1)ethyl glycosides [74],is transformed into an anomeric ester, e.g. propionate, by treatment with BF3.EtzO and propionic anhydride (other acyl anhydrides most likely can be used as well). From this glycosylating agents such as trichloroacetimidate can be subsequently prepared. The amidic bond between the valeric acid of the linker and the amino group of the functionalized polymer is sufficiently stable under most conditions used in oligosaccharide synthesis. Thioglycoside Linkers Thioglycosidic bonds are admirably stable under most conditions used for oligosaccharide synthesis and yet can be cleaved with many reagents, such as mercuric trifluoroacetate, N-bromosuccinimide with 1,6-di-t-butylpyridine, dimethylmethylthiosulfonium triflate or tetrafluoroborate, or oxidatively using dioxirane followed by NaOMe in methanol. Therefore cross-linked polystyrene utilizing thioglycoside [32a], or sulfide-based linkers are commonly used strategies [32h, i]. In fact, they were among the first linkers used in the two-phase oligosaccharide synthesis. 1,4Thiophenol, 1,3-propanedithiol, 1,3-thiopropanol, 2-(4-thiophenyloxy)acetamide have also been linked to the functionalized polystyrene support.
260
10 Polymer-Supported Synthesis of Oligosaccharides
1
THF J.
Reducing Oligosaccharide
ProtectedGlycoside
Scheme 9. A photochemically removable linker (top) can be combined with an ester funcionality (bottom) to increase versatility of the tether.
Linkers Cleavable by Photolysis The o-nitrobenzyl protection group has been used in oligosaccharide syntheses infrequently [14e], since it requires a long period (15-20 hours) of irradiation in a photochemical (e.g. Rayonet) reactor at 350 nm. The photoredox process can be substantially accelerated by the presence of a methoxy group in p-position to the nitrogroup [75]. However, the scope of the reaction is limited to furnishing upon photolysis a reducing saccharide when the tether is hooked to the anomeric carbon. The versatility of this type of linker, first used for an oligosaccharide synthesis by Zehavi in 1973, was recently expanded by Nicolaou's group (Scheme 9). In this novel form of the linker the nitrobenzyl moiety was connected to the anomeric carbon via 4-hydroxy benzoyl ester; therefore photolysis results in removal of the polymer producing a glycoside of p-hydroxy benzoyl ester. Alternatively, treatment with trimethylsilyl thiophenol/zinc iodide converts the complete linker assembly into a thioglycoside, an excellent glycosylating reagent.
10.6 Examples of Syntheses It would be of interest to evaluate different polymer-supported synthetic strategies for the preparation of the same oligosaccharides. To date, two oligosaccharides have been synthesized using both the one-phase system on MPEG and the two-
10.7 Combinatorial Libraries
261
phase system using crosslinked polystyrene. One example is the synthesis of the heptasaccharide 1. One method [32c] utilized phenolic polystyrene and the photolabile 5-hydroxy-2-nitro-benzyl alcohol tether, while the other [491 employed MPEG and the succinoyl linker. Because of the different synthetic designs a meaningful comparison of the two supports, such as to the advantages of one support over the other, is not possible. The second example is the synthesis of the pentamannoside [Manp(al-2)I4Manp (2). Mannose oligosaccharides are superbly suited to these studies since a-anomers are formed exclusively under appropriate glycosylation conditions. The one-phase (solution) system used MPEG and the DOX linker [54] and the two-phase system (solid state) utilized crosslinked polystyrene and a thioglycoside linker [32h]. To monitor reactions, the one-phase strategy took advantage of the support’s solubility to use NMR spectroscopy; the two-phase strategy employed cleavage of a small sample from the solid support, and analyzed the sample by either NMR spectroscopy or mass spectrometry (MALDI-TOF) [32h]. The results of these pentamannoside syntheses are not fully comparable since the perbenzylated pentamannoside obtained by the two-phase method was not deprotected. As noted before, the use of cross-linked polystyrene support for syntheses of oligosaccharides by the glycal strategy is discussed elsewhere in this Volume. In Ogawa’s group the p-alkoxybenzylether linker, used in one phase polymer supported synthesis, was utilized in combination with Wang’s resin to synthesize polylactoseamines of various lengths [ 32eI. Finally, polymer-supported synthesis has been used to synthesize uncommon monosaccharides [761. Controlled pore glass has been used to synthesize an a1-2 trimannoside [36a], several disaccharides [36b], and glycosylated oligonucleotides [77]. The examples confirm that CPG-supported oligosaccharide synthesis is feasible, although its limited loading capacity and scale-up may eventually become a restricting factor. TentaGel-N, linked via the succinoyl linker to a monosaccharide acceptor was utilized using trichloroacetimidate chemistry to prepare disaccharides [34] and trisaccharides [78]. It was observed that TentaGel is slightly unstable under the acidic conditions required by trichloroacetimidate chemistry [78], most likely due to the acid sensitivity of PEG [79]. No similar sensitivity was observed in similar applications employing Argogel [ 781.
10.7 Combinatorial Libraries Polymer-supported strategies of oligosaccharide syntheses are ideally suited to preparations of combinatorial oligosaccharide libraries since purification of all products of a particular synthetic step is done at the same time and it would not be expected that individual library members would be lost during the purification process. Both one-phase and two-phase polymer supported strategies have been examined, and a review was published recently [go]. More recent articles that were not discussed in this review include: syntheses of disaccharide [81] and trisaccharide 1821 libraries leading to the discovery of novel antibacterial agents [83], and prepa-
262
I0 Polymer-Supported Synthesis of Oligosaccharides
ration of libraries on TentaGel beads as polyvalent molecules [84]. Often carbohydrates (including oligosaccharides) form a scaffold for a library [85],and the variations in carbohydrate structure may have an effect on biological activity of the constructs in the library [86].
10.8 Capping Capping, or protection of the unreacted hydroxyl to prevent it from reacting in the next condensation step, is a very important operation in oligonucleotide automatic synthetic protocols. In oligosaccharide synthesis capping seems to be much less important, although sometimes unreacted hydroxyls are acetylated to prevent their glycosylation in the next step [49]. However, it seems more useful to repeat the glycosylation step, in analogy with syntheses of “difficult sequences” in oligopeptide synthesis, rather than to use capping. It should be noted that important variants of polymer supported synthesis of oligosacharides, such as the “self-policing” glycal strategy (Scheme 3 ) or strategies involving a hydrophobic handle (Scheme 5), do not require capping. The last mentioned strategy eventually may become more generally adopted.
10.9 Concluding Remarks The polymer-supported synthesis of protected oligosaccharides has attracted considerable attention of organic chemists during the last decade of the 20th century. Full reviews dedicated to the polymer supported techniques of oligosaccharide synthesis were published recently [26, 871, significant parts of reviews on oligosaccharide synthesis have been reserved for polymer-supported methods [5g, 88, 891 and parts of reviews on polymer-supported methodology have been dedicated to oligosaccharide syntheses [ 31. As well, a review on combinatorial carbohydratebased libraries was recently published [90]. It appears to be a suitable methodology for the synthesis of oligosaccharides in quantities required for biomedical applications; for instance, fragments of the capsular polysaccharide of Huemophilus inflenzae were synthesized both by one-phase [91] and two-phase [92] polymer supported methods. It is clear that the methodology, while not perfect, will become more widely used, as better and more efficient reaction conditions are discovered and the essential components become less expensive. References 1. D. Zopf, S. Roth, The Lancet, 1996,347, 1017-1021. 2. 0.-T. h u n g , S. P. Douglas, D. M. Whitfield, H. Y. S. Pang, J. J. Krepinsky, New J. Chem., 1994, 18, 349-363.
References
263
3. D. J. Gravert, K. D. Janda, a) Chem. Revs., 1997, 97, 489-509, b) Current Opin. Chem. Biol., 1997, I , 107-113. 4. For recent reviews, see e.g. a) K. Jarowicki, P. Kocienski, J. Chem. Soc. Perkin Trans. I , 1999, 1589-1615; b) 0. Seitz, Angew. Chem. Int. Ed. Engl. 1998,37, 3109-3111. 5. For reviews, see e.g. H. Paulsen, Angew. Chem. Int. Ed.Eng1. a) 1982,21, 155-224; b) 1990, 29, 823; c) H. Paulsen, Revs Chem. Soc. 1984, 13, 15-45; d) R. R. Schmidt, Angew. Chem. Int. Ed. Engl. 1986, 25, 212; e) P. Sinay, Pure Appl. Chem. 1991, 63, 519-528; f ) K. Toshima and K. Tatsuta, Chem. Revs., 1993, 93, 1503-1531; g) R. R. Schmidt, J. C. Castro-Palomino, 0. Retz, Pure Appl. Chem. 1999, 71, 729-744; h) R. R. Schmidt, Pure Appl. Chem. 1998, 70, 397-402. 6. E. g. Modern Methods in Carbohydrate Synthesis (Eds.: S. H. Khan, R. A. O’Neill), Harwood Academic Publishers, Amsterdam, 1996. 7. For instance: a) K. J. Kaur, G. Alton, 0. Hindsgad, Carbohydr. Res., 1991,210, 145; b) C.-H. Wong, Y. Ichikawa, T. Krach et al. J. Am. Chem. Soc., 1991, 113, 8137; c) S. Roth, US Pat. 5,180,674, Jan 19, 1993; d) Y. Ichikawa et al. J. Am. Chem. Soc., 1992, 114, 9283; (e) for a review, see Y. Ichikawa, G. C. Look, C.-H. Wong, Anal. Biochem., 1992,202, 215. 8. a) J. Thiem, B. Sauerbrei, Angew. Chem. Int. Ed. Engl., 1991, 30, 1503-1505; b) B. Sauerbrei, J. Thiem, Tetrahedron Lett., 1992, 33, 201-204. 9. For instance, G. C. Look, Y. Ichikawa, G.-J. Shen, P.-W. Cheng, C.-H. Wong, J. Org. Chem., 1993, 58,4326-4330. 1994, 116, 1135-1136. 10. M. Schuster, P. Wang, J. C. Paulson, C.-H. Wong, J. Am. Chem. SOC., 11. a) U. Zehavi, M. Herchman, Carbohydr. Rex, 1984, 128, 160-164; b) U. Zehavi, S. Sadeh, M. Herchman, Carbohydr. Res., 1983, 124, 23-34; c) U. Zehavi, M. Herchman, Curbohydr. Res., 1986, 151, 371-378. 12. T. Wiemann, N. Taubken, U. Zehavi, J. Thiem, Carbohydr. Res., 1994,257, C1LC6. 13. S.-I. Nishimura, K. Matsuoka, Y. C. Lee, Tetrahedron Lett., 1994, 35, 5657-5660. 14. a) J. M. J. Frechet, in Polymer-Supported Reactions in Organic Synthesis (P. Hodge, D. C. Sherrington, Eds.), Wiley, Chichester, 1980, pp. 293 and 407; b) J. M. J. Frechet, C. Schuerch, J. Am. Chem. Soc., 1971, 93, 492-496; c) Carbohydr. Res., 1972, 22, 399-412; d) N. K. Mathur, C. K. Narang, R. E. Williams, Polymers as Aids in Organic Chemistry, Academic Press, New York, 1980, Chapter 6; e) U. Zehavi, Adv. Carbohydr. Chem. Biochem., 1988, 46, 179-204; f ) J. M. J. Frechet, Tetrahedron 1981, 37, 663-683. 15. R. Eby, C. Schuerch, Carbohydr. Res., 1975, 39, 151-155. 16. a) P. Westerduin, G. H. Veeneman, Y. Pennings, G. A. van der Marel, J. H. van Boom, Tetrahedron Lett., 1987,28, 1557-1560; b) G. H. Veeneman, S. Notermans, R. M. J. Liskamp, G. A. van der Marel, J. H. van Boom, Tetrahedron Lett., 1987,28, 6695-6698. 17. G. H. Veeneman, H. F. Brugghe, H. van den Elst, J. H. van Boom, Carbohydr. Rex, 1990,195, C1-C4 (after p. 308). 18. a) U. Zehavi, A. Patchornik, J. Am. Chem. Soc., 1973, 95, 5673-5677; b) S.-H. L. Chiu, L. Anderson, Carbohydr. Rex, 1976, 50, 227-238; c) G. Excoffier, D. Gagnaire, J.-P. Utille, M. Vignon, Tetrahedron, 1975, 31, 549-553; d) for a review see; A. Malik, H. Bauer, J. Tschakert, W. Voelter, Chemiker Ztg., 1990, 114, 371-375. 19. a) R. D. Guthrie, A. D. Jenkins, J. Stehlicek, J. Chem. Soc. ( C ) , 1971, 2690-2696; b) R. D. Guthrie, A. D. Jenkins, G. A. F. Roberts, J. Chem. Soc. Perkin Trans. I, 1973, 2414-2417. 20. S. P. Douglas, D. M. Whitfield, J. J. Krepinsky, J. Am. Chem. Soc., 1991, 113, 5095-5097. 21. For example, J. T. Randolph, K. F. McClure, S. J. Danishefsky, J. Amer. Chem. Soc., 1995, 117, 5712-5719. 22. S. J. Danishefsky, K. F. McClure, J. T. Randolph, R. B. Ruggeri, Science, 1993,260, 1307-1309. 23. C. M. Timmers, G. A. van der Marel, J. H. van Boom, Red. Trav. Chim Pays-Bas, 1993, 112, 609-6 10. 24. J. T. Randolph, S. J. Danishefsky, Angew. Chem. Znt. Ed. Engl., 1994, 33, 1470-1473. 25. a) P. H. Seeberger, M. Eckhardt, C. E. Gutteridge, S. J. Danishefsky, J. Am. Chem. Soc, 1997, 119, 10064-10072; b) C. Zheng, P. H. Seeberger, S. J. Danishefsky, J. Org. Chem., 1998, 63, 1126-1 130. 26. P. H. Seeberger, S. J. Danishefsky, Acc Chem. Res., 1998, 31, 685-695. 27. Y. Ito, 0. Kanie, T. Ogawa, Angew. Chem. Int. Ed. Engl., 1996,35, 2510-2512. 28. a) M. M. Palcic, L. D. Heeze, M. Pierce, 0. Hindsgaul, Glycoconjugate J., 1988,5,49-63; b) P. Stangier, M. M. Palcic, D. R. Bundle, Curbohydr. Res., 1995, 267, 153-159.
264
10 Polymer-Supported Synthesis of Oligosaccharides
29. a) J. M. Harris, J. Macromol. Sci.-Rev. Macromol. Chem. Phys., 1985, C25, 325-373; b) J. M. Harris in Poly(Ethy1ene Glycol) Chemistry: Biotechnical and Biomedical Applications (J. M. Harris, Ed.) Plenum Press, New York 1992, pp. 2. 30. a) E. Bayer, M. Mutter, The Peptides (E. Gross, J. Meienhofer, Eds.), Academic Press, New York, 1980, 2, 286; b) G. M. Bonora, C. L. Scremin, F. P. Colonna, A. Garbesi, Nucl. Acids Res., 1990, 18, 3155-3159; c) G. M. Bonora, G. Biancotto, M . Maffini, C. L. Scremin, Nucl. Acids Res., 1993, 21, 1213-1217. 31. J. J. Krepinsky, S. P. Douglas, D. M. Whitfield, in Methods in Enzymology, 242, Neoglycoconjugates, Pt. A: Synthesis (Y. C. Lee, R. T. Lee, Eds.), Academic Press, San Diego, CA. 1994, pp. 280-293. 32. a) L. Yan, C. M. Taylor, R. Goodnow, Jr., D. Kahne, J. Am. Chem. Soc., 1994, 116, 69536954; b) J. A. Hunt, W. R. Roush, J. Am. Chem. Soc., 1996, 118, 9998-9999; c) K. C. Nicolaou, N. Winsinger, J. Pastor, F. DeRoose, J. Am. Chem. Soc., 1997, 119, 449-450; d) K. C. Nicolaou, N. Watanabe, J. Li, J. Pastor, N. Winssinger, Angew. Chem. Znt. Ed. Engl., 1998,37, 1559-1561; e) H. Shimizu, Y. Ito, 0. Kanie, T. Ogawa, Bioorg. Med. Chem. Lett., 1996, 6, 2841-2846; f ) K. Fukase, K. Egusa, Y. Nakai, S. Kusumoto, Mol. Divers., 1996,2, 182-188; g) J. Rademann, R. R. Schmidt, Tetrahedron Lett., 1996, 37, 3989-3990; h) J. Rademann, R. R. Schmidt, J. Urg. Chem., 1997, 62, 3650-3653; i) J. Rademann, A. Geyer, R. R. Schmidt, Angew. Chem. Znt. Ed. Engl., 1998, 37, 1241-1245; j) D. Weigelt, G. Magnusson, Tetrahedron Lett., 1998, 39, 2839-2842. 33. L. Szabo, B. L. Smith, K. D. McReynolds, A. L. Parrill, E. R. Morris, J. Gervay: J. Urg. Chem., 1998, 63, 1074-1078. This series of sialooligomers (up to the octamer) are linked by (1+ 5 ) amidic bonds between two units of neuraminic acid and not by (2+) glycosidic bonds. 34. M. Adinolfi, G . Barone, L. De Napoli, A. Iadonisi, G. Piccialli, Tetrahedron Lett., 1996, 37, 5007-5010. 35. S. P. Douglas, J. J. Krepinsky, to be published. 36. a) A. Heckel, E. Moss, K.-H. Jung, J. Rademann, R. R. Schmidt, Synlett, 1998, 171-173; b) M. Adinolfi, G. Barone, L. De Napoli, A. Iadonisi, G. Piccialli, Tetrahedron Lett., 1998, 39, 1953-1956. 37. R. Rodebaugh, S. Joshi, B. Fraser-Reid, H. M. Geysen, J. Org. Chem., 1997, 62, 5660-5661. 38. S. Manabe, Y. Ito, T. Ogawa, Synlett, 1998, 628-630. 39. G. G. Cross, D.M. Whitfield, Synlett, 1998, 487-488. 40. Suitable MPEG mw = 5,000 was obtained from Fluka AG, Buchs, Switzerland. 41. a) Technical Bulletin: Polyglycols Hoechst. Polyethylene Glycols: Properties and Applications. Hoechst, Frankfurt (1983); b) J. Dale, Zsr. J. Chem., 1980, 20, 3. 42. t-Butylmethyl ether of sufficient quality and degree of dryness for this purpose is available inexpensively in bulk quantities from A R C 0 (Atlantic Richfield) Chemicals. 43. S. P. Douglas, D. M. Whitfield, J. J. Krepinsky, unpublished observations. 44. L. Jiang, R. C. Hartley, T.-H. Chan, J. Chem. Soc. Chem. Commun., 1996, 2193-2194. 45. S. P. Douglas, D. M. Whitfield, H. Y. S. Pang, J. J. Krepinsky, unpublished observations. 46. a) D. M. Whitfield, S. P. Douglas, J. J. Krepinsky, Tetrahedron Lett., 1992, 33, 6795-6798; b) S. P. Douglas, H. Y. S. Pang, J. J. Krepinsky, unpublished results. 47. M. Y. Meah, D. M. Whitfield, S. P. Douglas, J. J. Krepinsky, unpublished results. 48. J. J. Krepinsky, in Modern Methods in Carbohydrate Synthesis (S. H. Khan, R. A. O’Neill, Eds.), Hanvood Academic Publishers, 1996, pp. 194-224. 49. R. Verduyn, P. A. M. van der Klein, M. Douwes, G. A. van der Marel, and J. H. van Boom, Recl. Trav. Chim. Pays-Bas, 1993, 112,464-466. 50. R. Verduyn, M. Douwes, P. A. M. van der Klein, E. M. Mosinger, G. A. van der Marel, J. H. van Boom, Tetrahedron, 1993, 49, 7301-7311. 51. C. M. Dreef-Tromp, H. A. M. Willems, P. Westerduin, P. van Veelen, C. A. A. van Boeckel, Bioorg. Med. Chem. Lett., 1997, 7, 1175-1180. 52. L. 0. Kononov, Y. Ito, T. Ogawa, Tetrahedron Lett., 1997, 38, 1599-1602. 53. Y. Wang, H. Zhang, W. Voelter, Chem. Lett., 1995, 213-274. 54. S. P. Douglas, D. M. Whitfield, J. J. Krepinsky, J. Am. Chem. Soc., 1995, 117, 2116-2117. 55. S. Mehta, D. M. Whitfield, Tetrahedron Lett., 1998, 39, 5907-5910. 56. D. M. Whitfield, H. Y. S. Pang, J. P. Carver, J. J. Krepinsky, Can. J. Chem., 1990,68,942-952.
References
265
57. S. Manabe, Y. Ito, T. Ogawa, Molecules Online, 1998,2, 40-45. 58. G. Hodosi, J. J. Krepinsky, Synlett, 1996, 159-162. 59. a) R. U. Lemieux, Chemistry in Canada, 1964, 16, 14; b) A. Berces, I. Chrzczanowicz, A. Obuchowska, D. M. Whitfield, J. J. Krepinsky, J. Am. Chem. Soc., 1999, manuscript to be submitted, and references therein. 60. Z . G . Wang, S. P. Douglas, J. J. Krepinsky, Tetrahedron Lett., 1996, 37, 6985-6988. 61. F. Barresi, 0. Hindsgaul, a) J. Am. Chem. Soc., 1991, 113, 9376; b) Can. J. Chem., 1994, 72, 1447- 1465. 62. Y. Ito, T. Ogawa, J. Am. Chem. Soc., 1997, 119, 5562-5566. 63. W. B. Li, B. Yan, J. Org. Chem., 1998, 63, 4092-4097. 64. P. H. Seeberger, X. Beebe, G. D. Suckenick, S. Pochapsky, S. J. Danishefsky, Angew. Chem. Int. Ed. Engl., 1997, 36, 491-493. 65. B. P. Zhao, G. B. Panigrahi, P. D. Sadowski, J. J. Krepinsky, Tetrahedron Lett., 1996, 37, 3093-3096. 66. M. Hori, D. J. Gravert, P. Wentworth, Jr., K. D. Janda, Biorg. Med. Chem. Lett., 1998, 8, 2363-2368. 67. K. Fukase, Y. Nakai, K. Egusa, J. A. Porco, Jr., S. Kusumoto, Synlett, 1999, 1074-1078. 68. Rapp Polymere, GmbH, 7072 Tubingen, Germany. 69. Argonaut Technologies, 887 Industrial Blvd., Suite G, San Carlos, CA 94070, USA. 70. a) E. Bayer, Angew. Chem. Znt. Ed. Engl., 1991, 30, 113-129; b) E. Bayer, W. Rapp in Poly (Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications (J. M. Harris, Ed.) Plenum Press, New York 1992, pp. 325-345. 71. M. E. Wilson, K. Paech, W.-J. Zhou, M. J. Kurth, J. Org. Chem., 1998, 63, 5094-5099. 72. T.-H. Chan, W.-Q. Huang, J. Chem. Soc. Chem. Commun., 1985,909-911. 73. T. Doi, M. Sugiki, H. Yamada, T. Takahashi, Tetrahedron Lett., 1999, 40, 2141-2144. 74. K. Jansson, T. Noori, G. J. Magnusson, J. Org. Chem., 1990,55, 3181-3185. 75. H. Venkatesan, M. M. Greenberg, J. Org. Chem., 1996, 61, 525-529. 76. S. Kobayashi, T. Wakabayashi, M. Yasuda, J. Org. Chem., 1998, 63, 4868-4869. 77. M. Adinolfi, G. Barone, L. De Napoli, L. Guariniello, A. Iddonisi, G. Piccialli, Tetrahedron Lett., 1999, 40, 2607-2610. 78. S. P. Douglas, J. J. Krepinsky, unpublished observations. 79. A. A. Patel, I. Chrzczanowicz, J. J. Krepinsky; manuscript in preparation. 80. Z.-G. Wang, 0. Hindsgaul, Adv. Exptl. Med. Biol., 1998, 435, 219-236. 81. D. J. Silva, H. Wang, N. M. Allanson, R. K. Jain, M. J. Sofia, J. Org. Chem., 1999, 64, 59265929. 82. T. Zhu, G.-J. Boons, Angew. Chem. Int. Ed. Engl., 1998, 37, 1898-1900. 83. M. J. Sofia, N. Allanson, N. T. Hatzenbuhler, R. Jain et al, J. Med. Chem., 1999, 42, 31933198. 84. R. Liang, J. Loebach, N. Horan, M. Ge, C. Thompson, L. Yan, D. Kahne, Proc. Natl. Acad. Sci. USA, 1997, 94, 10554-10559. 85. a) T. Wunberg, C. Kallus, T. Opatz, S. Henke, W. Schmidt, H. Kunz, Angew. Chem. Int. Ed. Engl., 1998,37,2503-2505; b) M. J. Sofia, R. Hunter, T. Y. Chan, A. Vaughan, R. Dulina, H. Wang, D. Gange, J. Org. Chem., 1998, 63, 2802-2803. 86. R. Liang, L. Yan, J. Loebach, M. Ge, Y. Uozumi, et al, Science, 1996,274, 1520-1522. 87. U. Zehavi, Reactive & Functional Polymers, 1999, 41, 59-68. 88. a) H. M. I. Osborn, T. H. Khan, Tetrahedron, 1999,55, 1807-1850; b) Y. Ito, S. Manabe, Curr. Opin. Chem. Biol., 1998,2, 701-708. 89. a) G.-J. Boons, Tetrahedron, 1996, 52, 1095-1121; b) G.-J. Boons, Contemp. Org. Synth., 1996, 3, 173-200; c) D. M. Whitfield, S. P. Douglas, Glycoconj. J., 1996, 13, 5-17. 90. M. J. Sofia, Molec. Diversity, 1998, 3, 75-94. 91. A. A. Kandil, N. Chan, P. Chong, and M. Klein, Synlett, 1992, 555-557. 92. S. Nilsson, M. Bengtsson, T. Norberg, J. Carhohydr. Chem., 1992, 11, 265-285.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
11 Glycopeptide Synthesis in Solution and on the Solid Phase Hovst Kunz and Michael Schultz
11.1 Introduction Glycopeptides are defined as fragments of glycoproteins consisting of saccharide units which are covalently linked to a peptide backbone. The structural diversity emerging from the combination of amino acid and carbohydrate functionality is immense [l-51. Table 1 summarizes the known types of natural linkage types between amino acid side-chains and the first carbohydrate residue. The most abundant carbohydrates are highlighted and the corresponding structural details are depicted. In nature, especially in eucaryotic organisms, most proteins are glycosylated. This observation suggests that such a sophisticated result of evolution should be accompanied by increased functional complexity [6]. In fact, the last decade has revealed numerous fascinating insights into glycoprotein biology. In the course of this introduction both the typical biological role of such a complex glycoprotein and the characteristic preparative problems one faces will be demonstrated by the example of P-selectin glycoprotein ligand-1 (PSGL-1) [7]. Organisms defend themselves against inflammation by deploying white blood cells (leukocytes) which infiltrate the inflamed tissue [8-121. One of the first signals for patrolling leukocytes to migrate from the blood vessel into neighboring tissue is presented on the endothelium of the vessel. This signal is the expression of the glycoprotein receptor P-selectin. Initial weak adhesion to the ligand PSGL-1 located on the surface of leukocytes to this selectin leads to retarded leukocyte movement (‘rolling’). Several subsequent adhesion processes mediated by other recognition molecules finally lead to migration of the leukocytes through the endothelium. In chronic diseases such as rheumatoid arthritis or reperfusion injury a promising treatment could involve inhibition of the aforementioned selectin-ligand interaction [ 111. In this context, the selectin ligand itself is the first lead structure. Biological investigations have revealed the critical binding segment of PSGL-1 to be a sulfated oligopeptide located in the N-terminus and carrying an oligosaccharide terminated with sialyl Lewis’ on a defined threonine residue (Scheme 1) [7, 13, 141.
268
11 Glycopeptide Synthesis in Solution and on the Solid Phase
Table 1. Linkage regions in natural glycoproteins. Amino acid
Carbohydrate
Asparagine
N-Acetylgalactosamine N-Acetylglucosamine D-Glucose L-Rhamnose
Hydroxylysine
D-Galactose
Hydroxyproline
L-Arabinose D-Galactose
Serine
Galactose Glucose Xylose Fucose
Threonine
Linkage region
I HO
HNJ 0
+O+
Fucose
HO
Serine or Threonine
N-Acetylgalactosamine N-Acetylglucosamine Mannose
AcHN
R
R=H, CH3
Tyrosine
Glucose Galactose
,O
O
11.1 Introduction
s0:so:-
I /
so?-
269
HO
I
Q A T E Y E Y L D Y D F L P E T E P I
I
kHo..0H
' T O H
OH
COH
AcHN
OH
1
Sialyl Lewis' (%ex)
1 n=2-6 Scheme 1.
If PSGL-1 is not sulfated, the molecule is no longer recognized by P-selectin but it still serves as a ligand for another adhesion molecule, E-selectin [15]. Because PSGL-1 is a glycosylated protein the over-expression of this factor in a suitable cell-line is not a possible method for the synthesis of an effective compound. Such an approach would not deliver the sulfated and glycosylated form. On the other hand, PSGL-1 cannot be isolated from natural sources as a homogeneous compound in a suitable quantity for medicinal purposes. At this point recent developments of glycopeptide synthesis justify closer retrosynthetic analysis of compound 1, although in terms of size and complexity 1 constitutes quite a challenging target. Thus, the first step is simplification of the structure in such a way that biological activity and experimental feasibility are both taken into account. Biological results suggest that mainly the terminal tetrasaccharide (sialyl Lewis") is required for recognition. Furthermore, only one of the sulfated tyrosine residues is considered essential for binding. Thus, the minimum target molecule is represented by a sulfated heptapeptide carrying sialyl Lewis" (2) (Scheme 2) [ 161. Further retrosynthetic analysis of this structure splits the target into the corresponding amino acid building blocks required for solid-phase synthesis (Scheme 2). N- and side-chain-protected amino acid derivatives are commercially available except for the glycosylated threonine. The glycosylated key building block can be substituted by the more readily available glycosylated asparagine derivative which is constructed from suitably protected monosaccharides 3-6 and an aspartic acid moiety ready for glycosylation. For the realization of such a synthetic approach a detailed knowledge of glycosylation chemistry and protecting group strategy becomes important [4, 17, 181. Fundamental methods in both fields relevant to the synthesis of 2 shall therefore be outlined in the following paragraphs. 11.1.1 Which Protecting Groups are Suitable for Carbohydrates (Table 2)?
For 0-glycosylation, at least, the answer to this question depends mainly on the desired stereochemistry of the products. Ether type hydroxy-protecting groups which do not exert a neighboring group-effect, e.g. benzyl (Bzl), in the 2-position lead almost exclusively to glycosides with axial linkages (usually a ) to the used glycosyl acceptor if respective reaction conditions are applied (see below) [ 191. The
270
I 1 Glycopeptide Synthesis in Solution and on the Solid Phase H-Tvr(S032-)-Asp-Phe-Leu-Pro-Glu-Asn-OH ,OH
/
Fmoc-Amino Acids-OH and Fmoc-Asn(SLeX)-OH ======3 SLeX-N3
Scheme 2.
target molecule 2 contains two a-linked saccharides and, indeed, the fucose donor 4 is an ideal example of the rule of preferred cis-glycosylation employing benzylprotected sugars. The formation of the a-sialoside, however, cannot be influenced by an assisting group neighboring the anomeric center. Thus, acyl type protection of hydroxy groups, which is readily introduced into the valuable neuraminic acid, can be applied after protecting the carboxy group as the methyl ester (6). The remaining donors galactose 5 and glucosamine 3 should be equipped with a combination of ether- and acyl-type protection. This pattern ensures the orthogonal stability of protecting groups during the synthetic protocol. Benzyl ethers can be removed by hydrogenation and ester type groups are cleaved by use of catalytic amounts of base (table 2).
Table 2. Common protecting groups in carbohydrate chemistry. Protecting group
Introduction
Removal ~~
CsHsCH20CH3COO-COOCH3
NaH/benzyl bromide Pyridine/acetic anhydride H+/methanol
H2/Pd-C cat. NaOMe/MeOH dil. NaOH
11.1 Introduction
27 1
11.1.2 Which Glycosylation Methods are Useful for the Formation of Glycopeptides?
Again the range of methods useful for the formation of the different types of linkage will be illustrated using compound 2 as an example [4, 18, 20-231. Formation of Asparagine N-Glycosides The asparagine-glucosamine linkage is the most common type of linkage found in natural glycoproteins. Because asparagine cannot be used directly as a glycosyl acceptor the linkage is usually formed by coupling suitable glycosylamine (saccharide) and aspartic acid derivatives. The amino function of the saccharide is accessible either from unprotected saccharides and ammonium hydrogen carbonate [24] or via the hydrogenation of an intermediate azide which is prepared, for instance, from glycosyl halides in phase-transfer-catalyzed reactions with sodium azide [25]. Coupling of the glycosylamine to aspartic acid can be accomplished by use of established peptide-coupling reagents such as isobutoxyisobutyldihydroquinoline (IIDQ, Scheme 3) or TBTU [ 171. a-Fucosylation
A very efficient introduction of a-fucosyl moieties, e.g. into the 3- or 4-position of a glucosamine acceptor, is possible via the in situ anomerization procedure developed by Lemieux and coworkers [26]. Tri-0-benzylated fucosyl bromide 7 is a useful glycosyl donor in these cases [27]. Activation of this donor by catalytic amounts of Br- results in the intermediate formation of the reactive P-halide (Scheme 4).
4 HZ Ra-Ni
Acoq Aliquat
AcHN CI NaN3
N H ~IIDQJDIPEA
N)
AcHN
AcHN
T-
Fmoc-Asp(0H)OtBu
Scheme 3.
Scheme 4.
TNH
L~
272
I I Glycopeptide Synthesis in Solution and on the Solid Phase
OBzl 5
OBzl 8
Scheme 5.
The P anomer spontaneously reacts with nucleophiles to give the desired afucoside. On the other hand, so-called disarmed donors such as Ac3FucBr carrying electron-withdrawing protecting groups do not react under these conditions. Formation of the P-Lactosamine Linkage
P-Galactosylation of the 4-hydroxy group of glucosamine is difficult because of the significantly reduced nucleophilicity of this acceptor group in the vicinity of protected or glycosylated neighboring hydroxy functions, as was shown in earlier investigations. The powerful trichloroacetimidate methodology [28, 291 is most recommendable to achieve the desired conversion (Scheme 5). Trichloroacetimidate donors are easily prepared from the corresponding 1-OH precursors and are stable towards basic conditions [30]. a-Sialylation
N-Acetylneuraminic acid contains no C-3 substituent but carries a carboxy group at position 2. Both factors complicate the stereoselective and efficient formation of the desired a-2,3-sialoside in example 2 [31]. A neighboring group at C-3, required for stereoselective synthesis, can be introduced temporarily (SPh, SePh) and removed after completion of the sialylation [32]. This strategy, however, requires additional steps and results in reduced overall yield. Recently, thioalkyl/aryl sialosides and Ssialosyl xanthates have been used to achieve regio- and stereoselective formation of a-glycosides of N-acetyl neuraminic acid derivatives (Scheme 6) [33-351. The 2-SR sialosides can be activated by use of N-iodosuccinimide (NIS) and traces of TfOH or with alkyl sulfenyl triflate [33, 36, 371. Under these conditions the desired a-product 10 can be obtained in yields above 50% without the need for purification by HPLC. The regioselectivity of the NeuNAc-SR donor for the 3-OH group of the galactose residue of the acceptor 9 is particularly advantageous [38].
11.1.3 Glycopeptides Containing Particularly Sensitive Linkages
Because of the particular sensitivity of glycopeptides, precautions must be taken during the assembly of the target molecule, in particular during the deblocking reactions.
273
11.1 Introduction AcO
Ac0w0cH3
L
SEt
AcHN 6
AcO
NIS/TfOH
N3 c
AcO
OBzl
10
NHAc
OBzl
NHAc
9
Scheme 6.
Acid Sensitivity In general, the 0-glycosidic linkages between saccharide residues and also between saccharides and amino acids are sensitive towards acids. For instance, the a-fucosidic linkage is only compatible with the trifluoroacetic acid-promoted cleavage of side-chain protection or anchoring groups if acyl protection is used (Scheme 7). In the presence of benzylated or methoxy benzylated (OMpm) carbohydrate moieties, treatment with trifluoroacetic acid can lead to extensive destruction of the corresponding saccharide linkage [39, 401. Base Sensitivity If strongly basic conditions (pH > 11) are used, the a-CH linkages of amino acids in peptides and, in particular, in peptide esters are likely to epimerize (Scheme 8). In
OBzl 1 OBzl
-0BA
A
C*o--
Peptide
I
NHAc
COOtBu
1
1
Peptide
I 0
NHAc
COOtBu
TFA TFA
Complete loss of the fucose residue
Scheme 7.
4
nNH
AcO
1
Selective cleavage of the rert-butyl ester
274
I 1 Glycopeptide Synthesis in Solution and on the Solid Phase
addition, with serine/threonine glycosides the removal of the a proton can result in complete elimination of the glycan portion by an E 1cB mechanism:
Scheme 8.
The risk of side-reactions is particularly increased if ester protecting groups (e.g. the methyl ester in 10) are cleaved using, for example, dilute sodium hydroxide. The progress of these deblocking reactions should, therefore, always be carefully monitored [41]. Fortunately, the basic conditions required for the removal of the Fmoc group (morpholine, short treatment with piperidine) and the 0-acetyl groups (catalyst NaOMe, pH 8.5) are so mild that undesired reactions can be minimized. For this reason the N-Fmoc and the 0-acetyl groups are well established as standard protecting tools in glycopeptide synthesis [ 171.
11.2 Synthesis of Glycopeptides in Solution Despite the successful development of a routine methodology for the solid-phase synthesis of peptides and glycopeptides (see below) a synthesis in solution is still the method of choice, in particular if glycopeptides with demanding saccharide components are to be synthesized. Short sequences with a relatively high content of complex saccharide residues can be prepared efficiently in solution avoiding the loss of valuable building blocks required in excess in syntheses on the solid phase. The development of new coupling reagents or protecting groups, moreover, often requires an initial verification in solution. Particularly difficult sequences are advantageously prepared in solution by coupling preformed fragments [42]. Finally, synthesis of glycopeptides in solution is required, not only if a few milligrams of the product are required for a first attempt at biological evaluation (and claiming) but also if preparative amounts of a glycopeptide with valuable biological properties are to be obtained. 11.2.1 0-Glycopeptides
Glycopeptides Carrying N-Acetylgalactosamine (Tn-Antigen)
N-Acetylgalactosamine (Tn-antigen) is usually found a-glycosidically linked to serine or threonine. The Ser/Thr(GalNAc) residue also represents the key building
11.2 Synthesis of Glycopeptides in Solution
215
block of the important glycoprotein family of the mucins [6, 43, 441. Mucins are highly glycosylated proteins produced by a wide variety of epithelial and endothelial cells. Despite their normal cell-protecting function, the appearance of mucin molecules with diminished carbohydrate chains has also been correlated with a negative prognosis in the development of tumors. In particular, Tn (aGalNAc), T (PGal-1,3-aGalNAc), STn (aNeuNAc-2,6-aGalNAc) and the regioisomeric ST [ PGal-I,3-(aNeuNA~-2,6)-aGalNAc or aNeuNAc-2,3-PGal-l,3-aGalNAc] antigens have been described as tumor-associated antigens. [8, 45, 461. The synthesis of partial structures of these biologically relevant cell surface components has, therefore, become a major target in glycopeptide synthesis. Some results of these efforts have already been applied in the fields of immunology (vaccination) and diagnosis
WI.
MUCl is the mucin which has been most thoroughly investigated [48]. It consists of numerous glycosylated tandem repeats of 20 amino acids. A bisglycosylated decapeptide of this repeating unit was synthesized in solution by use of enzymatic protecting group methodology [49]. The key building blocks for this synthesis were the Fmoc-protected Ser/Thr-aGalNAc residues which were accessible in eight steps starting from galactose via the azidonitrate 11 [50]and the thioethyl glycoside 12 [51] (Scheme 9). Because the azido group in the 2-position does not exert a neighboring-group effect, a mixture of a- and P-glycoside with prevailing a-anomer is obtained by reaction with the Fmoc-protected serine heptyl ester after activation with a soft electrophile (DMTST) [52].Subsequently, the C-terminal heptyl ester in 13 was cleaved in a lipase-catalyzed reaction [53] to give building block 14. Coupling of 14 with preformed glycosylated dipeptide 15 using water-soluble carbodiimide/HOBt gave the bisglycosylated tripeptide which was converted into the N-acetyl galactosamine derivative by use of thioacetic acid. After N-terminal enzymatic cleavage of the C-terminal heptyl ester and coupling with tripeptide 16 (+17), the Fmoc-protected N-terminus was removed and the amino component condensed with tetrapeptide 18 to give the bisglycosylated decapeptide (Scheme 10).
I ) AczOiHC104 2) PBr, 3) Zn/CuS04NaOAc AcOWH20 4) ceric ammonium AcO nitrateNaN3
Gal
-
1 ) EtOCSSK AcO ONo2 2) NaVacetone
OAC
AcO
11
AcO @SE' 12
Fmoc-Ser(OH)-OHep DMTST
\
FmocHN---r-COOHep
Aco+0213 AcO
Scheme 9.
OAc
276
I1 Glycopeptide Synthesis in Solution and on the Solid Phase 1) EDC/HOBt
FmocHN-cH-cooH II 13
*
7H2
pH7,3I0C
go
AcO OAc OAC 14
H-TF-Ala-OHep
I
aAc3GalN3
15
-
aAc3GalNHAc
I
FmocSer-Thr-Ala-Pro-Pro-Ala-OHep
I
2) CH3C(O)SH 3) LipaseM 4) EDCRIOBt aAc3Ga NHAc H-Pro-Pro-Ala-OHep 16
l7
\
1)Morpholine/Ac20 2)Ac-Pro-Ala-Pro-Gly-OH 18 3)Lipase N 4)NaOMe/MeOH GalNHAc
AcPro-Ma-Pro-Gly-Ser-Thr-Ala-Pro-Pro-Ala-OH GalNHAc
19
Scheme 10.
The target molecule 19 was obtained by lipase-catalyzed heptyl ester cleavage and employing sodium methanolate for the removal of the 0-acetyl groups. In summary, this result showed a set of three base-labile protecting groups (Fmoc, OAc, OHep) to be removable selectively and thus useful for the synthesis of complex glycopeptidic structures. A conformational investigation of the target compound 19 was conducted by use of state-of-the-art NMR techniques [49]. The results indicate that carbohydrate residues can influence the conformation of the peptide backbone. Further studies on lipase-catalyzed ester hydrolysis revealed that more hydrophilic structures such as the methoxyethoxy ethyl ester, were generally better substrates for the enzymes, in particular when the peptides and glycopeptides were sterically hindered (Scheme 11) [54, 551. A different approach to obtaining potential Tn antigen-containing vaccines is to assemble dimeric and trimeric structures from Ser(aGa1NAc) building blocks, which are equipped with an aminobutyric acid spacer molecule: Ac-Ser (aGalNAc)-Ser (aGalNAc)-Ser (aGalNAc)-CO-NH-( CH2) X O O H The resulting dimeric antigen was coupled to a lipopeptide and was successfully used to stimulate antibody (IgM and IgG) production in mice [ 5 6 ] . Glycopeptides Carrying the T-Antigen (Gal-GalNAc)
The key steps in the formation of the T antigen linked to serine or threonine usually involve azidonitrate 11 which is converted into the methyl glycoside, then deacety-
lipase
Aloc-Ser(GalNAc)-Val-Phe/O~O~O\
Scheme 11.
Aloc-Ser(GalNAc)-Val-Phe
277
11.2 Synthesis of Glycopeptides in Solution Ph
I
AcO
&q OAc
\n
2) 1) AcOH NIS/TfOH
I+
.+
Hok&
3) AczO/pyridine 4)ACZ"'H2SU4 * 5 ) TiBr4
N3
20
&;% OAc
21
N3
Br
Scheme 12.
lated and regioselectively benzoylated at the 4- and 6-OH groups [57]. This acceptor molecule can be efficiently and stereoselectively galactosylated in position 3 to give the desired disaccharide. Conversion into a suitable donor can be achieved after acetolysis of the anomeric position using TiBr4. Important disadvantages of glycosyl halides as donor molecules are their instability and the need for silver or mercury salts as activating agents. In this context an improvement was achieved by introduction of thioalkyl/aryl donors which can be stored and easily activated by means of a soft electrophile [58]. A combination of thioglycoside methodology with pentenyl glycoside activation [59] led to thiopentenyl glycosides as glycosyl donors (e.g 20) [60]. Attack of iodonium reagents at the double bond and subsequent exo-trig cyclization forms a thiotetrahydrofuran as the leaving group, which can be substituted by the glycosyl acceptor. After conversion of the intermediate methyl glycoside into a bromo glycoside 21, glycosylation of the Fmoc-protected threonine phenacyl (Pac) ester, reduction of the azido group and reductive elimination of the Pac ester [61], a building block for convergent glycopeptide fragment condensations was obtained (Scheme 12). Use of phenacyl protection avoids the application of harsh conditions for the liberation of the C-terminal carboxy group (e.g. tBu/CF3COOH). Coupling of T antigen derivative 22 with pentapeptide 23 gave a fully protected fragment of the MUCl repeating unit. Removal of all remaining protecting groups was achieved in high overall yield by standard deblocking procedures. ROESY NMR analysis suggests that glycosylation of the threonine induces a cis peptide bond between Thr and Arg in 24 (Scheme 13) Fmoc-Thr-OH 1) IIDQ
H-Arg(Pmc)-Pro-Ala-Pro-Gly-OtBu 23
AcO&O& I ) Fmoc-Thr-OPac AgOTf 2)CHxCOSH f
AcA coAc 22
\
2) morpholine 3) AcZOlpyridine 4) TFA 5 ) NaOMeMeOH
Ac-Thr-Arg-Pro-Ala-Pro-GI y-OH 21
H
O OH
Scheme 13.
OH
e
O g 24 OH OH
218
I1 Glycopeptide Synthesis in Solution and on the Solid Phase
AcO
1) dimethyldioxirane 2) 6-O-triisopropylsilyIgalactal/ZnC12 3)AC20, EtjN, DMAP
-
1) TBAF
R=TIPS
26
3) NaN3lCAN 4) PhSWDEA 5 ) CCI&N/KzCO,
AcO
gNH \
2) TMSOTO27 _, ....-3TO27
OAc
0
OAc
N3
CCI3
28
27
Scheme 14.
Glycopeptides Carrying the Sialyl T Antigen (NeuAca2,6[Gal~l,3]GalNAc) The synthesis of a nonapeptide carrying three sialyl T residues was recently reported [62]. As for the preparation of Tn and T antigens, glycal and azidonitrate intermediates play key roles throughout the synthetic strategy. The glycal 25 was subjected to epoxidation. Activation of the epoxide with ZnC12 and reaction with a 6-0-silyl protected galactal gave disaccharide 26. After fluoride-mediated removal of the TIPS group, the 6-OH groups were sialylated by use of sialyl phosphite 27 [63, 641 and activated with TMSOTf. The resulting protected sialyl T glycal was converted into the azidonitrate which was subsequently hydrolyzed. The resulting hemiacetal can be transformed into the trichloroacetimidate 28 by treatment with K2C03 and trichloroacetonitrile (Scheme 14). Upon activation with BF30Et2 the P-trichloroacetimidate 28 gave high yields of predominantly a-glycosylated Fmoc-protected serine benzyl ester. Subsequent to the reduction of the 2-azido groups and the deprotection of the carboxy group the serine and threonine building blocks 29 carrying the sialyl T antigen were introduced into a convergent peptide synthesis. The peptide couplings were achieved with IIDQ, and Fmoc removal was performed with potassium fluoride in the presence of 18-crown-6 [65]. Finally, complete deprotection by standard procedures gave the mucin-type target 30 (Scheme 15). To date, the immunoreactivity of this glycopeptide has not been reported. A similar strategy also using glycals as the crucial carbohydrate building blocks was used in the successful synthesis of glycopeptides containing regioisomeric sialyl (2, 3) T antigen carbohydrate side-chains [66]. An alternative sequence of reactions also based on an early introduction of the sialic acid was applied for the construction of a sialyl T antigen serine/threonine building blocks [67].
279
11.2 Synthesis of Glycopeptides in Solution 28
1 ) BF30Etz/Fmoc-Se/Thr-OBzl 2) CH3COSH 3) Hz/lO% Pd-C AcO
t
a2,6NeuNAc(p I ,3Gal-aGalNAc)
-
'
I
AcPro-Ile-Val-Ser-Th-Ser-Asp-Pro-Val-OH a2,6NeuNAc(p 1,3Gal-aGalNAc)
I
30
I
Scheme 15.
Glycopeptides Carrying 0-GlcNAc The existence of natural glycoproteins containing 0-linked N-acetylglucosamine was unknown until 1984 [68]. Since then, numerous investigations have focused on this ubiquitous structural motif [69]. Recently, it was found that the 0-GlcNAc moiety might be connected with important phenomena such as Alzheimer's disease. The development of methods for a straightforward and efficient approach towards 0-GlcNAc-containing peptides followed evaluation of the biological background. The major synthetic problem is the formation of the b-configured GlcNAc-Ser/Thr linkage. Although the 2-N-acetamido group has the desired neighboring effect, the intermediate cation has a high tendency to form the rather unreactive oxazolidine ring (Scheme 16). To overcome these drawbacks the trichloroethyloxycarbonyl (Tcoc) group was used for temporary protection of the 2-amino group [70]. In combination with the anomeric thioethyl group an efficient donor for the construction of p-GlcNAc-Ser/ Thr building blocks was provided [71].The reactivity of 31 was even high enough to enable the glycosylation of dipeptides in acceptable yields. In particular, the sensitive tert-butyl ester as the C-terminal protection remained unaffected during both the mild activation procedure and the subsequent conversion of the Tcoc group into the acetamide (Scheme 17). With 32 in hand, the orthogonal stability of the tert-butyl and the Z protecting groups facilitates extension of the peptide chain in the N-terminal direction.
Nu-
Scheme 16.
+
""yo
Nu-
280
A AcOc
11 Glycopeptide Synthesis in Solution and on the Solid Phase
ONH G S E31 ~
1)DMTST/ZSerAlaOtBu 1) H2/Pd-C 2) AcOWZn 2) Z-Ala 3)Ac20/pyridine EDCBIEA 2-Ser-Ala-OtBu
-
I
PAC~GICNAC32 0~OCH*CCI,
NHAc
Scheme 17.
Other glucosamine donors based on the N-Tcoc and N-tetrachlorophthaloyl protecting groups in combination with the anomeric trichloroacetimidate also enabled other efficient syntheses of GlcNAc-Ser/Thr building blocks [72]. Despite these achievements the 0-GlcNAc-containing building blocks for the solution-phase synthesis of a bisglycosylated and phosphorylated hexapeptide from RNA polymerase I1 were obtained by the activation of the oxazoline 34 in the presence of urethane-protected serine 33 (Scheme 18) [73]. The unusual N-terminal protection used herein facilitates the subsequent deblocking of the N-terminus by penicillin G acylase under exceptionally mild conditions. For assembly of the target peptide the GlcNAc-Ser derivative 35 had to be modified: elongation at the C-terminus led to glycodipeptide 36 and introduction of the tert-butyl ester and liberation of the terminal a-amino function gave building block 37. After coupling of both GlcNAc compounds (-38)) N-terminal deprotection, condensation with a suitable Pro-Thr moiety, and another N-terminal deprotection, a phosphorylated serine residue was introduced at the N-terminus (-39). Simultaneous removal of the amino and phosphate protecting groups using Pd(0) ally1 transfer followed by standard deblocking procedures gave target 40: 11.2.2 N-Glycopeptides N-Glycopeptides Carrying Natural Saccharide Side-Chains Natural N-glycoproteins usually carry a central (‘core’) pentasaccharide 41 linked to aspartic acid (Scheme 19). The Asn-GlcNAc unit is, therefore, an ideal model compound for methodological investigations towards the preparation of N-glycopeptides. That is, it served for the development of C-terminal protecting groups cleavable by lipases [ 5 5 ] . This class of enzyme was chosen because it does not cleave peptide or other linkages present in glycopeptides. For the synthesis of 0-glycopeptides, heptyl esters as lipase substrates had already been successfully used (see above) [54].In this context it was found that the hydrophobicity of heptyl esters is not a prerequisite for a sufficient conversion. Instead, rates of hydrolysis by lipases were distinctly higher for esters derived from ethylene glycol.
281
11.2 Synthesis of Glycopeptides in Solution
OH
BF3-OEtz
1)
PhAcOZ-Ser-OH
iPrN F O t B u iprm HSer-OtBu
2) penicilline G acylaseMHS03
I
c
A & % !AcO
AcHN
PAc~GIcNAc
35
31 1 ) EDC/HOB~-Pro-OAll 2) Pd(O)/morpholine
EDC/HOBt
PhAcOZ-Ser-Pro-OH
I
PAc~GIcNAc 36
Ph AcOZ-Ser-Pro-Ser-0th
I
PAc~G~cNAc
OPO(OH),
PGlcNAc
\
38
OPO(OAlI),
PAc3GlcNAc
I
I
Aloc-Ser-Pro-Thr-Ser-Pro-Ser-OtBu PGlcNAc
40
Scheme 18.
Mana 1,6
\
Manal,3 /
Man-Pl,4-GlcNAc-P1,4-GlcNAc-P-Asn
\
/ " Z e " :41
Scheme 19.
I
PAc~GIcNAc
39
282
-
I 1 Glycopeptide Synthesis in Solution and on the Solid Phase
PeptdieyO-o,n-O ,. ,
Peptide O
v
OMEE
K 0
O 0’-
OME
42
Scheme 20.
COOMe Tcoc ,
H 43
1
l)AcjGlcNAc-NHZ45 IIDQ Tcoc-Asn-Ser-Ala-OH 2)Lipase N Lipase A6 Tcoc , I N COOMe m-Ser-Ala-OMe PAc3GlcNAc H IIDQ OME 44 4)Lipase A6 46
Scheme 21.
The methoxyethyl (ME) esters (e.g. 42, Scheme 20) combined high substrate acceptance with optimum purification properties and high solubility in aqueous solution. As a model target in the area of N-glycopeptides glycotetrapeptide 46 was synthesized. Starting from the bis-ME aspartic acid ester 43 the p-ester was selectively cleaved in a lipase-catalyzed reaction. Condensation of compound 44 with AqGlcNAc-NH2 45 gave the fully protected asparagine derivative. Upon treatment with a different type of lipase, the a-carboxy group could be deblocked and condensed with a dipeptide ME ester. After elongation at the N-terminus the resulting ME ester again could be cleaved by use of a lipase (+46, Scheme 21). In summary, the selectivity and mildness of the lipase-catalyzed reactions was established as a tool applicable to multifunctional and sensitive glycopeptide targets in particular. Variants of the ‘core’ 41 shown in Scheme 19 with increased structural complexity were found, which can be related to pathogenic phenomena. As a basis for further biological investigations, the complete N-glycan of the so-called complex type carrying a pentapeptide was recently synthesized using a chemoenzymatic strategy (Scheme 22) [741. This molecule contains several preparative challenges that warrant more detailed description. The synthetic strategy applied can be summarized as:
I
xxx
NeuSNAca-2,6-Galp-l,4-GIcNAc~-I ,2-Mana-l,
\
I
Man-P-l,4-GlcNAc-P 1,4ClcAc-P-Asn 2,6-Galp- 1,4-GlcNAc~-1,2-Mana-1,4
I
xxx I
Scheme 22.
283
11.2 Synthesis of Glycopeptides in Solution
1) 2) 3) 4)
assembly of heptasaccharide 47 [75]; introduction of aspartic acid at the anomeric center; selective deprotection and extension of the peptide chain; and sequential enzymatic transfer of galactose and neuraminic acid on to the nonreducing ends of the glycan chain
The synthesis started from chitobiosyl azide 48 which is available in 11 synthetic steps. The azido group herein is essential for the chosen strategy because it: 1) remains stable during the applied reaction conditions, and 2) enables the generation of the anomeric amino function required for introduction of aspartic acid [76]. Next, a P-mannoside linkage must be established and two major problems must be overcome because commonly used neighboring groups, and the anomeric effect lead to the formation of the undesired a-mannosides. During the past 20 years several efficient solutions to this key problem have been provided [22].The strategy used here is based on the epimerization of the 2-hydroxy group subsequent to the formation of a p-glucosidic linkage (Scheme 23) [77, 781. To this end, a phenylcarbamoyl group at 3-OH and a leaving group at 2-OH (OTf) were introduced (49). An intramolecular s N 2 reaction (DMF, 40-50 "C) resulted in
NPth
\
OBzl
AcO
+
48
&
o+
6"
OAc
49
1) BFtOEt,
2) K2603kIeOH 3) dimethoxytoluene/H+
phqo
4) 5 ) TfiOlpyridine DMFipyridine
0HO a B
6 ) AcOWdioxane 7) NaOMdMeOH
O
e
:
z
An-, UDLl
&
NPth N3 50
I
\ OAc
47
Scheme 23.
g
1) A-trichloroacetimidate BF30Et2 2) AcOW80"C 3) B-trichloroacetimidate BF30Et2 I
NPth OBzl
284
11 Glycopeptide Synthesis in Solution and on the Solid Phase
the formation of the 2,3-cis carbonate which upon treatment with NaOMe/MeOH gave the trisaccharide acceptor 50. Advantageously, the 2”,3”-diol 50 was now selectively glycosylated with the disaccharide donor A at position 3”. After removal of the 4”,6/’-benzylidene group a second selective glycosylation with B gave heptasaccharide 47 in high yield. This glycan was now subjected to a manipulation of protecting groups followed by the conversion of the azido group into the P-configured amino function, by use of propanedithiol, and condensation with a fully protected aspartic acid derivative. All remaining protecting groups were subsequently removed by Pd-catalyzed hydrogenation and the Fmoc group was introduced at the terminal amino function. The efficiency and selectivity of modern peptide-coupling reagents thereafter facilitated the extension of the peptide chain at C- and N-terminal functional groups in the presence of the totally deblocked heptasaccharide side-chain. All remaining protecting groups were removed before the final enzyme-catalyzed transfer of galactose and neuraminic acid on to both non-reducing ends of the glycan chains (Scheme 24). During these transformations the addition of alkaline phosphatase was essential to prevent product inhibition by UDP [79, 801. These final steps yielded the target undecasaccharide glycopentapeptide 51 in 91% yield for the last two steps. It is obvious why the authors of the aforementioned synthesis have preferred peptide assembly in solution to the solid-phase approach:
1) the synthetic effort required for peptide assembly is small compared with the construction of the saccharide chain
YOOHHo
I
s,, I
Ser
I
Ser I Asn
I
Phe
HO
Scheme 24.
51
11.2 Synthesis o j Glycopeptides in Solution
285
2) on the solid phase, a molar excess of building blocks must usually be used to ensure high coupling yields. For the saccharide-asparagine moiety this would have entailed tedious re-isolation of this valuable compound. In general, therefore, glycopeptides with a comparably short peptide sequence and complex and valuable carbohydrate parts can be efficiently prepared in solution. This is also true for N-glycopeptides carrying Lewis antigen-type side-chains. As an alternative to the chemical and chemoenzymatic routes, N-glycopeptides can be obtained by a convergent strategy [ 8 1] using glycans isolated from natural Nglycoproteins or oligosaccharides and presynthesized peptides 1821. The unprotected saccharide having the reducing terminal N-acetylglucosamine is converted into a glycosylamine by use of excess ammonium hydrogen carbonate [24] and then coupled with a peptide [82]. By this strategy, large oligosaccharide side-chains can be introduced in the molecules. This is also true for the construction of N-glycopeptides from N-acetylglucosamine-containing glycopeptides and extension of their glycan portion by trans glycosylation-catalyzed saccharide transfer from N-glycoproteins [ 831. Despite the possible microheterogeneity of the natural glycoprotein glycans, the limitation of these reactions is that only analytical amounts (<1 pmol) are obtained. N-Glycopeptides with Lewis-Type Saccharide Side-Chains
The biological importance of Lewis-type saccharide structures, and typical methods for their chemical preparation, have already been outlined in the introduction. In particular, the preparation of peptide backbones carrying a high density of Lewis type antigens is of increasing interest. Such clustered structures are often more immunoreactive than the corresponding monovalent antigens [84]. This observation was the motivation for a chemoenzymatic synthesis of the trivalent N-glycopeptide 52 (Scheme 25) [85]. Selectively protected GlcNAc-Asn units were prepared as suitable precursors and conjugated via an amino acid of variable length. The trivalent glycopeptide was converted into the corresponding tris-sialyl LewisX glycoconjugates by stepwise elongation with galactosyltransferase/UDP-galactose, sialyltransferase/CMP-sialic acid, and fucosyltransferase/GDP-fucose. By this approach, 20 mg of target compound 52 were obtained and used for E-selectin binding assays. An inhibition four times stronger than that of the monovalent SLe"-Asn residue was achieved. Without the restrictions of enzymatic reactions, such as the strict specificity of glycosyltransferases and the limited availability of enzymes and nucleotide substrates, a purely chemical approach has its advantages in generating a broader structural variety and larger product quantities. For this purpose, the SLeXiaazides are ideal precursors (see Scheme 6). They can be: 1) produced on a gram scale; 2) easily converted into the corresponding P-glycosylamino derivatives and 3 ) efficiently coupled to form stable amide linkages.
286
I1 Glycopeptide Synthesis in Solution and on the Solid Phase 0
HO
0
HO
J
HO
J
Scheme 25.
In this manner, SLe" amine 53 was combined with a tetrapeptide partial sequence from fibrinogen required for binding to the integrin receptor GpIIb [29]. For this conjugation the C-terminal alanine of RGDA derivative 54 served the as coupling position (Scheme 26). The fully deprotected SLeX-RGDA glycopeptide 55 inhibited P-selectin binding with high affinity (1Cso 25 pmol), but had no affinity for E-selectin. Structurally even more sophisticated is the trivalent cycloheptapeptide 57 (861. The synthesis of this complex molecule involved the preformation of the cyclic peptide 56 containing three aspartic acid residues (Scheme 27). After removal of the tert-butyl side-chain protecting groups simultaneous condensation with three equivalents of SLeX-lactone amine 53 using HATU/HOAT/DIEA gave the cycloheptapeptide carrying three SLeX side-chains. This was fully deprotected in two further steps. The resulting trivalent antigen proved to inhibit the binding of human tumor cells (HL60) to E-selectin in the low millimolar range (IC50 0.35 mM), but in contrast with 55 had no affinity for P-selectin.
11.3 Glycopeptide Synthesis on the Solid Phase In contrast with the construction of glycopeptides in solution, automated assembly on solid supports promises rapid access to the desired target compounds [87-891.
11.3 Glycopeptide Synthesis on the Solid Phase
53
OH
287
I OBzl
nn-1
OH
NH
I
Scheme 26.
This only holds true, however, if certain structural and methodological prerequisites are fulfilled: 1) glycosylated amino acids must be provided in sufficient quantity as N-Fmocprotected compounds containing a free a-carboxylic function and 2) reaction conditions and polymer-anchoring systems applied during the solidphase procedure and the cleavage and deprotection steps must be adapted to the sensitive glycosylated compounds. Throughout the following section, solutions of these problems will be presented in connection with recently published solid-phase 0- and N-glycopeptide syntheses. 11.3.1 O-Glycopeptides Glycopeptides Carrying N-Acetylgalactosamine (Tn-Antigen)
The biological relevance of a-GalNAc-carrying glycopeptides has been outlined above. For the preparation of a glycopeptide from the homophilic recognition domain of Epithelial Cudherin 1 (E-CAD, 58, Scheme 28) the particular importance of the anchoring structure and of adapted coupling conditions can be demonstrated [901.
288
I 1 Glycopeptide Synthesis in Solution and on the Solid Phase
51
Scheme 27.
58
Scheme 28.
11.3 Glycopeptide Synthesis on the Solid Phase
289
58
Scheme 29.
The depicted target efficiently was constructed with the help of the allylic anchoring principle and a newly developed coupling reagent. To this end, the starting amino acid (Glu) was linked to the polymeric support (Tentagel@)via the Hycron anchor comprising the internal standard p-alanine, a flexible and polar oligo(ethy1ene glycol) spacer, and a 1,4-dihydroxybut-2-ene unit (59, Scheme 29). Subsequent initial attempts to synthesize the E-CAD partial structure with the help of TBTU [91] as coupling reagent gave only low yields although TBTU had already proved efficient in glycopeptide synthesis. Obviously, this sequence is prone to back-folding, and particularly efficient activation of the corresponding amino acids building blocks had to be achieved. This problem was finally overcome by the development of a new coupling reagent: PfPyU ( N ,N , N ’ , N’-bis(tetramethylene)-O-pentafluorophenyluronium hexafluorophosphate) 60 [92]. Compared with TBTU, activation with PfPyU in model reactions resulted in eightfold increased reaction rates. This observation was confirmed during the E-CAD synthesis. First, Fmoc-AlaVal-OH was coupled to the starting glutamic acid. This step was advisable to prevent diketopiperazine formation on the stage of the resin-bound dipeptide. All coupling steps were conducted using Pf PyU and were followed by morpholine-
290
-
11 Glycopeptide Synthesis in Solution and on the Solid Phase "Rink"
BzO Fmoc-He-Ser-Gly-Ile-GI
OH
NH
2) TFA/H20
Fmoc-Ile-Ser-Gly-lle-Gly-NH2
I
aBz3GalN3
Scheme 30.
mediated Fmoc removal. Finally, the fully assembled glycopeptide was detached from the resin by Pd(0)-catalyzed ally1 transfer to N-methylaniline and gave the glycododecapeptide 58a in an overall yield of 55%. After removing all acid labile protecting groups, the 0-acetyl and Fmoc groups were cleaved by Zemplen transesterification. The high overall yield and purity of 0-glycododecapeptide 58a demonstrated the efficiency of the Hycron/Pf PyU methodology. The approach described above employed conventional insertion of a preformed GalNAc-Ser building block into the peptide chain to be synthesized. A method presented recently performs solid-phase glycosylation subsequent to peptide assembly [93]. Obviously, with this strategy the construction of glycopeptide libraries containing different glycan side-chains becomes feasible. The improvement achieved by the authors concerned the nature of the polymer support. A polyoxyethylenepolyoxypropylene (POEPOP) resin with balanced polarity and hydrophobicity was developed. In combination with an acid-labile RINK linker [94] this system served for the synthesis of pentapeptide 62 containing one serine residue (Scheme 30). Moreover, the properties of the POEPOP resin enabled the solid-phase glycosylation of the serine hydroxy group using (among others) perbenzoylated 2-azidogalactosyl donor 61. The target Tn glycopeptide 63 was obtained in high yield after detachment from the resin. 0-Glycopeptides Carrying the T Antigen (Gal-GalNAc)
Similar to the above mentioned on-resin glycosylation of hydroxy amino acids, the solid-phase elongation of saccharide chains linked to peptides has also been described. With the help of this method the preparation of T antigen carrying MUC2
11.3 Glycopeptide Sjmthesis on the Solid Phase
291
Ac-Pro-Thr(tBu)-Thr-Thr(tBu)-Pro-ile-Ser(tBu)-Thr(tBu) -RINK-
BIo%0Yc13 BzO 64
NH
Ho+
G-" Ph
Ac-Pro-Thr-Thr-Thr-Pro-Ile-Ser-Thr-NH2
66
HO OH
Scheme 31.
fragments was achieved [95]. To this end, a suitably protected a-azidogalactosyl threonine unit was used in a solid-phase glycopeptide synthesis (Scheme 31). In the resulting glycooctapeptide 65 the unsubstituted 3-OH group of azidogalactose served as an acceptor for a solid-phase P-galactosylation using galactosyl trichloroacetimidate 64 [30]. After optimization of the reaction conditions and reagent purity, the disaccharide octapeptide 66 was obtained in 67% yield after cleavage from the resin with TFA and complete deprotection. It should again be noted that the resin employed had a decisive influence. 0-Glycopeptides Carrying the Sialyl Tn Antigen (NeuNAc-a2,6-GalNAc)
The sialyl Tn antigen is considered to be one of the most important tumor-associated carbohydrate antigens [46, 961. Because of its immunoreactivity the application of clustered STn conjugates as cancer vaccines is already being investigated in clinical studies [47]. Because it is assumed that both the peptide and carbohydrate parts of the corresponding conjugates are important for a tumor-selective cellular and humoral immune response [97], the synthesis and application of tumor related glycopeptides are other major areas currently being investigated. Only recently has the successful incorporation of a suitable STn-Ser/Thr building block into a solidphase glycopeptide synthesis been achieved [98]. The key element of the strategy used here consisted in the construction of the disaccharide threonine unit 70. This was achieved starting from the known GalNAc-Thr moiety already carrying the required Fmoc and the tert-butyl ester protecting group (Scheme 32). Despite the base-sensitive Fmoc group, the removal of the 0-acetyl groups was selectively accomplished by careful treatment with NaOMe in MeOH at pH 8.2.
292
I1 Glycopeptide Synthesis in Solution and on the Solid Phase
YOAc
COOtBu HNFmoc
I ) AC20 1 p p d ~ n e 88% , 2) TFA,anaole, quant
69
B
OAc
OAc
70
Scheme 32.
The resulting trio1 acceptor 68 was then sialylated by use of sialyl xanthate 67 [34] and methylsulfenyl triflate/AgOTf [37] as the promoter. At -62 "C the reaction proceeded almost diastereoselectively and regioselectively and the glycosylated threonine derivative 69 was obtained in 32% yield after purification. The desired building block 70 for solid-phase synthesis was provided after O-acetylation and cleavage of the tert-butyl ester group. Glycosyl threonine 70 was subsequently incorporated into a partial sequence of the MUCl repeating unit known to be tumorrelevant [99]. To this end, the starting amino acid proline attached to the Hycron anchor (see above) was amino-deprotected and coupled with Boc-Ala-OH. Changing to the Boc methodology at this stage prevented diketopiperazine formation on the resin-linked dipeptide strongly favored for an Ala-Pro sequence (Scheme 33). This intermittent change of protecting groups was possible because of the stability of the anchor towards acids and bases. Further chain extension and Pd (0)-catalyzed detachment from the resin gave the protected glycoundecapeptide. After acidolytic removal of the side-chain protecting groups the O-acetyl groups and the methyl ester were cleaved by carefully optimized treatment with dilute aqueous NaOH in MeOH and aqueous NaOH (pH 11.5) (-71). At pH < 11.5 no methyl ester cleavage was achieved whereas above this pH epimerization and b-elimination of the carbohydrate occurred. The attention paid to the preparation of STn and other sialic acid-containing glycopeptides of biological importance became apparent with the appearance of another publication reporting the successful synthesis of a STn-carrying decaheptapeptide from HIV at almost the same time [loo].
11.3 Glycopeptide Synthesis on the Solid Phase
293
aAcgNeu(OMe)-(2-6)-aAc2GalNac
I
Ac-Ala-Pro-Pr~Ala-His(Trt)-Gly-Val-Thr-Ser(tBu)-Ala-Pro-HYCRON-~Ala-AMPS
I)[Pd(PPh3)4], DMSO I DMF, rnorpholine 2) TFA, anisole, EtSMe 3) NaOH, MeOH 4) 5 mM NaOH
H
*’HO HO
71
Scheme 33.
0-Glycopeptides Carrying the 2,3-Sialyl T Antigen
Another biologically important saccharide found in nature and resulting from incomplete glycosylation (see Tn, T, STn) is the sialyl T antigen [ 1011. Only recently, a 27 amino acid peptide carrying the 0-glycosidically linked a2,3NeuNAcGalpl, 3GalNAc structure was synthesized [102]. Again, a glycosylated serine had to be prepared before the solid-phase procedure (Scheme 34). Starting with di-0-benzyl galactose residue 72 sialylation of the 3-OH was achieved by use of the perbenzylated sialic acid donor 73 [103]. This donor was temporarily equipped with a thiophenyl residue in the 3 position to ensure high stereoselectivity in the mercury salt-promoted a-sialylation. Upon desulfurization and treatment with DBU a disaccharide was obtained wherein the lactone serves as an internal protecting group. After oxidative cleavage of the anomeric methoxyphenyl group and conversion into the trichloroacetimidate 74, the fully protected trisaccharide serine was obtained by BF3 .OEt2-mediated reaction of 74 with TBDMS-protected azidogalactosyl serine residue 75. Protecting group manipulation and carboxy deprotection using Pd(O)/N-methylaniline gave building block 76 which was used for the solid-phase assembly of the peptide employing dicyclohexylcarbodiimide/HOBt as coupling reagent and an acid-labile anchoring group.
294 HO HO
I 1 Glycopeptide Synthesis in Solution and on the Solid Pliase OBzl
Bzlo
BzlO
72
+
BzlO
W AcHN C
’
OMe
O
BzlO
O
BSPh z
I
73
I ) HgO)
2) Ph3SnH. AIBN 3) DBWTHF 4) CAN 5 ) CCI3CNDBU I ) BF30Etz
76
75
Fmoc-Ser-OAll
NeuNAca2,3Gal!31,3GalNAca 1
I
H-Thr-Val-Val-Gln-Pro-Ser-Val-Gly-Ala-~a-Ala-Gl~~o-Val-Va~-Pro-Pro-Cys-Pro-Gl~~g-Ile-~g-His-Phe-Lys-Val-OH
77
Scheme 34.
Cleavage from the resin was followed by an unusual debenzylation step which was performed using TMSOTf and TFA [104]. By this procedure the hydrogenolytic cleavage of benzyl ethers-often found difficult in such complex molecules-was avoided. Finally, opening of the lactone ring with NaHC03/D20 and treatment with 1,4-dithiothreitol to reduce the undesired disulfide bridges gave the target glycoheptacosapeptide 77. 0-Glycopeptides Carrying 0-GlcNAc Side-Chains
The biological importance of 0-GlcNAc-carrying peptides has already been mentioned. Again, a prerequisite for the solid-phase approach is the availability of Fmoc-Ser/Thr(GlcNAc)-OH building blocks. Doubtlessly, the most rapid synthesis of these derivatives was achieved by Lewis acid-promoted activation of peracetylated glucosamine in the presence of Fmoc-Ser or Fmoc-Thr. Including the preparation of the starting compounds only three steps gave the desired glycosylated amino acid. In this instance the sugar oxazoline is the reactive intermediate [ 1051. A similar conversion can be performed starting from the corresponding N-Tcoc derivative [ 1061. The number of protecting-group manipulations can also be reduced by use of
11.3 Glycopeptide Synthesis on the Solid Phase
ACO&O&OQ; AcO
H *t
HNAC
R
O
F
R=Y CH3
/
295
OH
H-Ala-Val-Ser-Thr-GIu-Pro-Phe-Gly-Arg-NH~ I
I
F 78
79
HO HO HNAC
Scheme 35.
Fmoc-Ser/Thr pentafluorophenyl esters [ 1071. The latter remain stable during the glycosylation process and so suitable solid-phase building blocks 78 are obtained after the generation of the N-acetamido residue. Solid-phase synthesis with Pfp esters was performed on a poly(ethy1ene glycol)-derived resin equipped with an acidlabile linker [108]. By following a standard protocol for the coupling reactions, detachment from the resin, and deprotection, the bisglycosylated nonapeptide 79 was obtained and used for detailed NMR investigations (Scheme 35). A chemoenzymatic approach to O-GlcNAc peptides with less general applicability has recently been reported [ 1091. O-Glycopeptides Carrying O-Linked Fucose
Nothing was known about the appearance of O-fucosylated proteins until the early 90s. Then, the uncommon Fuc-Ser/Thr motif was found in the growth factor domains of several coagulation and fibrinolytic proteins [ 1101. The structure also occurs in a small serine protease inhibitor isolated from Locustu migratoriu (83; Scheme 36) [ 1 I I]. In the latter both the anomeric configuration of the fucose and its biological function remain unclear. To evaluate the influence of the single fucose moiety on the conformation and activity of this protein, the fucosylated and unfucosylated form were synthesized [ 1121. The required a-fucosylated threonine building block (which was assumed to be the more likely a isomer) was prepared in four steps from a suitable Mpm etherprotected 1-thioalkyl fucose 80 and the Fmoc/tBu-protected threonine derivative 79 [71]. Activation of the donor 80 was achieved with DMTST and resulted in the formation of the a-configured glycoside 81. Because of the acid-sensitivity of the afucosidic linkage, 2,6-di-tert-butylpyridine had to be added to the reaction mixture. The Mpm ethers were removed by oxidation with ceric ammonium nitrate followed by O-acetylation and cleavage of the tert-butyl ester with formic acid. The intermittent exchange of the hydroxy protecting groups was essential to enhance the acid stability of the fucoside before liberation of the terminal carboxy group and final peptide deprotection [40]. By use of the building block 82, the solid-phase synthesis was performed by means of an acid-sensitive resin and BOP [ 1 131 as coupling reagent. Release from the solid support and side-chain deprotection was achieved by
296
I1 Glycopeptide Synthesis in Solution and on the Solid Phase
-
Fmoc-Thr(0H)-OH tBuOWDCC CUCl Fmoc-Thr(0H)-OEJu
MpqFucSEt 80
Fmoc/'?OtBu
1 DMTST 2,643 -ferfbutylpyridine
@O ' MOMpm pm MpmO
1) CAN 2) AczO/pyridine
3, HCooH 81
-
Fmoc-Thr(aAc,Fuc)-OH 82
Fucose
I
HOOC -
Scheme 36.
treatment with TFA. Finally, disulfide formation and 0-acetyl removal were performed simultaneously in diethylmethylamine in the presence of air. Careful HPLC purification gave the monofucosylated peptide 83 containing 36 amino acids and three disulfide bridges. A straightforward synthesis towards the fucosyl threonine block 82 was performed by direct activation of peracetylated fucose with BF3.0Et2 in the presence of Fmoc-Thr-OH [114]. Small amounts of 0-configured impurities had to be removed from the crude product by HPLC, however. A more laborious procedure for the construction of building block 82 and its incorporation into a undecapeptide has also been reported [ 1 151. 0-Glycopeptides Carrying a Sialyl Lewis Antigen Structure The first synthesis of a glycopeptide carrying 0-linked SLe" was recently described 11161. The major achievement therein consisted of the enzymatic assembly of the SLe" tetrasaccharide on a suitable solid support. This approach promises a rapid and variable construction of complex glycopeptides. The starting point was the preparation of the corresponding O-GlcNAc-containing octapeptide 85 linked to controlled-pore glass (CPG) via the aforementioned Hycron linker [ 1171 by use of standard protecting groups and coupling conditions. Because the ally1 ester unit herein proved unstable under the typical conditions used for the deacetylation of sugar side-chains, a building block with unprotected sugar
11.3 Glycopeptide Synthesis on the Solid Phase
%
OAc
AcO AcO
1) BF30Et*/Z-Thr 2) NaOMehleOH 3) Pd-C/MeOH 4) Fmoc-OSu
Fmoc-Thr-OH
NHAc
84
" - h o
/ w
NHAc
--
Ac-Lys-Pro-Pro-Asn-Thr-Thr-Ser-Ala-HYCRON-CPG ,H0&0,
297
85
Ac-Lys-Pro-Pro-Asn-Thr-Thr-Ser-Ala-HYCRON-CPG
H6p NHAc
NHAc
\
HO
Scheme 37.
hydroxy groups (84) had to be provided according to the pathway depicted in Scheme 37. After removal of the tert-butyl and trityl side-chain-protecting groups with TFA, galactosylation, sialylation, and fucosylation were successively performed using the corresponding glycosyltransferasesand donor substrates. Finally, the advantages of mild and reliable detachment from the solid support by use of the Hycron/Pd(O) system was demonstrated with this complex and sensitive structure (86).
11.3.2 N-Glycopeptides Compared with O-glycopeptides, access to N-glycopeptides on the solid phase is more readily possible via two different pathways [42]-conventional sequential incorporation of preformed and suitably protected building blocks into the solidphase synthesis or assembly of the peptide chain, selective removal of the aspartic acid side-chain protecting groups, and formation of the amide linkage between glycosyl amines and the peptide backbone [81] on the solid phase. The simplicity of amide formation and access to a great variety of glycosyl amines enables the use of the second method for the synthesis of N-glycopeptides whereas only single examples of solid-phase O-glycosylation have been reported in the synthesis of O-glycopeptides.
298
11 Glycopeptide Synthesis in Solution and on the Solid Phase H-Q(GkNAc)YGGFL-OH H-Q(Fuc)YGGFL-OH H-Q(GlcNAc(p 1,~GICNAC)YGGFL-OH H-Q(GalP 1,4Glc)YGGFL-OH H-Q(GalP 1,3(Fuca 1,4)GlcNAcP1,3GalB1,3Glc)YGGFL-OH H-Q(Gal6SO3)YGGFL-OH
f
H-N(Ga1)YGGFL-OH H-N(Glca 1,4Glcal,4Glca 1,4Glca 1,4Glca 1,4Glcla I,4Glc)YGGFL-OH H-N(GlcN2,6(S0332)YGGFL-OH H-N(Ga16SOi)YGGFL-OH H-N(GaL4)YGGFL-OH
Fmoc-E(OAIl)Y(tBu)GGFL-SASRIN
’/
’
Fmoc-D(OAII)Y(tBu)GGFL-SASIUN
-
Fmoc-Y(tBu)GE(OAII)GFL-SASRIN
H-Y GO(GlcNAcEFL-OH H-YGQ(Gal)GFL-OH H-YGQ(Glca 1,4Glc)GFL-OH
Fmoc-Y(tBu)GGFLE(OAII)-SASRM
H-QGQ(GIc!~I,~GIc)GFL-OH
\
Fmoc-Y(tBu)GGFLD(OAlI)-SASRIN \
H-QGY(GICNAC~SO~~)GFL-OH
\ H-YGGFLQ(MmNAc)-OH H-Y GGFLQ(Glc)-OH H-YGGFLQ(GalP 1,4Man)-OH H-YGGFLQ(Glca 1,4Glca 1,4Glca 1,4Glca 1,4Glca 1,4Glc1a 1,4Glc)-OH H-Y GGFLN(G1cNAc)-OH H-YGGFLN(Ga1NAc)-OH H-YGGFLN(Lm)-OH
Scheme 38.
The Construction of N-Glycopeptide Libraries on the Solid Phase The development of efficient methodology for on-resin N-glycopeptide formation has been described as a starting point toward the preparation of glycopeptide libraries (Scheme 38) [ 1181. Employing an acid-labile anchoring system (Sasrin resin [ 1191) five different hexapeptides containing either glutamic or aspartic acid allyl ester residues were synthesized. Pd(0)-catalyzed selective cleavage of the allyl ester and subsequent formation of the corresponding pentafluorophenyl esters enabled conversion into N-glycopeptides by use of unprotected glycosyl amines and a mixture of DIEA and HOBt. Detachment from the resin and removal of tert-butyl side-chain protecting groups were achieved by treatment with TFA. The combination of mono- and oligoglycosidic amines with five peptides resulted in 23 different N-glycopeptides (see Scheme 38) including interesting products with sulfated glycosyl residues [ 1201. Problems were encountered with fucosylated glycosides-this 0-glycosidic linkage was cleaved even by dilute TFA. Another difficulty that prevents general applicability is the tendency of activated aspartic acid derivatives to rearrange to aspartimides. A solution to this problem was provided by appropriate partial protection of
11.3 Glycopeptide Synthesis on the Solid Phase
299
the peptide backbone. With the help of the N-2-acetoxy-4-methoxybenzyl (AcHmb) group on the alanine residue, aspartimide formation throughout the convergent solid-phase synthesis of a chitobiosylated hexapeptide was suppressed [ 1211. The AcHmb group can be removed in two steps-hydrazine-mediated deacetylation and subsequent treatment with TFA. Sequential N-Glycopeptide Synthesis on the Solid Phase with Oligosaccharides from Natural Sources The use of oligosaccharide residues from natural sources in solid-phase peptide synthesis requires two steps, isolation in sufficient quantity and conversion into suitable building blocks. Whereas conversion of the reducing end of very complex glyco-structures into glycosyl amines has already been achieved [24, 1221, the primary step of detaching sufficient quantities of N-linked oligosaccharides from their natural precursors still requires simplification. Very recently a modified hydrazinolysis procedure has enabled the isolation of di- and trianntenary structures from fetuin and high-mannose type compounds from ribonuclease B on a 100-500-mg scale (Scheme 39) [123]. Upon treatment with aqueous ammonium bicarbonate the corresponding p-configured glycosylamines were obtained and coupled to activated Fmoc-Asp-OtBu. After tert-butyl removal the resulting building blocks were successfully used in solid-phase synthesis. Compounds such as the undecaglycodecapeptide 87 were obtained.
Val
I I Thr I Ile
OH
I<
OH O 'H 0,
OH AcHN
OH
flnu 5
P " LOH O H
Scheme 39.
87
300
I1 Glycopeptide Synthesis in Solution and on the Solid Phase
Certainly, this methodology holds great potential for particular saccharide sidechains that can be isolated from natural sources and are not readily accessible via chemical synthesis.
11.4 Conclusion The synthetic methods reviewed in this chapter and in earlier survey articles now make available glycopeptides of considerable complexity. These methods have been provided by the development of distinctly chemo-, regio-, and stereoselective chemical reactions supplemented by efficient techniques adopted from biochemistry and biotechnology. The step-by-step assembly of glycopeptides in solution is still the method of choice if larger quantities of the target compounds ( 2 1 mmol) are required. The synthesis of glycopeptides containing important but complex saccharide residues remains a major task of glycopeptide chemistry. The introduction of sialic acid residues-considered to be a limitation in the past-can now be achieved efficiently. Remarkable progress has also been made in the use of enzymatic methods. The latter have been shown to be of particular advantage for the assembly of carbohydrate side-chains in the context of linear or convergent synthetic strategies. In combination with improvements in solid-phase technology, e.g. adapted resins and coupling reagents, further progress in the preparation of biologically important glycopeptides can be expected. Abbreviations AgOTf All
Bn Boc BOP Bz Bzl CAN DCC DIEA DMAP DMF DMSO DMTST EEDQ
Fmoc
silver triflate ally1 benzyl t-butyloxycarbonyl benzotriazole-1-yl-oxy-tris(dimethylamin0)phosphoniumhexafluorophosphate benzoyl benzyl ceric ammonium nitrate N ,N' -dicyclohexylcarbodiimide
diisopropylethylamine dimethylaminopyridine dimethylformamide dimethylsulfoxide dimethylthiomethylsulfonium triflate 2-ethoxy-1-ethoxycarbonyl-1,2-dihydrochinoline 9-fluorenylmethoxycarbonyl
References
301
Fuc Gal GalNAc GlcNAc HATU
fucose galactose 2-acetamido-2-desox y-galactose 2-acetamido-2-desox y-glucose 2-( 1H-9-azobenzotriazole-1-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate hydroxyazabenzotriazole HOAt hydroxybenzotriazole HOBt Sheptadecanoyl HYCRON (E)-17-hydroxy-4,7,1O,I3-tetraoxa-l IIDQ 1-isobutyloxycarbonyl-2-isobutyloxy1,2-dihydrochinoline lactose Lac mannose Man p-methoxyphenylmethy1 MPm N-acetyl neuraminic acid NeuNAc PEGA polyethylene glycol-dimethylacrylamide co-polymer pentafluorphenyl PfP PhAcOZ p-(phenylacetoxy)benzyloxycarbonyl POEPOP polyoxyethylene-polyoxypropylene copolymer rotating frame nuclear overhauser spectroscopy ROESY super acid sensitive resin SASRIN sialyl-Lewis-)< SLe" 2-(1H-benzotriazole-1-yl)- I, 1,3,3-tetramethyluroniumtetrafluoro-borate TBTU 'Bu tert-butyl Tcoc N-trichloroethoxycarbonyl TFA trifluoroacetic acid TMSOTf trimethylsilyl triflate benzyloxycarbonyl Z
References H. Lis, N. Sharon, Eur. J. Biochem., 1993, 218, I . A . Varki, Glycohiol., 1993, 3 , 97. R. A. Dwek, Chem. Rev., 1996, 96, 683. G. Arsequell, G. Valencia, Tetrahedron Asym., 1997, 8, 2839. B. K. Hayes, G. W. Hart, Curr. Opin. Struct. Biol., 1994, 4, 692. N. Jentoft, TZBS, 1990, 15, 291. D. Sako, K. M. Comess, K. M. Barone, R. T. Camphausen, D. A. Cumming, G. D. Shaw, Cell, 1995, 83, 323. 8. T. A. Springer, Cell, 1994, 76, 301. 9. M. L. Phillips, E. Nudelman, F. C. A. Gaeta, M. Perez, A. K. Singhal, S.4. Hakomori, J. C. Paulson, Science, 1990, 250, 1130. 10. M. Larkin, T. J. Ahern, M. S. Stoll, M. Shaffer, D. Sako, J. O'Brien, C.-T. Yuen, A. M. Lawson, R. A. Childs, K. M. Barone, P. R. Langer-Safer, A. Hasegawa, M. Kiso, G. R. Larsen, T. Feizi, J. Biol. Chem., 1992, 267, 13661. 11. E. E. Simanek, G. J. McGarvey, J. A. Jablonowski, C.-H. Wong, Chem. Rev, 1998, 98, 833. 12. H. Lis, N. Sharon, Chem. Rev., 1998, Y8, 637. 13. W.-j. Liu, V. Ramachandran, J. Kang, T. K. Kishimoto, R. D. Cummings, R. P. McEver, J. Biol. Chem., 1998, 273, 1078.
I. 2. 3. 4. 5. 6. 7.
302
11 Glycopeptide Synthesis in Solution and on the Solid Phase
14. P. P. Wilkins, R. P. McEver, R. D. Cummings, J. Biol. Chem., 1996, 271, 18732. 15. F. Li, H. P. Erickson, J. A. James, K. L. Moore, R. D. Cummings, R. P. McEver, J. Biol. Chem., 1996,271, 6342. 16. M. DeLuca, L. C. Dunlop, R. K. Andrews, J. V. Flannery, R. Ettling, D. A. Cumming, G. M. Veldman, M. C. Berndt, J. Biol. Chem., 1995, 270, 26734. 17. H. Kunz, M. Schultz, in D. G. Large, C. D. Warren (Eds.): Glycopeptides and Related Compounds, Marcel Dekker. Inc., New York 1997, p. 23. 18. H. G. Garg, K. v. d. Bruch, H. Kunz, Adu. Carbohydr. Chem. Biochem., 1994,50,277. 19. H. Paulsen, Angew. Chem. Int. Ed. Engl., 1982, 21, 155. 20. H. Kunz, Angew. Chem. Znt. Ed. Engl., 1987,26, 294. 21. K. Toshima, K. Tatsuta, Chem. Rev., 1993, 93, 1503. 22. 0. Lockhoff, in H. Hagemann, D. Klamann (Eds.): Methoden der Organischen Chemie, Vol. E14a/3, G. Thieme, Stuttgart 1992, p. 621. 23. R. R. Schmidt, Angew. Chem. Znt. Ed. Engl., 1986,25, 212. 24. L. M. Likhosherstov, 0. S. Novikova, V. A. Derevitskaja, N. K. Kochetkov, Carbohydr. Rex, 1986, 146, C1. 25. H. Kunz, H. Waldmann, Angew. Chem. Znt. Ed. Engl., 1985,24, 883. 26. R. U. Lemieux, J. I. Haymi, Can. J. Chem., 1965,43,2162. 27. H. Lonn, Carbohydr. Res., 1985, 139, 105. 28. R. R. Schmidt, J. Michel, Angew. Chem. Znt. Ed. Engl., 1980, 19, 731. 29. U. Sprengard, G. Kretzschmar, E. Bartnik, C. Huh, H. Kunz, Angew. Chem. Znt. Ed. Engl., 1995, 34, 990. 30. R. R. Schmidt, in H. Ogawa, A. Hasegawa, T. Suami (Eds.): Carbohydrates-Synthetic Methods and Applications in Medicinal Chemistry, Verlag Chemie, Weinheim 1992, p. 66. 31. R. R. Schmidt, ASC Symposium Series, 1994, 560, 276. 32. K. Okamoto, T. Goto, Tetrahedron, 1990, 46, 5835. 33. A. Hasegawa, H. Ohki, T. Nagahama, H. Ishida, M. Kiso, Carbohydr. Res., 1991,212, 277. 34. A. Marra, P. Sinay, Carbohydr. Res., 1989, 187, 35. 35. H. Lonn, K . Stenvall, Tetrahedron Lett., 1992, 33, 115. 36. T. Murase, H. Ishida, M. Kiso, A. Hasegawa, Carbohydr. Res., 1988, 184, C1. 37. F. Dasgupta, P. J. Garegg, Carbohydr. Rex, 1988, 177, C13. 38. W. Stahl, U. Sprengard, G. Kretzschmar, D. W. Schmidt, H. Kunz, J. Prakt. Chem., 1995, 337, 441. 39. H. Kunz, C. Unverzagt, Angewj. Chem. Znt. Ed. Engl., 1988, 27, 1697. 40. C. Unverzagt, H. Kunz, Bioorg. Med. Chem., 1994,2, 1189. 41. B. Liebe, H. Kunz, Helv. Chim. Actu, 1997, 80, 1473. 42. D. G. Large, I. J. Bradshaw, in D. G. Large, C. D. Warren (Eds.): Glycopeptides and Related Compounds, Marcel Dekker, Inc., New York 1997, p. 295. 43. P. L. Devine, I. F. C. McKenzie, BioEssays, 1992, 14, 619. 44. J. J. R. Gum, Biochem. SOC.Trans., 1995, 23, 795. 45. I. Brockhausen, J.-M. Yang, J. Burchell, J. T.-Papadimitriou, Eur. J. Biochem., 1995, 233, 607. 46. S. H. Itzkowitz, M. Yuan, C. K. Montgomery, T. Kjeldsen, H. K. Takahashi, W. L. Bigbee, Y. S. Kim, Cancer Rex, 1989, 49, 197. 47. R. R. Koganty, M. A. Reddish, B. M. Longenecker, in D. G. Large, C. D. Warren (Eds.): Glycopeptides and Related Compounds, Marcel Dekker, Inc., New York 1997, p. 707. 48. S. Gendler, J. Taylor-Papadimitriou, T. Duhig, J. Rothbard, J. Burchell, J. Biol. Chem., 1988, 263, 12820. 49. P. Braun, G. M. Davies, M. R. Price, P. M. Williams, S. J. B. Tendler, H. Kunz, Bioorg. Med Chem., 1998, 6, 1531. 50. R. U. Lemieux, R. M. Ratcliffe, Can. J. Chem., 1979, 57, 1244. 51. H. Paulsen, W. Rauwald, U. Weichert, Liebigs Ann. Chem., 1988, 75. 52. M. Ravenscroft, R. M. G. Roberts, J. G. Tillett, J. Chem. Soc., Perkin Trans., 1982, 1568. 53. A. Reidel, H. Waldmann, J. Prakt. Chem., 1993, 335, 109. 54. J. Eberling, P. Braun, D. Kowalczyk, M. Schultz, H. Kunz, J. Org. Chem., 1996, 61, 2683. 55. M. Gewehr, H. Kunz, Synthesis, 1997, 1499.
References
303
T. Toyokuni, S.4. Hakomori, A. K. Singhal, Bioorg. Med. Chem., 1994, 2, 1119. H. Paulsen, M. Paal, Carbohydr. Res., 1984, 135, 71. P. Fugedi, P. J. Garegg, H. Lonn, T. Norberg, Glycoconjugate J., 1987, 4, 97. D. R. Mootoo, P. Konradsson, B. Fraser-Reid, J. Am. Chem. Soc., 1989, 111, 8540. M. Leuck, H. Kunz, J. Prukt. Chern., 1997,339, 322. B. Liining, T. Norberg, J. Tejbrant, Glyeoconj. J., 1989, 6, 5. D. Sames, X.-T. Chen, S. J. Danishefsky, Nature, 1997, 389, 587. T. J. Martin, R. Brescello, A. Toepfer, R. R. Schmidt, Glycoeonj. J., 1993, 10, 16. M. M. Sim, H. Kondo, C. H. Wong, J. Am. Chem. Soc., 1993,115,2260. J. Liang, W.-R. Li, M. Joullie, Synth. Conzmun., 1994, 24, 187. J. B. Schwarz, S. D. Kuduk, X.-T. Chen, D. Sames, P. W. Glunz, S. J. Danishefsky, J. Am. Chem. SOC., 1999, 121, 2662. 67. S. Komba, M. Meldal, 0.Werdelin, T. Jensen, K. Bock, J. Chem. Soc., Perkin Trans. 1,1999, 415. 68. G. W. Hart, R. S. Haltiwanger, G. D. Holt, W. G. Kelly, Annu. Rev. Biochem., 1989, 58, 785. 69. G. W. Hart, D. M. Snow, Int. Rev. Cytol.; 1998, 181, 43. 70. J. Banoub, P. Boullanger, D. Lafont, Chem. Soc. Rev., 1992, 92, 1167. 71. M. Schultz, H. Kunz, Tetrahedron Asymmetry, 1993, 4, 1205. 72. U. K. Saha, R. R. Schmidt, J. Chem. Soc., Perkin Trans. I , 1997, 1855. 73. T. Pohl, H. Waldmann, J. Am. Chern. Soc., 1997, 119, 6702. 74. C. Unverzagt, Tetrahedron Lett., 1997, 38, 5627. 75. C. Unverzagt, Angew. Chem. Int. Ed. Engl., 1994: 33; 1102. 76. C. Unverzagt, H. Kunz, J. Prakt. Chem., 1992,334, 570. 77. H. Kunz, W. Giinther, Angew. Chern. Int. Ed. Engl., 1988, 29, 1068. 78. W. Gunther, H. Kunz, Curbohydr. Rex, 1992, 228, 217. 79. C. Uuverzagt, S. Kelm, J. C. Paulson, Carbohydu. Rex, 1994, 251, 285. 80. C. Unverzagt, H. Kunz, J. C. Paulson, J. Am. Chem. Soc., 1990, 112, 9308. 81. S. T. Cohen-Anisfeld, P. T. Lansbury, J. Am. Chem. Soc., 1993, 115, 10531. 82. S. Y. C. Wong, G. R. Guile, W. T. Rademacher, R. A. Dwek, Glycoconj. J., 1993, 10, 227. 83. M. Mizuno, K. Haneda, R. Iguchi, I. Muramotot, T. Kawakami, S. Aimoto, K. Yamamoto, T. Inazu, J. Am. Chem. Soc., 1999, 1999, 284. 84. T. Feizi, Curr. ,Opin. Stuuct. B i d , 1993, 3, 701. 85. G. Baisch, R. Ohrlein, Angew. Chem., Int. Ed. Engl., 1996, 35, 1812. 86. U. Sprengard, M. Schudok, W. Schmidt, G. Kretzschmar, H. Kunz, Angew. Chem., Int. Ed. Engl., 1996, 35, 321. 87. S. J. Danishefsky, J. Y. Roberge, in D. G. Large, C. D. Warren (Eds.): Glycopeptides and Reluted Compounds, Marcel Dekker, Inc., New York 1997, p. 245. 88. M. Meldal, Curr. Opin. Struct. Biol.,1994, 4, 710. 89. G. Barany, N. Kneib-Cordonier, D. G. Mullen, Int. J. Peptide Protein Res., 1987, 30, 705. 90. J. Habermann, H. Kunz, Tetrahedron Lett., 1998, 39, 265. 91. R. Knorr, A. Trzeciak, W. Bannworth, D. Gillessen, Tetrahedron Lett., 1989, 30, 1927. 92. J. Habermann, H. Kunz, J. Prukt. Chem., 1998, 340, 233. 93. A. Schleyer, M. Meldal, M. Renil, H. Paulsen, K. Bock, Angew. Chem., Int. Ed Engl., 1997, 36, 1976. 94. H. Rink, Tetrahedron Lett., 1987,28, 3787. 95. H. Paulsen, A. Schleyer, N. Mathieux, M. Meldal, K. Bock, J. Chem. Soc., Perkin Trans. I , 1997, 281. 96. S. H. Itzkowitz, E. J. Bloom, W. A. Kokal, G. Modin, S.4. Hakomori, Y. S. Kim, Cancer, 1990, 66, 1960. 97. H. Kunz, in C. H. Schneider (Ed.): Peptide3 in Immunology, J. Wiley and Sons, Ltd., ChiChester 1996, p. 10. 98. B. Liebe, H. Kunz, Helv. Chim. Acta, 1997, 80, 1473. 99. T. R. E. Stadie, W. Chai, A. M. Lawson, P. G. H. Byfield, F . G . Hanisch, Eur. J. Biochenz., 1995,229, 140. 100. M. Elofsson, L. A. Salvador, J. Kihlberg, Tetrahedron, 1997, 53, 369. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66.
304
11 Glycopeptide Synthesis in Solution and on the Solid Phase
101. J. U. Yang, J. C. Byrd, B. B. Siddiki, Y. S. Chung, M. Okumo, M. Sowa, Y. S. Kim, K. L. Matta, I. Brockhausen, Glycobiology, 1994, 4, 873. 102. Y. Nakahara, Y. Nakahara, Y. Ito, T. Ogawa, Tetrahedron Lett., 1997,38, 721 1. 103. Y. Nakahara, H. Iijima, T. Ogawa, Tetrahedron Lett., 1994, 35, 3321. 104. N. Fujii, A. Otaka, 0. Ikemura, K. Akaiji, J. Chem. Soc., Chem. Comm., 1987, 1987, 274. 105. G. Arsequcll, C. Krippner, R. A. Dwek, S. Y. C. Wong, J. Chem. Soc., Chem. Commun., 1994, 1994, 2383. 106. L. A. Salvador, M. Elofsson, J. Kihlberg, Tetrahedron, 1995, 51, 5643. 107. E. Meinjohanns, M. Meldal, K. Bock, Tetrahedron Lett., 1995, 36, 9205. 108. U. K. Saha, L. S. Griffith, J. Rademann, A. Gcyer, R. R. Schmidt, Carbohydr. Rex, 1997, 304, 21. 109. K. Witte, 0. Seitz, C.-H. Wong, J. Am. Chem. Soc, 1998, 120, 1979. 110. R. J. Harris, M. W. Spellman, Glycobiology, 1993, 3, 219. 111. N. Nakakura, H. Hietter, A. v. Dorssclear, B. Luu, Eur. J. Biochem., 1992, 204, 147. 112. H. Hietter, M. Schultz, H. Kunz, Synlett, 1995, 1219. 113. D. Nguyen, A. Heitz, B. Castro, J. Chem. Soc., Perkin Trans. I , 1987, 1915. 114. M. Elofsson, S. Roy, L. A. Salvador, J. Kihlberg, Tetrahedron Lett., 1996, 37, 7645. 115. S. Peters, T. L. Lowary, 0 . Hindsgaul, M. Meldal, K. Bock, J. Chem. Soc., Perkin Trans. I, 1995, 3017. 116. 0. Seitz, C.-H. Wong, J. Am. Chem. Soc, 1997, 119, 8766. 117. 0. Seitz, H. Kunz, Angew. Chem. Int. Ed. Engl., 1995, 34, 803. 118. D. Vetter, D. Tumelty, S. K. Singh, M. H. Gallop, Angew. Chem., Znt. Ed. Engl., 1995,34, 60. 119. M. Mergler, R. Tanner, J. Gostcli, P. Grogg, Tetrahedron Lett., 1988, 29, 4009. 120. C. Galustian, R. A. Childs, C. T. Yucn, A. Hasegawa, M. Kiso, A. Lubineau, G. Shaw, T. Feizi, Biochem., 1997, 36, 5260. 121. J. Offer, M. Quibell, T. Johnson, J. Chem. Soc., Perkin Trans. I , 1996, 175. 122. A. Lubineau, J. Auge, B. Drouillat, Carhohydr. Rex, 1995, 266, 211. 123. E. Meinjohanns, M. Meldal, H. Paulsen, R. A. Dwck, K. Bock, J. Chem. Soc., Perkin Trans. I, 1998, 549.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
12 Glycolipid Synthesis Hideharu Ishida
12.1 Introduction Glycolipids are classified into three groups, glycosphingolipids, glycoglycerolipids and others which include lipid A of lipopolysaccharide. Gangliosides, sialylated forms of glycosphingolipids, are most intensively studied because of their biological as well as chemical interest. As shown Table 1, gangliosides are further classified into several subclasses depend on the structures of oligosaccharide backbone. In the first section, the synthesis of ganglioside G D l a as a example of ganglioseries ganglioside is described, including the topics which are common even to other series gangliosides, for example preparation of building blocks and transformation of oligosaccharides into glycolipids. In the following section, the synthesis of polysialo ganglio-series gangliosides is described as the extension of this methodology.
12.2 Synthesis of Ganglio-Series Gangliosides In this section, synthesis of ganglioside G D l a as the example of a-series gangliosides which carry sialic acid at 0-3’ of lactose, is described. 12.2.1 Retrosynthetic Analysis of Ganglioside GDla
Retrosynthetic analysis of ganglioside G D l a (Scheme 1 [l]) shows two import concepts which can be applied for all of gangliosides in common. First, lipid part should be incorporated to the sialo-oligosaccharide after complete construction of the sugar chain. When the lipid is coupled with the glucose,
306
12 Glycolipid Synthesis
Table 1. Fundamental carbohydrate structure of gangliosides. hematoside-series Galp(l+I)Cer Galp(1 +4)GlcP( 1+1)Cer ganglio-series
GalNAc~(1+4)Gal~(l+4)Glc~(l+l)Cer Galp( 1+3)GalNAcP( 1+4)Galp( 1-+4)Glcp(1+1)Cer lacto- and neolacto-series [GalP(l+3)GlcNAc~(l+3)]nGal~(1+4)Glc~(l+l)Cer
[Gal~(l+4)GlcNAc~(l+3)]nGal~(l-+4)Glc~(l+l)Cer globo- and isoglobo-series
GalNAc~(l+3)Gala(l+4)Gal~(l+4)Glc~(l+l)Cer GalNAc~(l+3)Gala(l+3)Gal~(l+4)Glc~(l+l)Cer
which is the reducing terminal of oligosaccharide, at the first stage of the synthetic route, the resulting glucosyl ceramide has too low reactivity to serve as a glycosyl acceptor for further elongation of oligosaccharide chain, because mainly of the steric hindrance caused by the lipid. Second, sialic acid should be introduced to the oligosaccharide in the form of building block, e.g. sialylgalactose or sialyllactose. Because of the structural feature, sialic acid affords the corresponding glycoside in good yields only on the coupling with reactive acceptors. When reacted with less reactive acceptors, like oligosaccharides, sialic acid is easily transformed into the 2,3-dehydro derivative which is the major by-product of the reaction and lower the yields. The detail of this matter is discussed separately in this book. It is also of importance to note that the stereochemistry of the anomeric center was used to be determined by the chemical shifts of NMR of H-3ax, H-3eq, H-4 by the empirical rule [2]. This means that the stereochemistry of a novel sialoside synthesized by a coupling of sialic acid and complex sugar chain can not be determined absolutely by NMR. This problem can be solved by employing the building block, the stereochemistry of which has already been determined. 12.2.2 Preparation of Sialylgalactose Donor as Building Block
The sialylgalactose 1 [2] synthesized by the established procedure can be readily transformed into the corresponding glycosyl donor as the building block which is required to synthesize gangliosides in a tailor-made manner (Scheme 2 [3]). Compound 1 was converted by (1) benzoylation of the remaining hydroxyls, followed by either (2) selective transformation of 2-(trimethylsily1)ethyl (SE) group at the reducing end into the corresponding p-acetate by treatment [4] with boron trifluoride etherate-acetic anhydride and (3) introduction of the methylthio group by treatment [51 with (methy1thio)trimethylsilane and boron trifluoride, or (4) chemo-
12.2 Synthesis of' Ganglio-Series Gangliosides
301
,."* OH
AcHN HO "'OH HO
I OBZ
HO-Cl3H2,
HN
x-
C17H35
or AcO
OAc
AcO
OBz
"OAc AcO
HO+CI3H2, N3
SMe
AcO
OAc AcO
I .
n
__
< - s S M e NPhth AcO AcO
"'OAc
Scheme 1.
selective deprotection of SE group by treatment [4]with trifluoroacetic acid and (5) activation as trichloroacetimidate [6] to the desired donors, respectively. The use of resulting donors excludes any possibility of the P-sialyl glycoside formation. The same procedure can be applied for the conversion of disialylgalactose to the corresponding donor (see 3-(2)).
308
12 Glycolipid Synthesis ACO
OAc
OSE
AcHN AcO
’1
AAcOc O U 0 + OAc C02Me OSE
AcHN AcO
2)s 3)
BzO
OBz
2
1 AcO A c
OAc C02Me O U O *
SMe
AcHN
BzO
OBz
3
4 1) Bz,O, DMAP, Pyr., 2) Ac20, BF3*OEt2,3) TMSSMe, TMSOTf, 4) TFA, 5 ) CCISCN, DBU
Scheme 2.
12.2.3 Construction of Oligosaccharide The most important synthetic problems in the systematic synthesis of a-series ganglio-series gangliosides is a formation of GM2 structure, GalNAcb1-3(NeuAca23)Galbl-4Glc, synthesis of which has been found to be difficult because of its compact and lipid structure [7]. As shown in Fig. 1, retrosynthetic analysis of GM2 gives two synthetic strategies; (A): GA2 + NeuAc and (B): GM3 GalNAc. Because introduction of NeuAc into the isolated hydroyxl at 0-3’ of GA2 trisaccharide was found to be difficult by the conventional procedures reported, the strategy B is recommended for use.
+
A GalNAcP1, 4 GalPl+4Glc 3
’
NueAca2
GM2
GalNAcp,
P
4 GalPl+4Glc
NeuAc
+
GalNAc
GA2
\ B NueAca2’
Gal01-4Glc 3 GM3
Figure 1.
+
309
12.2 Synthesis of Gunglio-Series Ganyliosides A
OAc
C
O
AcHN
S
OSE
0 En0 AcO
~
&ty
OAc
~
SMe NPhth
5 A
~
-k
BnO
OBn
HO
~
6 C
AcHN
O
~
0 BnO OBn
~
~
~
,OSE OBn
NPhth
7
BzO 4cHN
+
OBz SMe
ACO""' AcO
C02Me
OBz
OAc
3
OAc
9
1) NIS-TfOH, 2) Lil, 3) N2H4*H20,4) Ac20, Pyr., 5) CH2N,,
6)80% aq. AcOH, 7) NIS-TfOH Scheme 3.
For the systematic synthesis of ganglio-series gangliosides, sialyllactose derivative 5 [8] and galactosamine derivative 6 [8] were selected as the glycosyl acceptor and the glycosyl acceptor, respectively (Scheme 3). The glycosylation of 5 with 6 in the presence of NIS-TfOH as the glycosyl promoter [9]gave the desired P-glycoside 3 in 68% yield. Methyl ester of the sialic acid was cleaved by treatment with LiI [ 101 to give the carboxyl group free derivative to present the side-product formation during
310
12 Glycolipid Synthesis
the treatment with hydrazine monohydrate to cleave the phthaloyl group. Schmidt et al. [ 1 I] employed the N-trichloroethoxycarbonyl group in place of phthaloyl group to protect the galactosamine donor to skip this tedious steps. After conversion of phthalimide into acetamide, isopropylidene group was removed to give the tetrasaccharide acceptor 8. The glycosylation of 8 with 3 in the presence of dimethyl(methy1thio)sulfonium triflate [ 121 gave the expected hexasaccharide in 66% yield.
12.2.4 Transformation of Oligosaccharide into Glycolipid Transformation of oligosaccharides into glycolipids can be accomplished be following our standard methods (Scheme 4). First, benzyl group used in oligosaccharide must be replaced with acetyl group, because final deprotection must be accomplished without affecting the double bond of ceramide. Hydrogenolysis over Pd/C, followed by treatment with Ac2O in pyridine led to replacement of all 0benzyl groups by 0-acetyl groups. Second, SE group at the anomeric position is removed by treatment with trifluoroacetic acid in CH2C12 (41 in a quantitative yield. When the anomeric position was protected with acetyl group, converted form benzyl group as described abve, treatment with hydrazine monoacetate (N2H4.AcOH) [ 131 cleaves the anomeric 0-acetyl group with high regioselectivity. And the following treatment with trichloroacetonitrile in the presence of 1,8-diazabicycl0[5.4.0]undec7-ene (DBU) 1141 afforded the hexasaccharide donor. The resulting glycosyl donor was then coupled with ceramide [15] or azidosphingosine [ 16, 171 which is a synthetic precursor of ceramide. Although the former strategy can shorten the synthetic route, the yields of coupling are rather low. Ogawa et al. [ 161 and Schmidt et al. [ 101 were successful in increasing the yield by employing the pivaloyl group for the protection of hydroxyl at the C-2 of the reducing terminal glucose, in place of acetyl group which causes the formation of the corresponding orthoester to decrease the coupling yield. The latter strategy, the coupling with azidosphingosine, is now popularly employed because of the good yield for coupling and the versatility to accept a series of fatty acids as components of ceramide, although the resulting glycosyl azidosphigosine nessitates the further transformation, reduction of azido to amine and introduction of fatty acids. The reduction was performed mainly by two procedures, treatment with H2S in aqueous pyridine [I81 or treatment with triphenylphoshine [19].When the hydroxyl at C-3 of azidosphingosine is protected with benzoyl group, both of the procedures often cause the migration of benzoyl from hydroxyl to amine formed. This problem can be solved by having the temperature during the reduction or by employing tertbutyldiphenylsilyl group [ 191 for the protection in place of benzoyl group. Finally, the complete deprotection of the acyl group, e.g. acetyl, benzoyl or pivaloyl group, followed by the saponification affords the desired gangliosides. Scheme 4 illustrates our standard methods to transform oligosaccharides into the corresponding gangliosides. When the silyl protection was employed, treatment [20] with tert-butylanmonium fluoride was required before the deacylation.
12.3 Synthesis of Polysiulo Gunglio-Series Gangliosides
3 11
OSE AcHN
OBn OAc
9 1) H2, Pd(OH)2I MeOH 2) AczO, Pyr.
3) CFjCOzH I CHzCl2 4) CCIICN, DBU I CH2CIz
TMSOTf I CH2C12
5) H O & C , ~ H ~ ~ OTBDPS 6) PPh3,HzO I C6Hs
7) stearic acid, WSC I CH2C12 8)l.OM TBAF I CH&N
9) MeONa I MeOH, 0.2M KOH V
OH
\ no-
\
OH
L..
C02H
GD1 a Scheme 4.
12.3 Synthesis of Polysialo Ganglio-Series Gangliosides Polysialo gangliosides are common in nature, having a(2+8)glycosidic linkage between two sialyl residues. In this section, the synthesis of ganglioside G Q l b as an example of polysialo ganglio-series gangliosides is described in comparison with that of GDla.
3 12
12 Glycolipid Synthesis
u1 \
W u)
?
12.3 Synthesis of Polysiulo Gunglio-Series Gangliosides
3 13
12.3.1 Retrosynthetic Analysis of GQlb An efficient method for obtaining disialyl glycoside is the use of dimeric sialic acid donor, which can be readily prepared from naturally available colominic acid. As shown in Scheme 5 [21], ganglioside GQlb can be synthesized in almost same procedure as that of G D l a by employing dimeric sialic acid in place of monomeric sialic acid, except for the construction of GD2 which is the key intermediate for the synthesis of GQlb. It is of worth to note that the dimeric sialic acid donor is enough reactive to glycosylate the isolated hydroxyl at 0-3' of GA2, which can not be glycosylated with the monomeric sialic acid. This surprising result made the synthetic route of GQlb short and practical.
12.3.2 Preparation of Building Block The disialylgalactose obtained by the glycosylation of the galactose acceptor with the dimeric sialic acid donor, was converted into the glycosyl donor as the same procedure as described for the sialylgalactose donor Scheme 6 [22].
11
Bz20, DMAP, Pyr., 2) Ac20, BF3*OEt2,3) TMSSMe, TMSOTf Scheme 6.
3 14
12 Glycolipid Synthesis OBn
AcO 4 0 A c
,OBn
co
+ OBn OBn
12 13
Aco O ,Ac
co
14
/
AcO o,Ac
co
/
11
3,
15 1) NIS-TfOH, 2) 80% aq. AcOH, 3) DMTST
Scheme 7.
12.3.3 Construction of Oligosaccharide
GD2 pentasaccharide acceptor 14 was selected as the key compound to achieve the systematic synthesis of polysialo ganglio-series gangliosides (Scheme 7). Glycosylation of GA2 acceptor 12 with a-sialyl-(2+8)-sialic acid donor 13 in the presence of NIS-TfOH in acetonitrile at -25 "C, afforded the desired pentasaccharide in 50% and no p-isomer was isolated. De-0-isopropylidenation of the pentasaccharide with aqueous 80% acetic acid afforded the acceptor 14 in 91% yield. It is noteworthy that the key intermediate 14 was prepared by one step, de-0-isopropylidenation, after the construction of GD2 pentasaccharide, in contrast to the synthesis of G D 1a. Glycosylation of 14 with sialyl a(2-8)sialyl galactose donor 11 by use of
315
12.4 Conclusion
+
Glc-Cer GaCGlc-Cer
GaCGlc-Cer Ned5Ac
GalNAc-Gal-Glc-Cer
GA2
+
+
Gal-GalNAc-GaCGlc-Cer
+
+ +
Gal-GalNAc-GaCGlc-Cer Ned5Ac
J.
NeyCAc Gal-GalNAc-Gal-Glc-Cer Ned5Ac GDclx I I
I' I/
GM2
+
GaCGalNAc-GaCGlc-Cer N e e Neu5Ac GDlb
GM1
f Gal-GalNAc-Gal-Glc-Cer Ned5Ac Ne@ GTlb Neu5Ac
GDla
I
I
f
GaCGalNAc-GaCGlc-Cer Ne&Ac NedsAc I
,
1
I I
I'
Gal-GalNAc-Gal-Glc-Cer Nei5Ac Ned5Ac Neu5Ac
GT1a
[a-series]
+
Gal-GalNAc-Gal-Glc-Cer Ned5Ac Ned5Ac Ned5Ac Ned5Ac
,
I
I
GT1act
Gal-Glc-Cer Nel(5Ac Neu5Ac GD3
GalNAc-GaCGlc-Cer Ne@ Neu5Ac GD2
Gal-GalNAc-Gal-Glc-Cer NedSAc
GM l b
I I I
GM3
GalNAc-Gal-Glc-Cer Ned5Ac
GA1
I I I I
~
I
GQ1b
[b-series)
NeukAc
GQlbcx Figure 2.
DMTST in CHZCl2 gave the octasaccharide 15 in 52% yield, which was transformed into the final compound by our standard methods described in the previous section.
12.4 Conclusion In this chapter, the methodology to synthesize a series of a-series as well as b-series ganglio-series gangliosides is described. By combining the two procedures, all of
3 16
12 Glycolipid Synthesis
ganglio-series (Fig. 2), GM2 [8, 91, GM1 [l, 91, G D l a [l],G T l a [23], GD3 [22, 241, GD2 [25], G D l b [Zl], G T l b [21], and G Q l b [21]. In addition, a series of novel ganglio-series gangliosides, GTlacl [27] and GQlbcl [28] (also see, Fig. 2) was successfully synthesized as the application of the procedures. Other series of gangliosides, sialyl Le" [29], sialyl dimeric Le" [30, 311, and sialyl Lea [32] of lacto- and neolacto-series gangliosides, and sialyl globopentasaccharide (SSEA-4) [33, 341 and sialyl isoglobopentaosyl ceramide [ 351 of globo- and isoglobo-series gangliosides in a tailor-made manner, respectively.
References 1. A. Hasegawa, H. K. Ishida, T. Nagahama, M. Kiso, J. Curbohydr. Chem. 1993,12, 703-718. 2. T. Murase, A. Kameyama, K. P. R. Kartha, H. Ishida, M. Kiso, A. Hasegawa, J. Curbohydr. Chem. 1989, 8, 265-283. 3. A. Kameyama, H. Ishida, M. Kiso, A. Hasegawa, Curbohydr. Res. 1990, 200, 269-285. 4. a) K. Jansson, T. Frejd, 3. Kihlberg, G. Magnusson, Tetrahedron Lett. 1986, 27, 753-756; b) K. Jansson, S. Ahlfors, T. Frejd, J. Kihlberg, G. Magnusson, J. Dahmen, G. Noori, K. Stenvall, J. Org. Chem. 1988, 53, 5629-5647. 5. V. Pozsgay, H. J. Jennings, Tetrahedron Lett. 1987,28, 1375-1376. 6. a) R. R. Schmidt, J. Michel, Angew. Chem. Int. Ed. Engl. 1980, 19, 731; b) R. R . Schmidt, J. Michel, M. Roos, Liebigs Ann. Chem. 1984, 1343. 7. Y.-T. Li, S.-C. Li, A. Hasegawa, H. Ishida, M. Kiso, A. Bernardi, P. Brocca, L. Raimondi, S. Sonnino, J. Biol. Chem. 1999,274, 10014-10018. 8. A. Hasegawa, T. Nagahama, H. Ohki, M. Kiso, J. Carbohydr. Chem. 1992, 11, 699-714. 9. a) P. Konradsson, U. E. Udodong, B. Fraser-Reid, Tetrahedron Lett. 1990, 31, 4313-4316; b) G. H. Veeneman, S. H. van Leeuwen, J. H. van Boom, Tetrahedron Lett. 1990, 31, 13311334. 10. M. Sugimoto, M. Numata, K. Koike, Y. Nakahara, T. Ogawa, Curbohydr. Rex 1986, 156, cl. 11. J. C. Castro-Palomino, G. Ritter, S. R. Fortanato, S. Reinhardt, L. J. Old. R. R. Schmidt, Angew. Chem. Int. Ed. Engl. 1997, 36, 1998-2001. 12. P. Fiigedi, P. J. Garegg, Curbohydr. Res. 1986, 149, c9-cI2. 13. G. Excoffer, D. Gagnaire, J. P. Utille, Curbohydr. Res. 1975, 39, 308-373. 14. R. R. Schmidt, J. Michel, Angew. Chem. Znt. Ed. Engl. 1980, 19, 731. 15. K. Koike, Y. Nakahara, T. Ogawa, Glycoconjugate J. 1986, 1, 107-109. 16. Y. Ito, M. Kiso, A. Hasegawa, J. Curbohydr. Chem. 1989,8,285-294. 17. R. R. Schmidt, P. Zimmermann, Angew. Chem. Znt. Ed. Engl. 1986,25, 725-726. 18. T. Adachi, Y. Yamada, 1. Inoue, M. Saneyoshi, Synthesis 1977, 45-46. 19. M. Mori, Y. Ito, T. Ogawa, Curbohydr. Res. 1990. 195, 199-224. 20. T. Ehara, A. Kameyama, Y. Yamada, H. Ishida, M. Kiso, A. Hasegawa, Curbohydr. Res. 1996.281.237-252. 21. H.-K. Ishida, H. Ishida, M. Kiso, A. Hasegawa, Tetrahedron: Asymmetry. 1994, 5, 24932512. 22. H.-K. Ishida, Y. Ohta, Y. Tsukadd, M. Kiso, A. Hasegawa, Carbohydr. Rex 1993,246, 75-88. 23. H.-K. Ishida, H. Ando, H. Ito, H. Ishida, M. Kiso, A. Hasegawa, J. Curbohydr. Chem. 1997, 16, 413-428. 24. Y. Ito, M. Numata, M. Sugimoto, T. Ogawa, J. Am. Chem. SOC.1989, 111, 8508. 25. Y. Matsuzaki, S. Nunomura, Y. Ito, M. Sugimoto, Y. Nakahara, T. Ogawa, Carbohydr. Res. 1993,242, cl. 26. H.-K. Ishida, Y. Ohta, Y. Tsukada, Y. Isogai, H. Ishida, M. Kiso, A. Hasegawa, Curbohydr. R ~ s 1994,252, . 283-290. 27. H. Ito, H. Ishida, M. Kiso, A. Hasegawa, Curbohydr. Res. 1997, 304, 187-190. 28. K. Hotta, H. Ishida, M. Kiso, A. Hasegawa, J. Curbohydr. Chem. 1995, 14, 491-506.
References
3 17
29. A. Kameyama, H. Ishida, M. Kiso, A. Hasegawa, J. Curbohydr. Chern. 1991, 10, 549-560. 30. A. Kameyama, T. Ehara, Y. Yamada, H. Ishida, M. Kiso, A. Hasegawa, J. Curbohydr. Chem. 1995, 14, 507-523. 31. G. Hammel, R. R. Schmidt, Tetrahedron Lett. 1997, 38, 1173-1 176. 32. A. Kameyama, H. Ishida, M. Kiso, A. Hasegawa, J. Curbohydr. Chem. 1994, 13, 641-654. 33. H. Ishida, R. Miyawaki, M. Kiso, A. Hasegawa, J. Curbohydr. Chern. 1996, 15, 163-182. 34. J. M. Lassaletta, K. Carlsson, P. J. Garreg, R. R. Schmidt, J. Urg. Chem. 1996,61, 6873-6880. 35. H. Ishida, R. Miyawaki, M. Kiso, A. Hasegawa, Carhohydr. Res. 1996, 284, 179-190.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
13 Stereoselective Synthesis of P-Mannosides Vince Pozsguy
13.1 Introduction P-D-Mannopyranosyl residues are frequently found in oligosaccharides and glycoconjugates in living systems. This unit is omnipresent in N-linked glycoproteins that contain a pentasaccharide core (1) in which the P-linked mannose residue can carry additional sugars [I] (Scheme 1). P-Mannopyranose moieties are often part of the 0-specific polysaccharides (OSPs) of Gram-negative bacteria [2-8] and of bacterial capsular and em-polysaccharides [6, 9-15]. P-Linked mannopyranose is also a common residue in plant polysaccharides [ 161. Further examples of the occurrence of the P-mannosidic linkage are the glycosphingolipids in wheat flour [ 171 and in the freshwater bivalve Hyriopsis schlegelii [ 181. Interestingly, this residue is absent from mammalian glycolipids [19]. Cell-wall phosphomannans of yeasts contain linear chains of up to fourteen P-1,2-linked mannose residues [20, 211. P-Mannopyranose units also occur in secreted small molecules. Examples include the phospholipase inhibitor caloporoside [22] and free oligosaccharides detected in a patient with mannosidosis [23] and isolated from ripening tomatoes [24]. The related P-linked 2-acetamido-2deoxy-D-mannopyranose also frequently occurs in OSPs [ 8, 251 and in capsular polysaccharides [26-3 11. The corresponding mannuronic acid is present in the enterobacterial common antigen [32] and in other bacterial polysaccharides [6, 331. The occurrence of the 0-mannosyl unit in many biological systems creates a biological rationale for its synthesis. Whereas the simple methyl [34-381 and phenyl PD-mannopyranoside [ 391 have been known for many years, the synthesis of complex oligosaccharides containing this unit is still a challenge for two reasons [40-441. First, the anomeric effect facilitates the formation of the 1,2-truns-mannopyranosyl (a) linkage thus it disfavors the P-linkage and, second, the 1,2-cis arrangement of the equatorial aglycone and the axial functionality at C-2 in P-mannopyranosides harbors repulsive steric effects [40]. Glycosyl donors with a participating group at 0 - 2 and halide-ion catalytic conditions promote the exclusive formation of (Y man-
320
13 Stereoselective Synthesis ojP-Mannosides
a-D-Man
1: fa
p-D-Man-(1 -+4)-p-D-GlcNAc-( 14 4 )--pD-GlcNAc-(1+Asn
1
a-D-Man
Scheme 1.
nosides. Once formed, the P-mannosidic linkage might be prone to anomerization under acidic conditions adding another factor to be considered in synthetic sequences involving this linkage [45, 461. Whereas anomeric pairs of galacto- and H-2 coupling constants, glucopyranosides can easily be differentiated by their 3JH-1. this is not so for mannopyranosides. Because of the gauche relationship between the H-1 and H-2 protons in either anomer, the 3JH-1,H-2 couplings (- 1-3 Hz) are not suitable for diagnostic purposes [47]. Reliable information can be gained from in, c-1 heteronuclear coupling traresidual NOE data [48] and from the one-bond, constants [49]. The chemical shift of the H-5 proton (- 3.3-3.4 ppm) is also of diagnostic value [50, 511. The specific rotation can also be used for configurational assignment, if both anomers are available [51]. This chapter reviews methods for the construction of P-mannosidic linkages. Together with the more popular protocols, less widespread and emerging approaches are also covered in the hope they will stimulate further progress.
'&-,
13.2 Chemical Methods 13.2.1 Glycosylation with Mannosyl Donors Mannosylation using Insoluble Promoters Silver cations on the surface of insoluble catalysts anchor mannosyl bromides from the a-side [52]. As a consequence, highly reactive alcohols can attack the anomeric center from the P-side in an S N type ~ reaction, thus leading to P-mannosidic linkages. The first application of this principle was reported by Perlin [53] in 1961 who condensed the cyclic carbonate-protected mannosyl bromide [541 2 and the reactive aglycone 3 under silver oxide activation to afford the first synthetic disaccharide (4) that contains a P-mannopyranosidic linkage (Scheme 2). Because of the strain imposed on the pyranose ring by the fused carbonate moiety in 2, the formation of a planar oxocarbonium ion from it is disfavored and an S N ~ type anomeric displacement predominates [ 551 when strongly nucleophilic primary [56]and secondary alcohols [57-591 are used as the aglycones. Condensations with moderate nucleophiles proceed in lower yields and with reduced P-selectivity. Interestingly, AgzO-assisted reaction of the carbonate 2 with weak nucleophiles gives, exclusively, a-mannosides [ 60, 6 11.
321
13.2 Chemical Methods FHzOAc
&
AcO
Br
CHzOH
HOH& 1 . Ag20.1,
2.Ba(OWzb -t
AcO OAc
2
3
Scheme 2.
+
H
O
OBn
c1 5
~
Ag2C03 O CH2CI2 Yield: 64%
~
E@&&o, Bn
6
7
Scheme 3.
The use of silver carbonate as the promoter has been successful for p-selective condensation between the 0-benzylated donor 5 and the reactive, non-carbohydrate primary alcohol 6 to give 7 (Scheme 3) [62] but it is less practical in other combinations [62-661. Because activation by Koenigs-Knorr promoters produces an equimolar amount of water, loss of the donor as a result of hydrolysis is difficult to prevent even in the presence of drying agents [52]. High yields have been achieved with Paulsen's silver silicate activator precipitated on aluminum oxide that can also act as a water scavenger 1641. A threshold level of acceptor reactivity is necessary for satisfactory p-stereoselectivity-whereas coupling of mannosyl bromide 8 with the anhydro derivative 9 proceeds in an excellent yield and high p-selectivity [67] to afford 10, mannosylation of the less nucleophilic disaccharide 11 with the same donor affords the trisaccharide 12 as a 1 : 1 a/P anomeric mixture (Scheme 4) [68].
-
10 R r -.
8
+ $ G e ONPhth B - & $CH20Bn J ) B n
-
AAll
c
~
&
o
~
o
B
n
All CH20Bn
NPhth
11
Scheme 4.
Yield: 76%
0 CHzOBn
12. cr/D - I
Bn NPhth
13 Stereoselective Synthesis of p-Mannosides
322
1
Br 13
-55 to -30°C Yield: 87%
14
Scheme 5.
+ Br 15
0~~ pphth 16
Ag silicate CH,CI, -15 to25 OC Yield: 22%
Bn
17
OMP NPhth
Scheme 6.
A more predictable approach to this problem has become possible by van Boeckel’s recognition that an electron-withdrawing, acyl substituent at 0-4 of mannosyl donors promotes P-selectivity whereas such substituents at 0 - 3 and 0-6 reduce it [52]. Highly reactive donor-acceptor combinations seem exempt from this rule [69, 701-silver silicate-promoted condensation of the mannosyl donor 13 with n-octanol affords the P-linked mannoside 14 in excellent yield even without an acyl substituent at 0-4 (Scheme 5) [69]. Paulsen’s activator has been used with success to synthesize a large number of P-mannosides related to N-glycoproteins [61, 62, 64, 67-79], glycolipids [ 80-831, and other natural products 1451. Among other insoluble promoters proposed for P-mannoside synthesis van Boeckel’sporous silver silicate [84] has emerged as a most efficient activator [85, 861. This catalyst even promotes 0-selective mannosylation by donor 15 at 0-4 of the disaccharide 16 to afford the 4,6-branched trisaccharide 17 that corresponds to an Asn-linked core structure (Scheme 6) 1861. Garegg’s silver zeolite [87] is also a powerful promoter [82, 88-90] and has been reported to afford increased yields and P-selectivity over silver silicate 182, 911. Other activators, including siluer imiduzolute [92], silver sulicylate [93], and thallium zeolite [88] have also been investigated. The Sulfonate Approach
The leaving group properties of sulfonate groups were first exploited in p-mannoside synthesis by Perlin [94, 951. In the first step of this approach mannofuranose 18 is treated with Tf20 to afford the anomeric triflate 19. This is converted in situ to the bromide 20 by Bu4NBr. Subsequent addition of MeOH led to a 2: 3 mixture of the methyl a- and P-mannosides 21 without heavy metal promoters (Scheme 7). Schuerch has synthesized O-benzyl-protected mannosyl brosylate, tosylate, tresylate, and triflate derivatives 23 from the corresponding chloride 22 and the respective silver sulfonate [96]. On exposure to MeOH, the sulfonates 23 give a nearly 1 : 1 mixture of methyl a- and P-mannosides 24 (Scheme 8).
13.2 Chemical Methods r
323
1
Scheme 7.
Scheme 8.
Scheme 9.
Improved P-selectivity has been obtained by using a mesyl [97-991 or a benzylsulfonyl group [I001 at 0 - 2 of the donor. Such groups are non-participating and by their electron-withdrawing capacity they disfavor anomeric oxocarbonium ion formation, thus favoring direct substitution. For example, reaction of the mesyl derivative 25 with cyclohexanol affords the p-mannoside 26 in 75% yield with excellent p-selectivity (Scheme 9) [99]. Condensation of the benzylsulfonyl-protected donor 27 with the moderately nucleophilic alcohol 28 has led to mannobiose 29 in 49% yield, in a 1 :2 alp ratio (Scheme 10) [loo]. In these studies Schuerch has shown that to achieve optimum pselectivity complete formation of the mannosyl sulfonate is essential before addition of the aglycone component [ 1001. A major development in the sulfonate approach was Crich's serendipitous observation that Kahne-activation of the mannosyl sulfoxide 30 with Tf20 in the presence of DTBMP, at -78 "C in CHzC12, followed by exposure of the intermedi-
z q n +f : Bn CF,CH2S020 27
Scheme 10.
!
M&N
HO
OSO2Bn
< )' ::
Yield:49%
Bn
OMe
28
R = p-BrC&
2 9 . a / p = 1:2
OMe
324
13 Stereoselective Synthesis ojp-Mannosides
phTq
Tf20, DTBML CH?CI?
phTw +
Bn
I
CF3S020
30
u
thenodC
AcOAMe
A20
31
I
OMe
rll
32
Yield: 95%
33, p/a > 25
Scheme 11.
ate triflate 31 to the alcohol 32 led to the disaccharide 33 in an excellent yield and very high P-selectivity (Scheme 11) [51].The a anomeric stereochemistry of the triflate 31 has been demonstrated by low-temperature NMR experiments [101, 1021. Hindered secondary and tertiary alcohols are also suitable acceptors [51, 1031071. Mannosyl triflates can also be prepared from thiomannosides and mannosyl bromides by reaction with PhSOTf, or silver triflate, respectively. As in Schuerch’s method [ 1001, the order of the addition of the reactants is critical for the success. The intermediate triflates should be prepared first before addition of the alcohol to prevent attack by the latter at the initially formed oxocarbonium ion. Additional requirements are the presence in the mannosyl donors of non-participating (benzyl) groups at 0 - 2 and 3 and a benzylidene-acetal moiety at 0 - 4 and 0-6. Crich’s method is likely to enjoy widespread popularity because of its simple experimental protocol combined with high yields and excellent P-stereoselectivity.
Intramolecular Mannosylation The previous examples demonstrate that intermolecular mannosylations proceed with high P-selectivity only with donors that are structurally disposed to direct sN2type substitution at the anomeric carbon and disfavor the formation of oxocarbonium ion intermediates. Because of the large number of variables, the steric outcome of intermolecular mannosylations is predictable only in closely related systems. Stork has proposed a conceptually new, intramolecular mannosylation protocol that has initially been implemented in the first stereocontrolled synthesis of a p-Cmannoside [ 1081. The starting compound is the phenylseleno mannoside 34 in which a 2-phenylethynyl group is anchored to 0 - 2 through a silicon tether. Radicalinitiated removal of the anomeric phenylseleno group (+35) in concert with cyclization of the phenylethynyl group on to the anomeric radical provides 36 from which fluoride-mediated detachment of the silicon connector affords 0-C-mannoside 37 (Scheme 12).
34
Scheme 12.
35
36
37 EK= 10.1
325
13.2 Chemical Methods
Me\
I.BuLi. THF, , S i ,C I 2. Me2SiC12
ye Bn SPh Yield: quant.
Yield: quant.
38
39
OMe
SPh 41
40
MCPBA -78 10 20 "c
-25 to 0 'C
OMe
S(0)Ph Yield: 73%
42
43
Scheme 13.
The principle of intramolecular C-mannosylation has been extended to the synthesis of 0-linked P-mannosides [ 1091. In the first step, a chlorodimethylsilyl moiety is introduced to the primary hydroxyl group of the 0-benzylated aglycone 38 to afford compound 39. Next, chlorosilane 39 is connected to the thiomannoside 40 to give the silaketal intermediate 41. Subsequent conversion to the sulfoxide 42 followed by Kahne-activation [ 1 101 affords the P-linked disaccharide 43 stereospecifically (Scheme 13). This version of the temporary connection method is not suitable for mannosylation of secondary alcohols because of the failure to fashion a silaketal tether between HO-2 of a thiomannoside and a weak nucleophile. More recently, Stork has demonstrated that the silaketal 46 involving a secondary alcohol can be prepared in a one-pot process by using equimolar amounts of the mannosyl sulfoxide 44 instead of the parent thiomannoside, the sugar 45 to be tethered and dichlorodimethylsilane [ l l l ] . Treatment of 46 with Tf20 affords the P(1-3) linked disaccharide 47 in a high yield (Scheme 14). A problem of the Stork method is signaled by a serendipitous finding-that Kahne-activation of the tethered sulfoxide 48 gives mostly the 1,6- (49) instead of the targeted 1,4-linked disaccharide [ 11 11 (Scheme 15). This observation indicates that the silicon-tethered oxygen is not always the most nucleophilic for reaction with the activated anomeric carbon. Hindsgaul has proposed the isopropylidene acetal as an alternative, reactive tether between 0 - 2 of the mannosyl donor and the linkage oxygen of the aglycone B n M Tf20, CH2CIz
OMe -100 10 20°C* %:& BnO nB
Bn
BnO
OMe
S(0)Ph
Yield 88%
44
Scheme 14.
45
B nO OMc
Yield 82% 46 S(O)Ph
41
326
13 Stereoselective Synthesis of p-Mannosides
Scheme 15.
[ 112, 1 131. In the first phase the vinyl ether 51 is prepared from the acetate 50 by methylene-transfer from the Tebbe-reagent. Next, the thioglycoside 51 and the aglycone 52 are connected by acid-catalyzed addition of the latter to the vinyl ether moiety to afford the acetal-tethered intermediate 53. In the final phase, NIS/TfOHinduced anomeric activation proceeds in concert with the attack of the tethered oxygen at the anomeric carbon to afford the P-linked disaccharide 54 (Scheme 16) [114]. Whereas both primary and secondary carbohydrate alcohols are suitable for the linking, the yield is very sensitive to structure and to experimental conditions. A further limitation is the acid-sensitivity of the acetal-tether; this can lead to hydrolysis and transacetalization [ 1141. Ogawa has introduced an interresidual 4-methoxybenzylidene-acetal moiety as the stereocontroling element for intramolecular P-mannosylation [ 1 151. This tether can be fashioned by oxidation of the 4-methoxybenzyl group at 0 - 2 of the mannosy1 fluoride 55 with DDQ to give an intermediate (56) that is stabilized by the aglycone alcohol 57 to form the mixed acetal 58 as a single stereoisomer (Scheme 17) [ 1161. Interestingly, oxidative coupling of the fluoride 59 and the methoxybenzyl ether 60 furnished 58 as a 3 :2 mixture of diastereoisomers [ 1161. The configuration of the acetal carbon atom is dictated by kinetic control. Anomeric activation of the acetal58 afforded the P-mannosyl disaccharide 61 in a stereospecific process that is rather insensitive to the configuration of the acetal (Scheme 17) [116]. The 4-methoxybenzyl-assisted P-mannosylation is also suitable for the blockwise synthesis of higher-membered oligosaccharides [ 117, 1181. For example, DDQ-
Cp2TiCHzClAlMez
SEI
Yield: 90% ,OBn
54
Scheme 16.
Bn'O I OMe
NIS. TfOH
p-TsOH
THF. C6H5CH,
+
DTBMP
OMe
S El 51
~
Bn
52
Yield: 57%
SE1
53
Yield: 77'0
13.2 Chemical Methods
327
1 DBMP
55
MeCN
%aYn B"
Bn
OUII
OBn
Yield: 67% 61
b
59
Bn
OBII
60
Scheme 17.
Scheme 18.
induced attachment of the branched trimannoside thioglycoside 62 to the chitobiose acceptor 63 followed by MeOTf-mediated anomeric activation afforded the pentasaccharide 64 in 42% overall yield (Scheme 18) [ 1181. This compound corresponds to the core oligosaccharide of Asn-linked glycoproteins and represents the most complex setting for which stereospecific pmannosylation has been achieved. The methoxybenzylidene-acetal procedure has also been implemented on a polymer support [ 1 191. In a related approach, studied by Ziegler, the precursor is a 'prearranged glycoside' (e.g. 65) constructed by connecting the mannosyl donor and the acceptor alcohol by means of a persisting succinyl bridge [120, 1211. Activation of 65 by NIS/ TMSOTf gives a 2: 1 mixture of the a- and the P-linked mannosyl disaccharide 66 (Scheme 19) [120]. Anomeric selectivity in this approach is sensitive to the activation procedure and also depends on the glycosyl acceptor component [122]. In contrast with the intramolecular mannosylation methods discussed above, in Ziegler's method the hydroxyl group to be mannosylated is unprotected and the tether remains intact in the glycosylation step.
Other Mannosyl Donor-Based Methods Starting from the epoxide 67 Kochetkov prepared the mannosyl thiocyanate 68 [123] which reacts with the trityl ether 69 in the presence of trityl perchlorate to afford the disaccharide 70 with an a/p ratio of 2 : 1 (Scheme 20) [ 1241.
328
13 Stereoselective Synthesis of P-Mannosides
66, a/p = 2:l
65
Bzo'OBn
Scheme 19.
1. NH4SCN, 18-crown-6 C6HS.acetone, -20 'C 2. CCI3COBr,CSH5N -20 OC Yield: 40%
Bn
CHf.212 SCN
Bn
OMe Yield: 58%
67
68
70, a/p = 21
69
Scheme 20,
Inazu's procedure [125] uses the mannosyl phosphinothioate 72 as the mannosyl donor; it can be prepared from the hemiacetal71 with Me2P(S)C1under basic conditions. Condensation of 72 with the alcohol 73 in the presence of iodine and trityl perchlorate affords the mannosyl disaccharide 74 in 45% yield, with an a/P ratio of 1 : 3 (Scheme 21). Tatsuta has examined the glycosyl donor properties of thiomannosides under NIS/TfOH activation and found that the best P-selectivity can be obtained with the bulky donor 75 that in the presence of the alcohol 76 has afforded the mannoside 77 as a 3 : 1 mixture of the P and a anomers (Scheme 22) [22]. Hashimoto found that low-temperature activation of mannosyl chlorides with silver triflate in the presence of alcohols leads to varying ratios of a- and P-linked mannosides [45, 126, 1271. A study of the factors controlling stereoselectivity in silver triflate-mediated mannosylation has been published [ 1281. Activation of 0benzyl-protected mannosyl fluorides by a combination of Sn(OTf)2 and La(C104)3 has been reported by Shibasaki to afford mixtures of a- and P-mannosides [129]. The large proportions of a-mannosides formed in the homogeneous reactions outlined in this section indicate the involvement of oxocarbonium-ions enabling the anomeric effect to dictate the steric outcome. Because the stereoselectivity of these approaches is crucially dependent on the reacting partners and conditions their scope remains limited.
I . BuLi
OH THF. -30 "C 71
Scheme 21.
Yield: 75%
BnO Bn
-z w
2. Me2P(S)CI
Bn
OP(S)Me2+
72
73
13.2 Chemical Methods OBn
+
gBn
H
o OBn
BnO
329
OTBDPS
NIS,TfOH ~
____C
Bn
OBn QBn
CHzCl2,40 O C Yield: 88%
BnO 15
16
77,a/p = I :3
Scheme 22.
13.2.2 Epimerization of P-Glucopyranosides at C-2 The Oxidation-Reduction Approach Extending Theander's finding [ 1301 that reduction of hexopyranosiduloses proceeds with high stereoselectivity, Lindberg has developed a powerful approach to Pmannosidic linkages [ 13I]. In the first phase, the glucopyranosyl donor 78 that carries a selectively removable, participating group at 0 - 2 is condensed with the aglycone to give the P-glucoside 79. Subsequently, the HO-2 group is deprotected (i 80). Glucal 1,2-epoxides can also be precursors to such intermediates [ 1321. In the next step C-2 is oxidized to give uloside 81. In the final phase, the uloside is reduced to afford the p-manno product 82 together with usually
Direct Inversion Intermolecular nucleophiles The first one-step conversion of P-glucosides to P-mannosides has been reported by Miljkovic [ 1501. In his approach a mesyloxy group at C-2 of glucoside 83 is displaced by a benzoyl group to afford the mannoside 84 (Scheme 24). In a related reaction 2-deoxy-2-fluoro-~-mannoside86 has been prepared [ 15I] by fluoride displacement of the 2-triflyloxy group in 85 (Scheme 25).
78
Scheme 23.
13 Stereoselective Synthesis of p-Munnosides
330 % o T & h +p
BzOK. DMF
Me
OMe
7
MsO
OMe
Yield: 62%
83
84
Scheme 24.
phTow BudNF
BZ
OMe
TfO
p BzOh TOMes
EiGzT Yield: 3540%
86
85
Scheme 25.
These substitutions proceed in moderate yields because of repulsive electrostatic interaction between the incoming nucleophile and the lone pair of the ring oxygen atom [I521 and non-bonded steric interactions in the transition state [150]. Extension of the repertoire of leaving groups has alleviated these problems and made the direct displacement a powerful approach to P-mannoside synthesis. David's variation uses an imidazolylsulfonyloxy group as the leaving group at C2 and Bu4NOBz as the nucleophile [153]. In a similar strategy, a C-2 triflyloxy group is displaced by a combination of CH3C02Cs and 18-crown-6 under sonication to give P-mannosides [ 1541. An analogous method reported by Furstner employs BmNOAc as the nucleophilic reagent in conjunction with ultrasound [ 155, 1561. David has also reported the conversion of double-triflylated P-galactosides to P-mannosides [157, 1581. According to this procedure, Tf0-4 of P-galactoside 87 is displaced by BuNOBz at room temperature to give the gluco compound 88. In the second phase heating at 100 "C causes displacement of Tf0-2 to afford mannoside 89 (Scheme 26). A variation of this approach uses CH3COzCs and CF3C02Cs in combination with 18-crown-6 [ 159, 1601. An improved protocol for the P-galacto--,p-manno conversion and identification of by-products has been reported by Sinay [161]. Intramolecular nucleophiles
Kunz has taken advantage of an entropically favored, intramolecular substitution reaction [ 1621 to construct P-mannosides by configurational inversion at C-2 of P-
Bz
OMe
All
TfO 87
Scheme 26.
25
OC
TfO
sn
Bu~NOBZ * OMe C6H3Me I00 O C Yield: 64%
89
13.2 Chemical Methods
'yo Ac%
ROH AgOTf AcO
C6%
OR 1 , K2C03. MeOH
7
Z.C&CH(OMe): 3 Tf20 Yldd ca 75%
Ac&NHPh -3OOC Yield: 65-808
NHPh
331
90
91
PhTO OO& o l 75 "C Yield: 65-808 93
NHPh 92
OK
PhT& i&, 94
Scheme 27.
glucosides [152, 1631. The first phase is assembly of a P-glucopyranosyl donor (90) with a urethane-type group at C-3. This donor is then condensed with the aglyconic component to form the P-glucopyranoside 91. Subsequently, a benzylidene-acetal moiety is installed at 0 - 4 and 0 - 6 and HO-2 is triflylated (+ 92). In the final stages, thermally induced intramolecular attack of the carbamoyl oxygen at C-2 affords the P-mannoside 93 with small amounts of the iminocarbonate 94 (Scheme 27). The acetal moiety is essential for the success of this approach. Using this procedure, Unverzagt has synthesized the core-region trisaccharide P-D-Manp-1,4-P-DGlcpNAc-l,4-P-~-GlcpNAcin multigram quantities [ 1641. C-2 epimerization by intramolecular substitution has also been achieved with the 3-0-benzoyl analog of the 0-glucoside 92 [165]. 13.2.3 The 2-Ulosyl Donor Method Introduced by Lichtenthaler [42, 1661, this method utilizes the limited tendency of 2-glycosulosyl a-bromides to form oxocarbonium ions upon anomeric activation, thus favoring an S N type ~ reaction at C-I. In the first phase the arabino-hexulosyl bromide 96 is prepared by exposure of the readily available acyloxyglycal 95 to NBS/EtOH [48, 166-1711. Next, the bromide 96 is activated by an insoluble silver promoter in the presence of the aglycone. Because the 2-0x0 function not only supports the sN2 mechanism by its electron-withdrawing capacity but also is a nonparticipating group, the product is almost exclusively the P-glycoside 97. The use of soluble promoters with bromide 96 or with the corresponding pentenyl and thioglycosides yields substantial amounts of a-linked products [42, 1671. In the final step, the 0x0 group is reduced as in Lindberg's method to afford the p-mannoside 98 (Scheme 28) [167]. The low anomeric reactivity of 2-ulosyl bromides can be a limitation when moderately nucleophilic alcohols are used as aglycones. Attempts
Yield: 92% 95
Scheme 28.
Yield: 93%
Yicld:Yi% 96
97
98
332
13 Stereoselective Synthesis of P-Mannosides
Scheme 29.
to overcome t h s problem include the use of ulosyl iodides and 0-benzyl protected ulosyl donors [48, 169, 1721 in combination with van Boeckel's silver silicate promoter [84]. 13.2.4 Anomeric 0-alkylation
Alkylation of 1-0-Metal Complexes Schmidt has developed a technically simple route to p-manno furanosides and pyranosides in which the sodium or potassium salt of mannose hemiacetals is reacted with strong alkylating agents derived from the aglyconic component [ 173-1781. For example, treatment of the sodium complex of mannofuranose 99 with the triflate 100 gives the corresponding mannofuranoside 101 as a 1 :2 ratio of the ct and the P anomers (Scheme 29) [ 1751. It is believed that intramolecular complexation between the alkali metal and the sugar oxygens locks the anomeric oxygen in the P position before the mannosyloxy anion attacks the aglycone. The Stannylene Acetal Method Five-membered cyclic dibutylstannylene acetals formed on vicinal cis-axialequatorial pairs of hydroxyl groups selectively enhance the nucleophilicity of the equatorial oxygen in 0-alkylation reactions [179, 1801. On the basis of this rule Schuerch surmised that alkylation of the tin complex 103, having the anomeric oxygen locked in the equatorial position, should lead to P-mannosides [181]. This assumption was proved by treating the mannose diol 102 with BuzSnO (+ 103) followed by in situ exposure to alkyl halides whereby P-mannopyranosides 104 were formed stereospecifically (Scheme 30) [ 1811.
B Bn :
102
Scheme 30.
103
Yield: 94-100%
~
o
= M e Or A" 104
R
13.2 Chemicul Methods
%fNH
333
Yield: 82%
Bn
Scheme 31.
107
108
OMe Yield: 67%
109
Scheme 32.
This strategy [99, 1821 has been used by Nicolaou for stereospecific synthesis of trehalose analogs [50]. The procedure involves reaction of the complex 103 with the a-mannosyl donor 105 under TMSOTf activation to furnish the 0,a-linked mannosy1 disaccharide 106 without the a,a-connected stereoisomer (Scheme 3 1). In another application of Schuerch's concept, 0-triflyl derivatives of monosaccharides are used as electrophiles [183]. For example, reaction of the 4-0-triflyl guluctoside 108 with a sixfold molar excess of the partially protected dibutylstannylene complex 107 in DMF affords the p-1,4-linked mannosyl-glucoside 109 in 67% yield. The scope of this protocol is limited by configurational inversion at the linkage carbon and solubility problems (Scheme 32) [ 1831. 13.2.5 Miscellaneous Methods Radical Inversion of the Anomeric Chirality of a-D-Mannopyranosides This approach was first reported by Kahne in 1988. He found that hydrogen atom transfer to hexopyranosyl radicals having an alkoxy group at C-1 occurs, preferentially, from the a-side giving rise to P-glycosides with high stereoselectivity [ 1841. For example, the anomeric radical 111 generated by photolysis of the mannose hemithio orthoester 110 is converted under reducing conditions to the mannoside 112 with an impressive 18 : 1 ratio of the and a anomers (Scheme 33).
zqsMe
Bu3SnH, AIBN C6H5Me
Bn
bMe 110
Scheme 33.
30 "C Yield: 85% 111
112,cdp=l:18
334
13 Stereoselective Synthesis of p-Mannosides
Scheme 34.
In Curran’s method 11851 the first step is homolytic cleavage of the Br-C bond in the radical translocating group in 113 to produce the aryl radical 114. Subsequent intramolecular 1,6-hydrogen transfer from the anomeric carbon translocates the radical to C-1 (-115). In the final phase the radical is quenched from the a side by intermolecular hydrogen transfer from Bu3 SnH to afford the P-mannoside 116 (Scheme 34). A major problem with this approach is epimerization at C-2 through 1,5-hydrogen abstraction producing the a-yluco isomer 117. Another competing reaction is the intermolecular replacement of bromine by hydrogen in the radical translocating group. Additional approaches capitalizing on stereospecific hydrogen translocation to the anomeric radical have been described by Crich 1186, 1871. Despite excellent stereoselectivity, the radical approach is “rendered impractical by the circuitous methods needed for generation of the key anomeric radical” [51]. Reductive Cleavage of Cyclic Orthoesters Inspired by Liptak’s regioselective transformations of hexopyranose 4,6-benzylidene acetals by hydrogenolysis [ 1881, Ikegami has developed a new entry to glycosidesynthesis 11891. In the first phase the mannopyranosylidene acetal 120 is prepared from lactone 118 and the diol 119. In the subsequent step the interresidual spiro orthoester is reduced with LiAlH4/AlCl3 to produce the P-linked disaccharide 121 regio- and stereospecifically (Scheme 35). This method is limited to the preparation of P(1-4) linkages.
De novo Syntheses Tietze has synthesized ethyl P-D-mannopyranoside from non-carbohydrate precursors [ 1901. The procedure involves [4 + 21 hetero Diels-Alder cycloaddition of the enol ether 122 and the chiral oxazolidinone 123 under promotion by MeZAlC1 to give the dihydropyran 124 in very high diastereoselectivity. Reductive cleavage of
118
Scheme 35.
119
Yield: 77%
120
Yield 98%
121
13.2 Chemical Methods
''3
V
+
Ac
op N
A
o
1. Ac,O, NaOAc 2. BH3 . Me2S
/
Yield: 95%
U"O ~.
Yield: 85%
Ulld
123
I22
335
Bn6
Yield: 82%
Bn6
126
125
I24
Scheme 36.
the oxazolidinone moiety (+125) followed by hydroboration with oxidative workup has provided P-mannoside 126 (Scheme 36). In a similar approach, Boger has prepared enantiomeric mixtures of P-mannopyranosides using achiral precursors (Scheme 36) [191].
13.2.6 2-Acetamido-2-deoxy-~-~-mannopyranosides Two main directions have emerged to this group of compounds. In the first an azido group is employed as the precursor to the acetamido group [ 192-1941. In Paulsen's method the 2-azido-mannosyl donor 127 is used as the ManNAc synthon that is condensed with the aglycone component under silver silicate activation to afford the P-linked mannoside 128 (Scheme 37). In the alternative route [159, 195, 1961 a P-glucopyranoside is synthesized first, followed by azide-displacement at C-2 to provide the 2-azido-2-deoxy-Pmannopyranoside unit in a process as reviewed for the parent compound in Section 13.2.2. The second direction is based on 2-ulosyl intermediates. In one approach [ 197, 1981 an uloside e.y. 129 is synthesized first, as reviewed in Section 13.2.2, then converted to the oxime 130. Subsequent reduction and N-acetylation affords the Plinked N-acetyl-mannosaminyl disaccharide 131 (Scheme 38).
$,
25'C
Yield: 83%
127
128, cc/p = 2:3
Scheme 37.
E - d k 2 O H .
0 129
Scheme 38.
&;?*::
HCI 0CH20Bn Yield: 80%
Bn
LiAIH,,
2. AczO
Bn
'IHL
:-;&
CH20Bn
HO"
130
BnOoMe
Bn 0 CHzOBn 131
13 Stereoselective Synthesis of p- Mannosides
336
\
B z d h Br
OBz Yield: 74%
132
BzON Yield: 93%
133
134
135
Scheme 39.
In Lichtenthaler's method [33, 42, 199-2011 the oximinoglycosyl bromide 133 is used as the p-mannosamine precursor that can be prepared from the benzoylated hydroxyglycal 132. Condensation of 133 with MeOH using A g ~ C 0 3as the promoter has provided the p-linked glycoside 134 from which N-acetyl mannosaminide 135 can be obtained by BH3 reduction and N-acetylation [199] (Scheme 39). This approach is also suitable for glycosylating less reactive, secondary hydroxyl groups and for blockwise oligosaccharide synthesis [42]. 13.2.7 Aryl P-D-mannopyranosides P-Mannopyranosides with a chromo- or fluorogenic aromatic aglycone, e.g. a 4nitrophenyl, naphthyl, or 4-methylumbelliferyl group, are valuable enzyme substrates. The 4-nitrophenyl derivative is also employed as a mannosyl donor in glycosidase-catalyzed P-mannosylation. These compounds can be prepared in 4070% yields by reaction of the mannosyl bromide 2 [54] with sodium [202] or potassium phenolates [203, 2041. Garegg has synthesized aryl P-mannosides in 50-650/0 yields by condensation of dicyclohexylidene-mannopyranose with phenols in the presence of diethyl azodicarboxylate and triphenylphosphine [44, 2051. Epimerization of C-2 of aryl P-D-glucopyranosides by the Lindberg protocol (Section 13.2.2) has also been described [206]. 13.2.8 1-Thio-P-D-mannopyranosides 4-Nitrophenyl 1-thio-P-D-mannopyranoside has been synthesized by reaction of acetochloromannose [207] or the mannosyl bromide 2 [202] with the sodium salt of 4-nitrothiophenol. A similar reaction between sodium thiophenoxide and acetobromomannose in HMPT gives phenyl 1-thio-P-D-mannopyranoside quantitatively [208]. In a related reaction the sodium salt of 1-thio-(3-D-mannopyranosehas been alkylated with benzyl bromide as in Schmidt's approach (Section 13.2.4) to give benzyl P-thiomannoside in 86%) yield [209]. P-Linked 1-thiomannopyranosides can also be prepared by BF3-catalyzed anomerization of the readily available a-anomers [46]. The Lindberg protocol has also been used for the synthesis l-thioP-D-mannopyranosides [ 146, 1491. Aryl 1-thio-p-mannofuranosides have been syn-
13.3 Enzymatic Synthesis
136
137
331
138
Scheme 40.
thesized by reaction of di-isopropylidene-mannofuranose 99 and diary1 disulfides in the presence of triethylphosphine [210]. 13.2.9 P-D-Mannopyranosylamines The only N-glycoside that contains a P-linked mannopyranosyl residue was synthesized by palladium-catalyzed condensation of the amine 136 with the chloropurine 137 to afford the nucleoside antibiotic analog 138 together with the a-linked isomer in a 6: 1 ratio [211]. It is of interest to note the exclusive formation of the P amine 136 upon catalytic reduction of the corresponding azide, with no trace of the c1 anomer (Scheme 40).
13.3 Enzymatic Synthesis Although chemical methods have provided access to many oligosaccharides containing the p-mannopyranoside linkage, their overall efficiency is quite low because of numerous protecting group manipulations and glycosylation steps. As an alternative enzymatic methods have been receiving increasing attention. Both exomannosidases (glycosyl hydrolases) [47, 196, 2 12-2 161 and mannosyl transferases [21, 217, 2181 might be suitable biocatalysts for stereospecific p-mannoside synthesis. Although mannosiduse-catalyzed P-mannosylation (reverse hydrolysis) uses relatively readily available enzymes and mannosyl donors, e.g. p-nitrophenyl p-Dmannopyranoside or even mannose [216],overall yields are low [215].An additional difficulty is that when a saccharide is used as the acceptor alcohol the regioselectivity is not always predictable [47]. In the mannosyl transjerase approach a pmannosyl unit is transferred from GDP-mannose to the acceptor in a regio- and stereospecific manner. The high cost of GDP-mannose, the restrictions that might be imposed by substrate specificity, and instability and limited availability and of mannosyl transferases can be serious difficulties that require considerable investment before this method can be used for preparative-scale synthesis of 0-mannosides. A more detailed account of the enzymatic approach can be found in Chapters 23 to 30.
338
13 Stereoselective Synthesis of P-Mannosides
13.4 Conclusions Numerous developments in the last decade have rendered the construction of the problematic P-mannoside unit a less formidable challenge. Many years after its introduction, Paulsen’s silver silicate procedure is still often used for P-mannoside synthesis. Of the new methods Crich’s version of the sulfonate approach stands out because of its scope, operational simplicity, high overall yields, and P-selectivity. The more laborious inter- and intramolecular inversions of the C-2 chirality of P-glucosides are also powerful methods but require more steps that reduce the overall yield. Of the intramolecular glycosylation approaches Ogawa’s 4-methoxybenzylidene-assisted intramolecular glycosylation is a generally applicable method that is compatible with a variety of manipulations and is uniquely suitable for block-synthetic operations. Schuerch’s stannylene acetal approach has proved particularly useful for the stereoselective synthesis of trehalose analogs. Rapid advances in biotechnology are likely to open up efficient biocatalytic approaches to P-mannosyl oligosaccharides.
References 1. J. F. G. Vliegenthart and J. Montreuil, in Glycoproteins (Eds. J. Montreuil, J. F. G. Vliegen-
thart, H. Schachter), Elsevier, Amsterdam, 1995, Vol. 1, pp. 13-28. 2. D. R. Bundle, Topics. Curr. Chem., 1990, 154, 1-37. 3. C. Erbing, L. Kenne, B. Lindberg, S. Hammarstrom, Curbohydr. Res., 1978, 60, 400-403. 4. B. Lindberg, K. Leontein, U. Lindquist, S. B. Svensson, G. Wrangsell, A. Dell, M. Rogers, Curbohydr. Rex, 1988, 174, 313. 5. M. Betley, J. W. Redmond, N. H. Packer, D. Liu, P. Reeves, Curbohydr. Rex, 1997, 303, 313-318. 6. L. Kenne, B. Lindberg, in The Polysucchurides (Ed. G. 0. Aspinall) Academic Press, New York, 1983, Vol. 2, pp. 287-363. I . L. Galbraith, S. G. Wilkinson, Carbohydr. Rex, 1997, 303, 245-249. 8. U. Zahringer, H. Rettenmaier, H. Moll, S. N . Senchenkova, Y. A. Knirel, Curbohydr. Res., 1997,300, 143- 151. 9. V. Gloagen, L. Vebret, L. Hoffmann, H. Morvan, Y. Plancke, J.-M. Wieruszeski, G. Strecker, XVIII Int. Curbohydr. Symp., 1996, Milan, Italy, AP075. 10. D. Garozzo, G. Impallomeni, E. Spina, L. Sturiale, XVIII Int. Carbohydr. Symp., 1996, Milan, Italy, A0017. 11. P. L. Hackland, H. Parolis, L. A. S. Parolis, Curbohydr. Res., 1988, 172, 209-216. 12. Y.-M. Choy, G. G. S. Dutton, A. M. Zanlungo, Can. J. Chem., 1973,51, 1819-1825. 13. S. Kalelkar, J. Glushka, H. Van Halbeek, L. C. Morris, R. Cherniak, Curbohydr. Res., 1997, 299, 119-128. 14. D. Garazzo, G. Impallomeni, E. Spina, L. Sturiale, Curbohydr. Rex, 1998, 307, 113-124. 15. N. A. Paramanov, L. A. S. Parolis, H. Parolis, I. F. Boan, J. Anton, F. Rodriguez-Valera, Carbohydr. Res., 1998, 309, 89-94. 16. J. F. Kennedy, C. A. White, in Carbohydrate Chemistry (Ed. J. F. Kennedy), 1988, Clarendon Press, Oxford, pp. 220-226. 17. M. Ohnishi, S. Ito, Y. Fujino, Agric. Biol. Chem., 1985, 49, 3609-3611. 18. M. Sugita, Y. Nakano, T. Nose, 0. Itasaka, T. Hori, J. Biochem. (Tokyo), 1984, 95, 47-55. 19. N. Hada, T. Takeda, Y. Ogihara, Carbohydr. Rex, 1994, 258, 93-104. 20. P. A. Trinel, G. Lepage, T. Jouault, G. Strecker, D. Poulain, FEBS Let., 1997, 416, 203-206.
References
339
21. A. Suzuki, N. Shibata, M. Suzuki, F. Saitoh, H. Oyamada, H. Kobayashi, S. Suzuki, Y. Okawa, J. Biol. Chem., 1997,272, 16822-16828. 22. K. Tatsuta, S. Yasuda, Tetrahedron Let., 1996, 37, 2453-2456. 23. N. Sharon, H. Lis, H. in Glycosciences (Eds. H.-J. Gabius, S. Gabius) 1997, Chapman and Hall, London, pp. 133-162. 24. B. Priem, R. Gitti, C. A. Bush, Plant. Phys., 1993, 102, 445-458. 25. N. A. Kocharova, 0. V. Bystrova, S. A. Borisova, A. S. Shashkov, A. S, Y. A. Knirel, XVIII Znt. Curbohydr. Symp., 1996, Milan, Italy, AP008. 26. P. Branefors-Helander, L. Kenne, B. Lindberg, K. Petersson, P. Unger, Carbohydr. Res., 1981, 97,285-291. 27. F.-P. Tsui, R. Schneerson, R. A. Boykins, A. D. Karpas, W. Egan, Carbohydr. Rex, 1981, 97, 293-306. 28. P. Branefors-Helander, L. Kenne, B. Lindberg, K. Petersson, P. Unger, Curbohydr. Res., 1981, 88, 77-84. 29. F.-P. Tsui, R. Schneerson, W. Egan, Carbohydr. Rex, 1981, 88, 85-92. 30. C. Adlam, J. M. K. Knights, A. Mugridge, J. C. Lindon, P. R. W. Baker, J. E. Beesley, B. Spacey, G. R. Craig, L. K. Nagy, J. Gen. Microbiol., 1984, 130, 2415-2426. 31. H. J. Jennings, Curr. Top. Microbiol. Zmmunol., 1990, 150, 97-127. 32. C. Lugowski, E. Romanowska, L. Kenne, B. Lindberg, Curbohydr. Res., 1983,118, 173-181. 33. E. Kaji, Y. Osa, K. Takahashi, M. Hirooka, S. Zen, F. W. Lichtenthaler, Bull. Chem. SOC. Jpn., 1994, 67, 1130-1140. 34. J. K. Dale, J. Am. Chem. Soc., 1924,46, 1046-1051. 35. H. S. Isbell, H. L. Frush, J. Research Natl. Bur. Standards, 1940,24, 125-151. 36. H. G. Bott, W. N. Haworth, E. L. Hirst, J. Chem. SOC.,1930, 2653-2659. 37. B. Helferich, G. Duve, Chem. Ber., 1958, Y I , 1790-1793. 38. W. A. Bonner, J. Am. Chem. SOC.,1958,80, 3372-3379. 39. B. Helferich, S. Winkler, Chem. Ber., 1933, 66, 1556-1558. 40. H. Paulsen, Angew. Chem. Znt. Ed., 1982, 21, 155-173. 41. P. J. Garegg, Chemtracts, Org. Chem., 1992, 5, 389-393. 42. E. Kaji, F. W. Lichtenthaler, Trends. Glycosci. Glycotechnol., 1993, 5, 121-143. 43. F. Barresi, 0. Hindsgaul, in Modern methods in carbohydrate synthesis (Eds. S. H. Khan, R. O’Neill), Harwood Academic Publishers, Amsterdam, 1996, pp. 25 1-276. 44. B. V. McCleary, Methods Enzymol., 1988, 160, 515-518. 45. J. Tamura, S. Horito, H. Hashimoto, J. Yoshimura, Curbohydr. Rex, 1988, 174, 181-199. 46. D. T. Connolly, S. Roseman, Y. C. Lee, Curbohydr. Res., 1980, 87, 227-239. 47. N. Taubken, B. Sauerbrei, J. Thiem, J. Curbohydr. Chem., 1998,12, 651-667. 48. F. W. Lichtenthaler, T. Schneider-Adams, J. Org. Chem., 1994, 59, 6728-6734. 49. R. Kasai, M. Okihara, J. Asakawa, K. Mizutani, 0. Tanaka, Tetrahedron, 1979, 35, 14271432. 50. K. C. Nicolaou, F. L. van Delft, S. R. Conley, H. J. Mitchell, Z. Jin, R. M. Rodriguez, J. Am. Chem. SOC.,1997,119, 9057-9058. 51. D. Crich, S. Sun, Tetrahedron, 1998, 54, 8321-8348. 52. C. A. A. van Boeckel, T. Beetz, S. F. van Aelst, Tetruhedron, 1984, 40,4097-4107. 53. P. A. J. Gorin, A. S. Perlin, Can. J. Chem., 1961,39, 2474-2485. 54. T. J. Tewson, M. Soderlind, J. Curbohydr. Chem., 1985, 4, 529-543. 55. M. A. E. Shaban, I. E. Ary, D. A. Jeanloz, R. W. Jeanloz, Carbohydr. Rex, 1975,45, 105-1 14. 56. R. K. Jain, K. L. Matta, Curbohydr. Rex, 1996, 282, 101-111. 57. G. M. Bebault, G. G. S. Dutton, Curbohydr. Res., 1974, 37, 309-319. 58. V. I. Betaneli, M. V. Ovchinnikov, I. V. Backinowsky, N. K. Kochetkov, Curbohydr. Rex, 1980,84,211-224. 59. N. K. Kochetkov, V. I. Torgev, N. N. Malysheva, S. A. Shashkov, Tetrahedron, 1980, 36, 1099-1105. 60. M. A. E. Shaban, R. W. Jeanloz, Carbohydr. Rex, 1976, 52, 103-114. 61. M. A. E. Shaban, R. W. Jeanloz, Curbohydr. Rex, 1976,52, 115-127. 62. T. Sugawara, K. Irie, H. Iwasawa, T. Yoshikawa, S. Okuno, H. Watanabe, T. Kato, M. Shibukawa, Y. Ito, Curbohydr. Rex, 1992,230, 117-149.
340
13 Stereoselective Synthesis of P-Mannosides
H. Paulsen, 0. Lockhoff, Tetrahedron Let., 1978, 402774030, H. Paulsen, 0. Lockhoff, Chem. Ber., 1981, 114, 3102-3114. P. J. Garegg, T. Iversen, Curbohydr. Rex, 1979, 70, C13-C14. P. J. Garegg, T. Iversen, R. Johansson, Acta Chem. Scund., 1980, B34, 505-508. H. Paulsen, M. Heume, Z. Gyorgydeak, R. Lebuhn, Carbohydr. Rex, 1985, 144, 57-70. T. Ogawa, T. Kitajama, T. Nukada, Carbohydr. Rex, 1983, 123, C5-C7. H. Paulsen, M. Springer, F. Reek, E. Meinjohanns, I. Brockhausen, H. Schachter, Justus Liebigs Ann. Chem., 1995, 53-66. 70. P. Grice, S. V. Ley, J. Pietruszka, H. M. I. Osborn, H. W. M. Priepke, S. L. Warriner, Chem. Eur. J., 1997, 3, 431-440. 71. T. Ogawa, K. Katano, K. Sasajima, M. Matsui, Tetrahedron, 1981,37, 2779-2786. 72. H. Paulsen, R. Lebuhn, Angew. Chem. Int. Ed., 1982,21,926-927. 73. T. Ogawa, T. Kitajima, T. Nukada, Curbohydr. Res, 1983, 123, C8-Cll. 74. H. Paulsen, R. Lebuhn, Justus Liebigs Ann. Chem., 1983, 1047-1072. 75. H. Paulsen, R. Lebuhn, Carbohydr. Res., 1984, 130, 85-101. 76. T. Ogawa, M. Sugimoto, T. Kitajima, K. K. Sadozai, T. Nukada, Tetrahedron Let., 1986,27, 5739-5742. 77. F. Yamazaki, T. Nukada, Y. Ito, S. Sato, T. Ogawa, Tetrahedron Let., 1989, 30, 4417-4420. 78. H. Paulsen, M. Heume, H. Nurnberger, Curbohydr. Res., 1990,200, 127-166. 79. T. Nukada, T. Kitajima, Y. Nakahara, T. Ogawa, Curbohydr. Rex, 1992, 228, 157-170. 80. K. Koike, M. Mori, Y. Ito, Y. Nakahara, T. Ogawa, Agric. Biol. Chem., 1990,54,2931-2939. 81. M. Mori, Y. Ito, T. Ogawa, Carbohydr. Rex, 1990, 195, 199-224. 82. 0. Kanie, T. Takeda, N. Hada, Y. Ogihara, J. Curbohydr. Chem., 1991,10, 561-581. 83. K. Koike, M. Mori, Y. Ito, Y. Nakahara, T. Ogawa, Biosci. Biotech. Biochem., 1993,57,698702. 84. C. A. A. van Boeckel, T. Beetz, A. C. Kock-van Dalen, H. van Bekkum, Rec. Truo. Chim. Pu~s-Bus,1987, 106, 596-598. 85. Z.-W. Guo, Y. Nakahara, T. Ogawa, Bioorg. Med. Chem., 1997,5, 1917-1924. 86. Z.-W. Guo, Y. Itoh, Y. Nakahara, T. Ogawa, Carbohydr. Rex, 1998,306, 539-544. 87. P. J. Garegg, P. Ossowski, Actu Chem. Scand., 1983, B37, 249-251. 88. D. M. Whitfield, R. N. Shah, J. P. Carver, J. J. Krepinsky, Synth. Commun., 1985, 15, 737747. 89. T. Takeda, N. Hada, Y. Ogihara, Chem. Pharm. Bull., 1992,40, 1930-1933. 90. R. N. Shah, D. A. Cumming, A. A. Grey, J. P. Carver, J. Krepinsky, Curbohydr. Rex, 1986, 153, 155-161. 91. P. J. Garegg, C. Henrichson, T. Norberg, P. Ossowski, Carbohydr. Rex, 1983, 119, 95-100. 92. P. J. Garegg, R. Johansson, B. Samuelsson, Actu Chem. Scand., 1981, B35, 635-636. 93. G. Wulff, J. Wichelhaus, Chem. Ber., 1979, 112, 2847-2853. 94. J. Leroux, A. S. Perlin, Curbohydr. Rex, 1976, 47, C8-CIO. 95. J. Leroux, A. S. Perlin, Carbohydr. Rex, 1978, 67, 163-178. 96. E. S. Rachaman, R. Eby, C. Schuerch, Carbohydr. Rex, 1978,67, 147-161. 97. V. K. Srivastava, C. Schuerch, Carbohydr. Res., 1980, 79, C13-CI6. 98. V. K. Srivastava, C. Schuerch, J. Org. Chem., 1981, 46, 1121-1126. 99. E. S. El Ashry, C. Schuerch, Carbohydr. Rex, 1982, 105, 33-43. 100. L. F. Awad, E. S. H. El Ashry, C. Schuerch, Bull. Chem. SOC.Jpn., 1986,59, 158771592, 101. D. Crich, S. Sun, J. Am. Chem. Soc., 1997,119, 11217-11223. 102. D. Crich, W. L. Cai, J. Org. Chem., 1999,64,4926-4930. 103. D. Crich, S. Sun, J. Am. Chem. Soc., 1998, 120,435-436. 104. D. Crich, S. Sun, J. Org. Chem., 1997, 62, 1198-1199. 105. D. Crich, S. Sun, J. Org. Chem., 1996, 61, 4506-4507. 106. D. Crich, Z. Dai, Tetrahedron Let., 1998, 39, 1681-1684. 107. D. Crich, Z. M. Dai, Tetrahedron, 1999, 55, 1569-1580. 108. G. Stork, H. S. Suh, G. Kim, J. Am. Chem. Soc., 1991,113,7054-7056. 109. G. Stork, G. Kim, J. Am. Chem. Soc., 1992, 114, 1087-1088. 110. L. Yan, D. Kahne, J. Am. Chem. Soc., 1996, 118, 9239-9248. 111. G. Stork, J. J. La Clair, J. Am. Chem. Soc., 1996, 118, 247-248. 63. 64. 65. 66. 67. 68. 69.
References
341
F. Barresi, 0. Hindsgaul, J. Am. Chem. Soc., 1991, 113, 9376-9377. F. Barresi, 0. Hindsgaul, Synlett, 1992, 759-761. F. Barresi, 0. Hindsgaul, Can. J. Chem., 1994, 72, 1447-1465. Y. Ito, T. Ogawa, Angew. Chem. Int. Ed., 1994, 33, 1765-1767. M. Lergenmuller, T. Nukada, K. Kuramochi, A. Dan, T. Ogawa, Y. Ito, Eur. J. Org. Chem., 1999, 136771376, 117. A. Dan, Y . Ito, T. Ogawa, J. Org. Chem., 1995, 60, 4680-4681. 118. A. Dan, Y. Ito, T. Ogawa, Tetrahedron Let., 1995, 36, 7487-7490. 119. Y. Ito, T. Ogawa, J. Am. Chem. Soc., 1997, 119, 5562-5566. 120. T. Ziegler, G. Lemanski, A. Rakoczy, Tetrahedron Let., 1995,36, 8973-8976. 121. T. Ziegler, G. Lemanski, Eur. J. Org. Chem., 1998, 163-170. 122. T. Ziegler, G. Lemanski, Angew. Chem. Int. Ed., 1998, 37, 3129-3132. 123. N. K. Kochetkov, N. N. Malysheva, A. V. Demchenko, N. G. Kolotyrkina, E. M. Klimov, Bioorg. Chem. (Russia), 1991, 17, 1655-1659. 124. E. M. Klimov, A. V. Demchenko, N. N. Malysheva, N. K. Kochetkov, Bioorg. Chem. (Russia), 1991, 17, 1660-1664. 125. T. Yamanoi, K. Nakamura, H. Takeyama, K. Yanagihara, T. Inazu, Bull. Chem. Soc. Jpn., 1994,67, 1359-1366. 126. J. Yoshimura, S. Horito, J. Tamura, H. Hashimoto, Chem. Let., 1985, 1335-1338. 127. J. Tamura, S. Horito, J. Yoshimura, H. Hashimoto, Carbohydr. Res., 1990, 207, 153-165. 128. J. Tamura, R. Terayama, T. Shimadate, J. Carbohydr. Chem., 1993, 12,893-902. 129. W.-S. Kim, H. Sasai, M. Shibasaki, Tetrahedron Let., 1996, 37, 7797-7800. 130. 0. Theander, Acta Chem. Scand., 1958, 12, 1883-1885. 131. G . Ekborg, B. Lindberg, J. Lonngren, Acta Chem. Scand., 1972,26, 328773292, 132. K. K. C. Liu, S. J. Danishefsky, J. Org. Chem., 1994, 59, 1892-1894. 133. A. Liptak, J. Hajko, Unpublished results, 1998. 134. H. Boren, G. Ekborg, K. Eklind, P. J. Garegg, A. Pilotti, C. Swahn, Acta Chem. Scand., 1973, 27, 2639-2644. 135. N. K. Kochetkov, B. A. Dmitriev, 0. S. Chizhov, E. M. Klimov, N. N. Malysheva, A. Y. Chernyak, N. E. Bayramova, V. I. Torgov, Carbohydr. Res., 1974, 33, C5-C7. 136. N. K. Kochetkov, B. A. Dmitriev, N . N . Malysheva, A. Chernyak, E. M. Klimov, N . E. Bayramova, V. I. Torgov, Carbohydr. Rex, 1975, 45, 283-290. 137. G. Ekborg, J. Lonngren, S. Svensson, Acta Chem. Scand., 1975, B29, 1031-1035. 138. E. E. Lee, G. Keaveney, P. S. O’Colla, Carbohydr. Res., 1977, 59, 268-273. 139. C. D. Warren, C. Auge, M. L. Laver, S. Suzuki, D. Power, R. W. Jeanloz, Carbohydr. Rex, 1980, 82, 71-83. 140. C. Auge, C. D. Warren, R. W. Jeanloz, M. Kiso, L. Anderson, Carbohydr. Res., 1980,82, 8595. 141. G. Ekborg, C. P. J. Glaudemans, Carbohydr. Rex, 1984,129,287-292. 142. P. J. Garegg, C. Hallgren, J. Carbohydr. Chem., 1992, 11, 425-443. 143. H. H. Lee, L. N. Congson, D. M. Whitfield, L. R. Radics, J. J. Krepinsky, Can. J. Chem., 1992, 70, 2607-2617. 144. J . Kerekgyarto, J. G. M. van der Ven, J. Kamerling, A. Liptak, J. F. G. Vliegenthart, Carbohydr. Res., 1993, 238, 135-145. 145. S. H. Khan, K. L. Matta, Carbohydr. Res., 1995,278, 351-362. 146. P. H. Seeberger, P. F. Cirillo, S. Hu, X. Beebe, M. T. Bilodeau, S. J. Danishefsky, Enantiomer, 1996, I , 311-323. 147. S. Sarbanja, A. K. Misra, N. Roy, Synth. Commun., 1998,28, 2559-2570. 148. J. Kerekgyarto, J. P. Kamerling, J. B. Bouwstra, J. F. G. Vliegenthart, A. Liptak, Carbohydr. Res., 1989, 186, 51-62. 149. S. J. Danishefsky, S. Hu, P. F. Cirillo, M. Eckhardt, P. H. Seeberger, Chem. Eur. J., 1997, 3, 161771628, 150. M. Miljkovic, M. Gligorijevic, D. Glisin, J. Org. Chem., 1974, 39, 3223-3226. 151. A. Olesker, A. Dessinges, T. T. Thang, G. Lukacs, C. R. Acad. Sci. Paris Ser. 2, 1982, 295, 575-517. 152. W. Gunther, H. Kunz, Carbohydr. Rex, 1992,228, 217-241.
112. 113. 114. 115. 116.
342
13 Stereoselective Synthesis of /3-Mannosides
153. S. David, A. Malleron, C. Dini, Curbohydr. Rex, 1989, 188, 193-200. 154. I. Matsuo, M. Isomura, R. Walton, K. Ajisaka, Tetruhedron Let., 1996, 37, 8795-8798. 155. A. Fiirstner, I. Konetzki, J. Org. Chem., 1998, 63, 3072-3080. 156. A. Fiirstner, I. Konetzki, Tetrahedron Let., 1998, 39, 5721-5724. 157. S. David, A. Fernandez-Mayoralas, Carbohydr. Res., 1987, 165, clILc13. 158. J. Alais, S. David, Carbohydr. Rex, 1990, 201, 69-77. 159. K. Sato, A. Yoshimoto, Y. Tdkai, Bull. Chem. SOC.Jpn., 1997, 70, 885-890. 160. K. Sato, H. Seki, A. Yoshimoto, H. Nanaumi, Y. Takai, Y. Ishido, J. Curbohydr. Chem., 1998, 17, 703-727. 161. Y.-M. Zhang, J.-M. Mallet, P. Sinay, Carbohydr. Res., 1992,236, 73-88. 162. H.-W. Hagedorn, R. Brossmer, Helv. Chim. Actu, 1986, 69, 2127-2132. 163. H. Kunz, W. Giinther, Angew. Chem. Znt. Ed., 1988,27, 1086-1087. 164. C. Unverzagt, Angew. Chem. Int. Ed., 1994,33, 1102-1104. 165. I. A. Ivanova, A. V. Nikolaev, J. Chem. Soc. Perkin Trans 1, 1998, 3093-3099. 166. F. W. Lichtenthaler, E. Kaji, Justus Liebigs Ann. Chem., 1985, 1659-1668. 167. F. W. Lichtenthaler, E. Kaji, S. Weprek, J. Org. Chem., 1985, SO, 3505-3515. 168. F. W. Lichtenthaler, U. Klares, M. Lergenmiiller, S. Schwidetzky, Synthesis, 1992, 179-184. 169. F. W. Lichtenthaler, T. Schneider-Adams, S. Immel, J. Org. Chem., 1994, 59, 6735-6738. 170. F. W. Lichtenthaler, U. Klares, Z. Szunnai, B. Werner, Carbohydr. Rex, 1997,305, 293-303. 171. F. W. Lichtenthaler, T. W. Metz, Tetrahedron Let., 1997, 38, 5477-5480. 172. A. Fiirstner, I. Konetzki, Tetrahedron, 1996, 52, 15071-15078. 173. R. R. Schmidt, M. Reichrath, U. Moering, Tetrahedron Let., 1980,21, 3561-3564. 174. R. R. Schmidt, U. Moering, M. Reichrath, Chem. Ber., 1982, 115, 39-49. 175. A. Terjung, K.-H. Jung, R. R. Schmidt, Justus Liebigs Ann. Chem., 1996, 1313-1321. 176. A. Terjung, K.-H. Jung, R. R. Schmidt, Curbohydr. Rex, 1997,297, 229-242. 177. J . Tamura, R. R. Schmidt, J. Curbohydr. Chem., 1995,14, 895-911. 178. W. Klotz, R. R. Schmidt, Justus Liebigs Ann. Chem., 1993, 683-690. 179. S. David, S. Hanessian, Tetrahedron, 1985, 41, 643-663. 180. David, S. in Preparative carbohydrate chemistry, (Ed. S. Hanessian), Marcel Dekker, Inc., New York, 1997, pp. 69-83. 181. V. K. Srivastava, C. Schuerch, Tetrahedron Let., 1979, 3269-3272. 182. A. Dessinges, A. Olesker, G. Lukacs, T. T. Thang, Carbohydr. Res., 1984, 126, C6-C8. 183. G. Hodosi, P. Kovac, Curbohydr. Res., 1998, 308, 63-75. 184. D. Kahne, D. Yang, J. J. Lim, R. Miller, E. Paguaga, J. Am. Chem. Soc., 1988, 110, 87168717. 185. N. Yamazaki, E. Eichenberger, D. P. Curran, Tetrahedron Let., 1994, 35, 6623-6626. 186. D. Crich, S. Sun, J. Brunckova, J. Org. Chem., 1996, 61, 605-615. 187. D. Crich, J.-T. Hwang, H. Yuan, J. Org. Chem., 1996, 61, 6189-6198. 188. A. Liptak, I. Jodal, P. Nanasi, Curbohydr. Rex, 1975, 44, 1-11. 189. T. Iimori, H. Ohtake, S. Ikegami, Tetrahedron Let., 1997,38, 3415-3418. 190. L. F. Tietze, A. Montenbruck, C. Schneider, Synlett, 1994, 509-510. 191. D. L. Boger, K. D. Robarge, J. Org. Chem., 1988, 53, 5793-5796. 192. H. Paulsen, J. P. Lorentzen, W. Kutschker, Carbohydr. Res., 1985, 136, 153-176. 193. H. Paulsen, B. Helpap, J. P. Lorentzen, Carbohydr. Rex, 1988, 179, 173-197. 194. T. Sugawara, K. Igarashi, Carbohydr. Res., 1988, 172, 195-207. 195. B. Classon, P. J. Garegg, S. Oscarson, A.-K. Tiden, Carbohydr. Rex, 1991, 216, 187-196. 196. M. Scigelova, S. Singh, D. H. G. Crout, J. Chem. SOC.Perkin I , 1999, 777-782. 197. E. Micheli, F. Nicotra, L. Panza, F. Ronchetti, L. Toma, Curbohydr. Rex, 1985, 139, C1-C3. 198. R. Andersson, I. Gouda, 0. Larm, M. E. Riquelme, E. Scholander, Curbohydr. Rex, 1985, 142, 141-145. 199. E. Kaji, F. W. Lichtenthaler, Y. Osa, K. Takahashi, S. Zen, Bull. Chem. Soc. Jpn., 1995, 68, 2401-2408. 200. Y. Osa, E. Kaji, K. Takahashi, M. Hirooka, S. Zen, F. W. Lichtenthaler, Chem. Let., 1993, 1567-1570. 201. E. Kaji, F. W. Lichtenthaler, T. Nishino, A. Yamane, S. Zen, Bull. Chem. Soc. Jpn., 1988, 61, 1291-1297.
References 202. 203. 204. 205. 206. 207. 208. 209. 210. 21 1. 212. 213. 214. 215. 216. 217. 218.
343
K. L. Mattd, J. J. Barlow, Curhohydr. Rex, 1976, 48, 294-298. M.-C. Courtin-Duchateau, A. Veyrieres, Curhohydr. Res., 1978, 65, 23-33. H. P. Kleine, R. S. Sidhu, Curbohydr. Rex, 1988, 182, 307-312. K. Akerfeldt, P. J. Garegg, T. Iversen, Actu Chem. Scund., 1979, B33, 467-468. N. Kashimura, K. Kawaguchi, Ayric. Biol. Chern., 1976, 40; 1621-1624. M. Blanc-Muesser, J. Defaye, H. Driguez, Tetruhedron Let., 1976, 4307-4310. V. Pedretti, A. Veyrieres, P. Sinay, Tetruhedron, 1990, 46, 77-88. P. L. Durette, T. Y. Shen, Carbohydr. Res., 1980, 81, 261-274. A. Fiirstner, Justus Liehigs Ann. Chem., 1993, 1211-1217. N. Chida, T. Suzuki, S. Tanaka, I. Yamada, Tetrahedron Let., 1999, 40, 2573-2576. H. Itoh, Y. Kamiyama, Journal of Fermentution and Bioengineering, 1995, 80, 510-512. H. Fujimoto, M. Isomura, K. Ajisaka, Biosci. Biotech. Biochem., 1998, 61, 164-165. T. Usui, M. Suzuki, T. Sato, H. Kawagishi, K. Adachi, H. Sano, Glycocorzj. J., 1994, 11, 105110. S. Singh, M. Scigelova, D. H. G. Crout, J. Chrm. SOC.Chem. Commun., 1996, 993-994. N. Kildemark, K. Nilsson, XIX. Znt. Curhohydr. Symp., 1998, San Diego, CA, USA, BP035. G. M. Watt, L. Revers, M. C. Webberley, I. B. H. Wilson, S. L. Flitsch, Curhohydr. Rex, 1998,305, 533-541. L. Revers, R. M. Bill, I. B. H. Wilson, G. M. Watt, S. L. Flitsch, Biochim. Biophys. Actu, 1999, 1428, 88-98.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
14 Special Problems in Glycosylation Reactions: Sialidations Mukoto Kiso, Hideharu Ishida and Hiromi It0
14.1 Introduction Sialoglycoconjugates such as gangliosides and sialoglycoproteins have been recognized to play important roles in many biological processes [ 11. Sialooligosaccharide chains on cell surfaces are exposed as ligdnds to the external environment, and are capable of various biological functions, not only serving as receptors for hormones, viruses, and bacterial toxins, but also as mediators in cell growth, cell differentiation, cell adhesion, immune responses, and oncogenesis, etc. The most representative sialic acid, N-acetylneuraminic acid (NeuSAc, 1) is usually attached to 0 - 3 or 0-6 of galactose (Gal) or 0 - 6 of N-acetylgalactosamine (GalNAc) with an a(2-3) or a(2-6) linkage, and to 0-8 of another Neu5Ac with an w(2-8) linkage, giving diverse structures and functions. Because of this diversity systematic understanding of structure-function relationships of sialoglycoconjugates at the molecular level necessitates an efficient method for regio- and a-stereoselective glycoside synthesis of sialic acids.
14.2 Sialidation by the Koenigs-Knorr Method Several successful preparations of a-sialyl glycosides have been achieved mainly by use of the classic Koenigs-Knorr method employing N-acetylneuraminyl halides 2 as sialyl donors [2]. Although coupling of 2 (X = C1) with primary sugar hydroxyls, such as 3, gave a mixture of a-glycoside (4) and 0-glycoside ( 5 ) in good yields (Scheme 1) [3], the yield and stereoselectivity of the glycosides with hindered sugar hydroxyls were generally poor. For example, the sialidation of secondary sugar hydroxyls, such as 6, afforded an a,P mixture in low yields accompanied by a major by-product 7 (Scheme 2) [4], and the similar results were also obtained in couplings
346
14 Special Problems in Glycosylation Reactions: Siulidutions HO AcHN HO\W' W
C
0
2
H
~AcHN ~
HO
~
C
O
&
AcO
1 (Neu5Ac)
2
X = CI, Br etc
+
2 ( X = CI)
BnO
sieves 4 8, CHzC12
R
3 (R = OBn, N3)
AcouOAc
AcHN
"BnO " O0F o B n
AcO
OBn BnO
4 (a-glycoside) 36 % (R = OBn) 42 % (R = N3)
5 (p-glycoside) 48 % (R = OBn) 36 % (R = N3)
Scheme 1.
2 (X = CI)
t
HO
sieves 4 8, CH2C12
OBn 6
AcHN
OBn
AcO
C02Me
AcHN AcO
a (6 -12 %)
(3 (6 -19 %)
Scheme 2.
7
l
e
14.3 Siulidution Using an Auxiliary Group at C-3
347
of 2 (X = C1 or Br) with the lactose derivatives [5a, 5b]. Some improvements in the yield and a-stereoselectivity have been achieved by employing modified KoenigsKnorr conditions [2, 5c]. Particularly annoying is the competitive elimination which results from the deoxy center at C-3, giving the 2,3-dehydro derivative 7 as the major by-product. As critical problems, there are several disadvantages to the stereoselective synthesis of a-sialyl glycosides: 1) the anomeric center (C-2) of sialic acid is not only sterically hindered, but the reactivity is also electronically disfavored by the carboxylic acid function, which destabilizes the oxonium ion intermediate necessary for glycosidation; 2) the lack of a substituent at C-3 precludes the suitable neighboring participation leading to a-glycoside; and 3) the thermodynamically favored product is the P-glycoside. These combined factors are considered to disfavor formation of the desired aglycoside.
14.3 Sialidation Using an Auxiliary Group at C-3 The use of 2-halo-3-P-substituted sialic acid derivatives 8 as sialyl donors is an efficient means of overcoming the disadvantages in a-sialyl glycoside syntheses (Scheme 3) [2].Early work employing 8 (X = Br, Y = OH, P = Ac) showed that primary sugar hydroxyls react smoothly with this sialyl donor to give the desired
7
-
PO C02Me
AcHN
p0\\lts' AcHN
PO
PO
8
9
X = Halides Y = OH, SPh, SePh P = Ac, Bn
Scheme 3.
ROH
PO*"'
348
14 Special Problems in Glycosylation Reactions: Siulidutions
+
8
H O f l0O BnO e O B n
(X = CI or Br, Y = SPh, P = Bn)
HO
OBn
OBn 11
Hg(CN)2 HgBr2
Brio
sieves Molecular cc144A
B nBnOO AcHN
OBn
C02Me k
f
64 % (X = CI)
p
OBn OBn
OBn
HO
78 % (X = Br)
Brio &
o
12
OAc 7-
PhSCl CHcC12 30 C 2 days
AcOu' W L AcHN AcO
O
z M SPh
+
e
BnO* BnO
Brio OMe
8
AgOTf
(X = CI, Y = SPh, P = Ac)
40
13
Mokcular sieves 4 A
Na2HP04 A
c OAc O U
~
&
AcHN AcO
BnO BnO BnO OMe 14
Scheme 4a.
a-glycosides 9 with modest stereoselectivity. Nevertheless, the total yield and astereoselectivity of the glycosides with secondary sugar hydroxyls were generally poor. Many attempts to solve this problem were examined, and the thiophenyl auxiliary proved to be superior, as shown in Scheme 4a [6]. In fact, coupling of 8 (X = C1 or Br, Y = SPh, P = Bn) with a benzyl-protected lactose acceptor 11 gave excellent yields of the sialyl a(2-3')lactose derivative 12 [6a]. This procedure was immediately applied to the preparation of the sialyl a(2-8)sialic acid building blocks toward the synthesis of ganglioside GD3 and GQlb (Scheme 4b) [ 7 ] . The acetyl-protected sialyl donor 8 (X = C1, Y = SPh, P = Ac) was also examined for coupling with 13 to form a-sialoside 14 in 40% yield [6b]. Additional multiple steps for preparation of glycosyl donor 8 and removal of the C-3 substituent to arrive at the desired products 10 are, however, required (see Scheme 3), even though improvements in the yield and a-stereospecificity of the glycosides are achieved.
-
14.4 Siulidution Using 2-Tltioglycosidrs
BnO 8
(X = Br, Y = SPh, P = Bn)
OH
+
349
Hg(CN)2 HgBr2 C02Me
AcHN
Molecular
sieves 4A
cc14
BnO
(64%)
."W
C02Me
Bnvn C02Me
Scheme 4b.
14.4 Sialidation Using 2-Thioglycosides, Xanthates, or Phosphites of Sialic Acids in Acetonitrile 14.4.1 Thioglycosides Facile and highly regio- and a-stereoselective sialyl glycoside syntheses have been achieved [S, 91 by using the 2-thioglycosides of NeuSAc (15, 16) or 3-deoxy-w glycero-~-gaZacto-2-nonulosonicacid (Kdn, 17) as sialyl donors, and the partially protected sugar acceptors (18-26) (Figure l), in the presence of thiophilic promoters such as dimethyl(methy1thio)sulfonium triflate (DMTST) (promoter A) [9c, lo] or N-iodosuccinimide (N1S)-trifluoromethanesulfonic acid (TfOH) (promoter B) [ l l ] in acetonitrile (Table 1, Figure 2). The sialyl donors (15-17) were readily prepared [9, 12, 131 in three steps from NeuSAc or Kdn. Early work employing methyl 2-thioglycoside 15 as the glycosyl donor and DMTST as the promoter indicated [S, 91 that glycosylation in acetonitrile gives predominantly a-sialyl glycosides. Also, the use of lightly protected sugar acceptors (Figure I), in which several OH groups are unprotected, efficiently gave the desired sialyl glycosides a(2-3)- or a(2-6)-linked to the Gal residue in 50-70% yields, even in large-scale reactions [13]. Later, it was found that the use of phenyl 2-thioglycoside 16 [ 121 as the sialyl donor, promoted by NIS/TfOH, gives higher yields (Table 1, entries 3-5). Sialidation of trisaccharide acceptors (25, 26) with 16 afforded the corresponding sLeX (36) [14] and sLea (37) [15] derivatives in good yields (Table 1, entries 18 and 19). Reaction with reactive hydroxyls, such as OH-6 of 22 or OH-3 of 24, however, resulted in anomeric mixtures (entries 12-17). A
350
14 Special Problems in Glycosylation Reactions: Sialidations
Sialyl donors:
AcHN AcO 15R=Me 16R=Ph
AcO AcO 17
Sugar acceptors:
18 R’ = H, R2 = BZ 19 R’ = R2 = Bn
21 R = B z 22 R = Bn
23
24
J @ o ) O B n NHAc OBz OBn
&
HO Me
OBz
OBn
OBn
BnO OBn 25
26
Figure 1. Sialyl donors (15-17) and sugar acceptors (18-26).
similar tendency was observed also in the reaction with OH-6 of the GalNAc residue. Thus, the gangliotriose derivative 38 reacted with 16, promoted by NIS/ TMSOTf in acetonitrile, to afford 39a and 39p in 65% and 15% yield, respectively (Scheme 5). The use of NIS/TMSOTf, instead of NIS/TfOH, as the glycosyl promoter might give higher a-selectivity for reactive hydroxyls. Modification of the leaving group at C-2 in 2-thioglycosides of sialic acid has been found to modulate the reactivity as sialyl donors [ 161. The N-acetoxyacetamido (40a) [ 171, N-benzyloxyacetamido (40b) [ 181, and Ntrifluoroacetamido (42) [ 191 derivatives of 15 or 16 were also successfully coupled
14.4 Sialidation Using 2-Thioglycosides Products:
AcopOAc
R'
R2
R3
27
H
H
Bz
28
Bn
H
Bn
29
H
30
benzylidene
z:N2$$ b ; 33R=Bz 34R=Bn
OBz
31 R = NHAc 32 R = OAC
OSE
AcO
RO
OSE
AcO
CO2Me
OH
.
c
OAc o b
p
o C02Me
AcHN AcO
0 HO
o&
BnO
OBn
OSE OBn
35
OSE
OBn OBn
Figure 2. Structures of sialidation products.
351
352
14 Special Problems in Glycosylution Reactions: Siulidations
Table 1. DMTST (A)”-or NIS/TfOH (B)b-promoted sialidation of sugar acceptors (18-26) with methyl or phenyl 2-thioglycoside of sialic acid (15-17) in acetonitrile [Sc].
Entry
Donor
Acceptor
Promoter
Isolated yield (%)
Product
a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
18 18 18 19 20 18 23 23 23 21 21 22 22 22 24 24 24 25 26
15 15 16 16 16 17 15 16 17 15 15 15 15 16 15 15 16 16 16
A B B B B B A B B A B A B B A B B B B
52 61 70 70 65 49 47 55 46 70 59 50 51 50 30 59 58 58 69
27 21 27 28 29 30 31 31 32 33 33 34 34 34 35 35 35 36 37
Reactions were performed at - 15 to -20 “ C . bReactions were performed at -30 to -40°C.
a
HO ,OH*
16 b
OBn
OBn
38
NIS / TMSOTf Molecular
sieves 3 A CH3CN -30‘C
AcHN
OSE OBn 39 a (65 %), p (15 %)
Scheme 5.
P 0 0 0 0 0
0 0 0 0 0 0 15 26 25 8 10 11 0 0
353
14.4 Sialidation Using 2-Thioglycosides
18
SPh
NIS I TMSOTf CH$N -35 c (54 %)
40a
0
41a
24
NH
'
BnO" C\
c o
SMe OAc
40b
NIS / TfOH CH&N -23"C
OAc
A
CQMe o
c
B n o Z C0y
OAc
Ho
(55 %)
~
~
20
c o
OAC
42
~
OBn
41b
NH
CF3-C:
o
BnO
OBn
Acow;: AcO
~
NIS I TfOH CH&N/CHzCI, -30 "C (61 %)
OAc
*
CF3-C;A NHc OAC 0 W 0
i
M
p OSE
0 \
Ph
43
Scheme 6.
with 18,24 or 20, promoted by NIS/TfOH in acetonitrile, providing the desired sialyl-u(2-3)Gal derivatives 41a,41b,and 43 in yields of 45, 55, and 61%, respectively (Scheme 6). The N-benzyloxycarbonyamino (Boc) derivative (44)of sialyl donor 15 reacted with the lactose derivatives 45 and 46,promoted by PhSeOTf/ TMSOTf in tetrahydrofuran, to give 47 (84%) and 48 (46%), u predominantly (Scheme 7) [20]. When this reaction was conducted in dichloroethane, the sialosides were preferred products [21]. An efficient method for obtaining oligosialyl glycosides is the use of dimeric or trimeric Neu5Ac glycosyl donors, which can be prepared from naturally available colominic acid [22]. The lactonized phenyl 2-thioglycosides of dimeric [23] or trimeric [24] sialic acids (49)were each coupled with sugar acceptors 18 and 19 by use of NIS/TfOH as the promoter in acetonitrile, as just described, giving 30-48% of the desired a-glycosides (50,51) selectively (Scheme 8) [9d]. The reactivity of monosialyl (15,16)and disialyl (52)donors for the highly hindered OH-3 of the internal Gal residue (53)was examined (Scheme 9) [25], providing di- and trisialooligosaccharides (54, 55), the key intermediates for the synthesis of u-series gangliosides GTlau [25] and GQlbu [26]. As summarized in Table 2, the lactonized disialyl donor (52)gave the best result (Table 2, entry 6; 44%, u only) promoted by NIS/TfOH, whereas the monosialyl donors (15,16) afforded 34% (entry 1) and 40% (entry 5) of the desired a-sialosides, respectively, promoted by DMTST or
o
s
E
354
-
14 Special Problems in Glycosylation Reactions: Sialidations OH
OBn
OBn
HO H o
c
OBn
45
PhSeOTf TMSOTf THF
1
g
;
*
OBn
OBn
OBn
46
AcO\\‘’ BocHN AcO
c
44
BocHN AcO OBn OBn
48 4 6 % ( a : f 3 = 8 : 1 )
47 8 4 % ( a : P = l l : l )
Scheme 7.
SPh
+
18or19
AcHN AcO
49 (n = 0,l)
N ISKfO H Molecular sieves 4 A (30-48 %)
OSE
50 R’ = H, R2 = BZ 51 R’ = R2 = Bn
Scheme 8.
14.4 Sialidation Using 2-Thioglycosides OAc AcO%
15116
AcHNAcO
2%
CO2Me
.O.
'OSE
AcNH
+
or
355
k1 '0
.t
53
O
G OBn
OBn
OAc A
c
O
k
C02Me
g
AcO * O AcO\\'''
SPh AcO
52
AcHN
Scheme 9.
Table 2. a-Glycosylation" of 53 with sialyl donors 15, 16, and 52 in acetonitrile. Entry
Donor
Promoter
Product
Isolated yield (%)
54 54
34 I
54 54 55
26 40 44
~~
1 2
15 15
DMTST NIS/TfOH
?
16
NT9ITMCnTf
4
16 16 52
NIS/TfOH NIS/TMSOTf NIS/TfOH
5
_I
6 a
All reactions were performed at
~
._I
-I-----.
- 15 "C.
9A --
in A"
356
14 Special Problems in Glycosylation Reactions: Siulidutions 60
Acow
1
RSOTf
Me
Act37 R
SR
C02Me
AcO
(4
I I
-
FH3
TMSOTf
70
+ a-sialosides
Figure 3. A proposed mechanism for a-predominant sialidation in acetonitrile.
NIS/TMSOTf. The yields and a-selectivity are quite appreciable, considering the bulkiness of the donor and the steric hindrance on the sialidation site. Taking into account all these results the critically important factors seem to be (1) the rate of generation of the oxocarbenium ion, (2) the preferred formation of the p-acetonitrilium ion intermediate, and (3) the nucleophilicity of sugar acceptors; this suggests the reaction mechanism [8c, 91 proposed in Figure 3. This highly practical sialidation method has been extensively applied [9] to the synthesis of numerous gangliosides, sialooligosaccharides, and their derivatives and analogs by using not only monosialyl (15-17, 40, 42 etc.), di- and trisialyl (49, 52) donors, but also building blocks such as 56-59 shown in Figure 4 (9; see also 'Glycolipids' in this book). 14.4.2 Xanthates and Phosphites
As alternative sialyl donors, the use of 2-xanthates 60 [27-291 or 2-phosphites 70 [30, 311 of NeuSAc has been examined (Schemes 10 and 11). Early work employing DMTST as the glycosyl promoter showed [27] that sialyl 2-xanthate 60 reacts with Gal acceptor 61 or 6 in acetonitrile to give predominantly
14.4 Siulidution Using 2-Thioglycosides AdqOAc
357
AmuoAc C02Me
C02Me
PO \op
OBz
57
56 AcO
4 0 A c
58
co
59
Figure 4. Building blocks for the systematic synthesis of sialooligosaccharides, gangliosides, and their derivatives and analogs. X = NHAc, NHCOCH~OAC,NHCOCF3, NHBoc, OAc, efc. Y = SMe, OC(= NH)CC13, etc. P = Ac, Bz, etc.
a-sialosides 63 or 64 (Scheme lo), similarly to the corresponding sialyl2-thioglycosides. Improvements in the yield and a-stereospecificity of the products were achieved [28] by using methylsulfenyl triflate [32] as the promoter and a lightly protected lactose acceptor 62 which corresponds to 23 in Figure 1 , to afford 65 in 63-82% yield. The reactive sugar hydroxyl, such as OH-6 of Gal or GalNAc, gave an a,p mixture, however, even at -70 "C [28]. Similar results were also obtained by using phenylsulfenyl triflate as promoter in acetonitrile (Scheme 11) [29]. Sialyl 2-phosphites 70 were also found to react with sugar acceptors (11, 13, 71) promoted by TMSOTf in acetonitrile (Scheme 12) [30, 311, giving a-sialosides (7274) predominantly. 14.4.3 Reaction Mechanism A possible reaction mechanism is shown in Figure 3. When the 2-thioglycosides (a) of sialic acids are specifically activated by the thiophilic promoter at low temperature, the cyclic oxocarbenium ion (d), necessary for almost all known glycosylation reactions, is formed by the initially produced intermediates (b) or (c), and subsequently, reacts with acetonitrile to generate the p-acetonitrilium ion (e), which then undergoes S Ndisplacement ~ at the anomeric center by sugar hydroxyls to give predominantly a-glycosides. The common oxocarbenium ion intermediate ( d ) is also thought to be formed by activation of 2-xanthate (60) or 2-phosphites (70), generating the p-acetonitrilium ion (e)which provides predominantly a-sialosides. It has been demonstrated that the a-D-glucopyranosyl acetonitrilium ions are stereospecifically generated from the corresponding oxocarbenium ions in dry ace-
358
14 Special Problems in Glycosylution Reactions: Siulidutions
BzO "-0~~
+
or
6
OH
AcHN
61 AcO 60
4 62
(A) DMTST CH$N -15
(6)AgOTf
c
t
MSB CH&N/CH
AcouOAc AcHN
C02Me
AcHN
A&
OMe
AcO
63 a (48 %), fi (16 %)
64 a (26%), p (4 %)
,OBz AcHN AcO
-
65 (63 82 Yo)
Scheme 10.
tonitrile [33-351. It seems most plausible, therefore, that stereospecific generation of the p-acetonitrilium ion of Neu5Ac from the oxocarbenium ion holds the key to the a-predominant formation of sialyl glycosides in acetonitrile medium as shown in Figure 3 . Thus, the reactive sugar hydroxyls have a chance to attack other reaction intermediates before the generation of the fi-acetonitrilium ion of NeuSAc is complete, giving an anomeric mixture. Coupling with highly hindered secondary alcohols, however, generally results in low yields (but high a-selectivity) of the desired sialyl glycosides with formation of 7 as a major by-product.
14.5 Furtlzev Solutions to the Problem
359
.OTBDMS
un
HO(
-0Bn
n
OBn
I OAc A AcH N
c
67
66
PhSCI/AgOTf/DTBP CH,CN/CH2CI,, -70 "C
,OTBDMS
C02Me O ~ 'OMe
AcO \OBn 68 74 % (a$ = 955)
or
35 (78%; a:p = 96:4)
Scheme 11.
14.5 Further Solutions to the Problem To solve the problem in sialidation (1) introduction of an auxiliary group at C-3 in sialyl donors, (2) specific activation of the anomeric carbon C-2 by use of thioglycosides, xanthates or phosphites in acetonitrile medium, and (3) the use of sterically less hindered sugar acceptors have been attempted and found to be highly effective for obtaining a-sialosides in a regio- and stereospecific manner. Combinations of these procedures have resulted in further improvements. 14.5.1 Combination of C-3 Auxiliary and Sterically less Hindered Sugar Acceptors Combination of C-3 auxiliary (SPh) in sialyl donor 8 and sterically less hindered sugar acceptors (75, 77) has been successfully applied to the synthesis of sialyl LeX [33] and ganglioside GM3 [34] to give the desired 76 and 78 in 63% and 72% yields, respectively (Scheme 13). This method has been extended to the synthesis of disialyl lactose derivative 81 by the coupling of 80 with 77 (Scheme 14) [35].
14 Special Problems in Glycosylation Reactions: Sialidutions
360
OAc
A AcHN C O w E : i e
+
11/13
or 0
AcO
Po
70 (R = Et, Bn)
Ph
71
TMSOTf Molecular sieves 3A
CH&N -40 "C
AcouoAc C02Me AcHN BnO
OBn BnO
OMe
72
73
70-80% (a:P= 4:l
- 5.6:l)
50-55% (aonly)
Ph
74 38 % (aonly)
Scheme 12.
14.5.2 Combination of C-3 Auxiliary and Specific Activation of the Anomeric Center C-2 Further improvements in yield and a-stereospecificity have been achieved [36, 371 by combining the C-3 auxiliary (SPh) and specific activation of the anomic carbon C-2 by use of 2-thioglycosides (82) in acetonitrile (Scheme 15). Thus the coupling of 82 with 24, promoted by (A) methylsulfenyl triflate [36] or (B) phenylsulfenyl triflate [37] in acetonitrile at -40°C, gave 83 in 71% and 83% yields, respectively. Even when coupling was performed with the sterically hindered acceptor 84, the desired a-sialoside 85 was obtained in 67% yield [36]. Coupling with 86, however, resulted in 28% yield.
14.5 Further Solutions to the Problem
8
(X = Br, Y = SPh, P = Bn)
i-
OBn
63%
1
75
Hg(CN)2kwr2 Molecular sieves 4A, CC4 40 “C, 2 days
AcHN
OBn
76
8 (X = CI, Y = SPh, P = Ac)
+ OPlV
72%
AgOTf Molecular sieves 3A,CH3CN 60 “C, 2 days
SEt
AcHN AcO OPIV
78
Scheme 13.
361
14 Special Problems in Glycosylution Reactions: Siulidations
362
AcO
I::!;
C02Me
AcHN
AgOTf Molecular sieves 4R Na2HP04 CHpC12/toluene
AcO
79
-*
AcHN
(X = CI, Y = 8SPh, P
77
AcHN AcO
80 (49 %)
AcHN
AgOTf SnC12 Molecular
SEt
AcHN
sieves 4R -AW300 Na2HP04 CH3CN
OPiV
81 (39 %)
Scheme 14. OAc AcHN A c
O
AcO%;i,i;e".OAc
CqMe S -
AcO
82
24
*
(A) MeSBdAgOTf (6)PhSCI/AgOTf CH+N -40
(R = Me, Et)
C02Me
AcHN AcO
OSE
HO
C
OBn
Brio
83 (71 %, 83 %)
OBn
84 85 (67 %)
AcHN ACo
MPMO
86 AcHN
BZO
87 (28%)
Scheme 15.
OBn
14.5 Further Solutions to the Problem
363
AcO'
Phd
90
8 8 X = CI 89 X = OP(OEt)2
(A) AgOTf, C2H4C12 (B) TMSOTf, CH3CN
AcO
AcHN A
c OAcO
C02Me G PhOBnO
AcHN AcO
OBn
Fs PhO HO
OBn
Brio
OBn
C02Me
AcHN AcO
92 (68%, 83 %)
91 (89 %, 88 %) Scheme 16.
Dramatic improvement in the yield of a-sialyl glycosides has been achieved [38] by using the 3-phenoxythiocarbonyloxy derivatives of the 2-chloride (88) and 2phosphite (89) as sialyl donors (Scheme 16). Even the chloride derivative 88 coupled smoothly with 11 or 90, promoted by AgOTf in dichloroethane, to afford 91 (89%) and 92 (68%) in high yields. Specific activation of C-2 by diethyl phosphite (89), promoted by TMSOTf in acetonitrile, improved the yield of 92 (83%).
14.5.3 Thioglycoside of N,N-Diacetylneuraminic Acid and Combination with C-3 Auxiliary A novel N,N-diacetyl sialyl donor 93, prepared by N-acetylation of 15, has been found to react with 18 more smoothly than 15, giving 94 in 72% yield (Scheme 17) [39]. This method was immediately combined with C-3 auxiliary [40]. In fact, a novel sialyl donor 95 was found to couple with 84 or 86 in higher yields than the corresponding Neu5Ac donor 82 (R = Et) shown in Scheme 15, affording 96 (83%) and 97 (44%).
14 Special Problems in Glycosylation Reactions: Siulidutions
364
18 __7
NIS/TfOH
#
AyN
Molecular 93 __
sieves 38, CH3CN -40 "C
94 (72%)
04
COzMe SEt
AcO
Molecular
I
sieves 3A CH3CN/CH&I, -45 "C
96 (83%)
97 (44 %)
Scheme 17.
References 1. (a) W. Reutler, E. Kottgen, C. Bauer, W. Gerok in Sialic Acids-Chemistry, Metabolism and Function, Cell Biology Monographs, Vol. 10 (Ed.: R. Schauer) Springer, New York, 1982, pp. 263-305; (b) S. Hakomori, J. Bid. Chem. 1990, 265, 18713-18716; (c) S. Kelm, A. Pelz, R. Schauer, M. T. Filbin, S. Tong, M.-E. de Bellard, R. L. Schnaar, J. A. Mahoney, A. Hartnell, P. Bradfield, P. R. Crocker, Curr. Biol. 1994, 4 , 965-972; (d) C. Mitsuoka, K. Ohmori, N. Kimura, A. Kanamori, S. Komba, H. Ishida, M. Kiso, R. Kannagi, Proc. Natl. Acad. Sci. USA 1999, 96, 1597-1602. 2. (a) K. Okamoto, T. Goto, Tetrahedron 1990, 46, 89-102; (b) M. P. DeNinno, Synthesis 1991, 583-593. 3. (a) H. Paulsen, H. Tietz, Carbohydr. Res. 1984, 125, 47-64; (b) H. Paulsen, U. von Deessen, H. Tietz, ibid. 1985, 137, 63-77. 4. (a) T. Ogawa, M. Sugimoto, Carbohydr. Res. 1985, 135, c5-c9; (b) M. Numata, M. Sugimoto, K. Koike, T. Ogawa, ibid. 1987, 163, 209-225. 5. (a) M. Sugimoto, T. Ogawa, Glycoconjugate J. 1985, 2, 5-9; (b) H. Paulsen, U. von Deessen, Carbohydr. Res. 1986, 146, 147-153; (c) S. J. Danishefsky, J. Gervay, J. M. Peterson, F. E. McDonald, K. Koseki, T. Oriyama, D. A. Griffith, J. Am. Chem. Soc. 1992,114, 8329-8331. 6. (a) Y. Ito, T. Ogawa, Tetrahedron Lett. 1988, 32, 3987-3990; (b) T. Kondo, H. Abe, T. Goto, Chemistry Lett. 1988, 1657-1660.
References
365
7. Y. Ito, S. Nunomura, S. Shibayama, T. Ogawa, J. Org. Chem. 1992,57, 1821-1831. 8. (a) 0. Kanie, M. Kiso, A. Hasegawa, J. Curbohydr. Chem. 1988, 7; 501-506; (b) T. Murase, H. Ishida, M. Kiso, A. Hasegawa, Curbohydr. Res. 1988, 184, cl-c4; (c) A. Hasegawa, T. Nagahama, H. Ohki, K. Hotta, H. Ishida, M. Kiso, J. Carbohydr. Chem. 1991, 10, 493-498. 9. (a) A. Hasegawa, M, Kiso in Carbohydrates-Synthetic Methods and Applications in Medicinal Chemistry (Eds.: H. Ogura, A. Hasegawa, T. Suami), Kodansha/VCH, Tokyofleinheim, 1992, pp. 243-266; (b) A. Hasegawa in Synthetic Oligosaccharides~lndispensableProbes f o r the Life Sciences (Ed.: P. Kovdc), ACS Symposium Ser. 560, ACS, Washington DC, 1994, pp. 184-197; (c) Hasegawa et al., Methods Enzymol. 1994, 242, 158-198; (d) A. Hasegawa in Modern Methods in Carbohydrate Synthesis (Eds.: S. H. Khan, R. A. O’Neill), Harwood Academic Publishers, The Netherlands, 1996, pp. 277-300; (e) A. Hasegawa, M. Kiso in Prepururive Carbohydrate Chemistry (Ed.: S. Hanessian), Marcel Dekker, Inc., 1997, pp. 357-379. 10. P. Fugedi, P. J. Garegg, Carbohydr. Rex 1986, 149, c9-cl2. 11. (a) G. H. Veeneman, S. H. van Leeuwen, J. H. van Boom, Tetrahedron Lett. 1990, 31, 13311334; (b) P. Konradsson, U. E. Udodong, B. Fraser-Reid, Tetrahedron Lett. 1990, 31, 43134316. 12. A. Marra, P. Sinay, Carbohydr. Res. 1989, 187, 35-42. 13. A. Hasegawa, H. Ohki, T. Nagahama, H. Ishida, M. Kiso, Carbohydr. Res. 1991, 212, 277281. 14. A. Hasegawa, K. Fushimi, H. Ishida, M. Kiso, J. Curbohydr. Chem. 1993, 12, 1203-1216. 15. Y. Makimura, H. Ishida, M. Kiso, A. Hasegawa, J. Curbohydr. Chem. 1996; 15, 1097-1 11 8. 16. R. Roy, F. 0. Andersson, M. Letellier, Tetrahedron Lett. 1992, 33, 6053-6056. 17. A. Hasegawa, A. Uchimura, H. Ishida, M. Kiso, Biosci. Biotech. Biochem. 1995, 59, 10911094. 18. T. Sugata, R. Higuchi, Tetrahedron Lett. 1996, 37, 2613-2614. 19. S. Komba, C. Galustian, H. Ishida, T. Feizi, R. Kannagi, M. Kiso, Angew. Chem. Int. Ed. Engl. 1999,111, 1131-1133. 20. S. Fujita, M. Numata, M. Sugimoto, K. Tomita, T. Ogawa, Carbohydr. Rex 1992, 228, 347370. 21. Y. Ito, T. Ogawa, Tetrahedron Lett. 1988, 29, 1061-1064. 22. R. Roy, R. A. Pon, Glycoconjugate J. 1990, 7, 3-12, 23. H.-K. Ishida, Y. Ohta, Y. Tsukada, M. Kiso, A. Hasegawa, Curbohydr. Res. 1993,246, 75-88. 24. (a) H.-K. Ishida, H. Ishida, M. Kiso, A. Hasegawa, J. Curbohydr. Chem. 1994, 13, 655-664; (b) H. Ando, H. Ishida, M. Kiso, A. Hasegawa, Curbohydr. Rex 1997, 300, 207-217. 25. H. Ito, H. Ishida, M. Kiso, A. Hasegawa, Carbohydr. Res. 1998, 306, 581-585. 26. K. Hotta, H. Ishida, M. Kiso, A. Hasegawa, J. Curbohydr. Chem. 1995, 14, 491-506. 27. A. Marra, P. Sinay, Curbohydr. Res. 1990, 195, 303-308. 28. H. Lonn, K. Stenvall, Tetrahedron Lett. 1992, 33, 115-1 16. 29. V. Matrichonok, G. M. Whitesides, J. Org. Chem. 1996, 61, 1702-1706. 30. T. J. Martin, R. R. Schmidt, Tetruhedron Lett. 1992,33, 6123-6126. 31. M. M. Sim, H. Kondo, C.-H. Wong, J. Am. Chem. SOC.1993,115, 2260- 2267. 32. F. Dasgupta, P. J. Garegg, Curbohydr. Res. 1988, 177, c13-c17. 33. K. C. Nicolaou, C. W. Hummel, N. J. Bockovich, C.-H. Wong, J. Chem. Soc. Chem. Commun. 1991, 870-872. 34. T. Tomoo, T. Kondo, H. Abe, S. Tsukamoto, M. Isobe, T. Goto, Carbohydr. Res. 1996, 284, 207-222. 35. T. Kondo, T. Tomoo, H. Abe, M. Isobe, T. Goto, J. Carbohydr. Chem. 1996, 15, 857-878. 36. T. Ercegovic, G. Magnusson, J. Org. Chem. 1995,60, 3378-3384. 37. V. Martichonok, G. M. Whitesides, Carbohydr. Res. 1997, 302, 123-129. 38. J. C. Castro-Palomino, Y. E. Tsvetkov, R. R. Schmidt, J. Am. Chem. Soc. 1998, 120, 54345440. 39. A. V. Demchenko, G.-J. Boons, Tetrahedron Lett. 1998,39, 3065-3068; Chem. Eur. J. 1999,5, 1278- 1283. 40. N. Hossian, G. Magnusson, Tetrahedron Lett. 1999, 40, 2217-2220.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
15 Special Problems in Glycosylation Reactions: 2-Deoxy Sugars Aluin Veyrihes
15.1 Introduction 2-Deoxy and 2,6-dideoxy glycosides are important structural units in many natural products including antitumor drugs (anthracyclines, aureolic acids, calicheamicin, esperamicin), antibiotics active against Gram-positive bacteria (erythromycins, orthosomycins), antibiotics inhibiting platelet aggregation (angucyclines), drugs used in the treatment of cardiac insufficiency (cardiac glycosides), antiparasitic agents (avermectins). Whereas the therapeutic effect of these drugs is usually mediated by their aglycone part, the glycosidic part is essential for biological activity by regulating the pharmacokinetic properties, not only transport, but also molecular recognition (interaction with nucleic acids, for example). The role of carbohydrates in biologically active natural products has been recently reviewed [ 11. Therefore, methods for the efficient and stereoselective construction of deoxyglycosidic linkages will have useful applications in medicinal and bioorganic chemistry by allowing to understand biological mechanisms and elaborate new and less toxic drugs. The lack of a stereodirecting neighboring group adjacent to the anomeric center makes 2-deoxyglycoside synthesis a particular challenge. Moreover, the absence of electron-withdrawing substituent at C-2 makes the glycosidic bond much more acid labile, giving rise to easy hydrolysis or anomerization. Glycals soon appeared as ideal precursors especially for the synthesis of 2-deoxy-a-glycosides which are thermodynamically more stable than the P-isomers. The most frequently employed strategy for accessing 2-deoxy-~-glycosidesuses temporary equatorial blocking groups at C-2 which can be removed after glycosylation. These groups should stabilize the glycosidic bond and provide the anchimeric assistance required for a good P-selectivity. The synthetic aspects of the chemical construction of 2-deoxyglycosides have been the subject of four recent reviews [2-51.
368
15 Special Problems in Glycosylation Reactions: 2-Deoxy Sugars
15.2 Electrophilic Additions to Glycals: Mechanistic Aspects and Applications to the Synthesis of 2-Deoxyglycosides Addition of an electrophile E+ to a glycal such as protected D-glucal 1 gives a positively charged species which can be viewed as an oxycarbenium ion (2 or 3) in resonance with a cyclic onium ion (4 or 5 respectively); the weight of each structure is highly dependent of the nature of the electrophile and the polarity of the solvent (Scheme 1). If the addition of an electrophile is made in the presence of an alcohol R'OH, one can easily conceive that the glycosylation stereoselectivity will be dictated by the face-selectivity of electrophilic attack, at least if a true onium ion is involved. Trans-diaxial opening of the cyclic ion will occur under assistance by a lone pair of the pyranose oxygen atom. An above-plane addition should lead to an a-D-manno pyranoside 6 (diaxial outcome); a below-plane addition should give a P-D-glucopyranoside, first as a boat conformer 7, then flipping to the usual chair conformer 8 (diequatorial outcome). If a true oxycarbenium ion is involved, one can expect that the stereoselectivity will be governed by the kinetic anomeric effect [6-81 leading to a predominant axial stereochemistry at C-1, the a/P ratio being of course modulated by steric effects (a 3-axial substituent can override the stereoelectronic effect). Oxycarbenium ions 2 and 3 lead to a-D-manno 6 and a-D-gluco 9 pyranosides respectively. Altogether, 2-deoxy-a-glycopyranosides can be predicted to be more easily attained after replacement of E by hydrogen.
RO
Scheme 1. Electrophile-promoted addition of an alcohol to protected D-glucal 1 .
-
15.2 Electrophilic Additions to Glycals Ph3P-HBr 0.05 e ROD 10
H
11
D
369
OR
12
Scheme 2. Ph3P-HBr promoted addition of a deuterated alcohol to 3,4,6-tri-0-acetyl-~-glucal.
15.2.1 Protonation of Glycals
The acid-catalyzed addition of an alcohol to an acetylated glycal appears to be the most direct method for the synthesis of a 2-deoxyhexopyranoside. However, the acid catalyst has to be carefully chosen in order to avoid the Ferrier allylic rearrangement. Bolitt et al. [9] have reported that triphenylphosphine hydrobromide allows mild and high-yield protonation, then subsequent glycosylation (Scheme 2). Glycals with all equatorial substituents, like 3,4,6-tri-O-acetyl-~-ghcal10, give mainly a-glycoside products. Sabesan and Neira [ 101 have disclosed another efficient procedure where a dehydrated strongly acid ion-exchange resin in acetonitrile is used; lithium bromide is added to generate traces of hydrogen bromide needed for the reaction. Both methods give good results only with primary alcohols and water. However, when TMS triflate-triethylamine is used [ 111 at low temperature instead of a protic acid, silylation of the secondary alcohol 14 generates in situ Et3NH+ TfO- which activates the glycal 13 and gives the 2’-deoxy-a-(1-4)-disaccharide 15 in excellent yield (Scheme 3 ) . Franck [12] has shown that the major axial stereochemistry at the anomeric center is not due to a trans addition. No strong bridging of the proton can be really expected and the oxycarbenium ion 11 should be the reactive intermediate (Scheme 2). The use of deuterated alcohols ROD with catalytic amounts of Ph3P-HBr showed that deuteron delivery occurs largely from below the plane of the double bond. The predominant axial stereochemistry at C-1 and C-I’ of the glycoside products 12 and 15 probably arises from the kinetic anomeric effect. These results are consistent with an A d ~ 2(addition-electrophilic-bimolecular) mechanism for a first step of protonation, followed by a glycosyl transfer usually directed by the kinetic anomeric effect. Theoretical calculations [ 131 have shown O h
Scheme 3. Protioglycosylation of a glycal
15 Special Problems in Glycosylation Reactions: 2-Deoxy Sugars
370
16
17
18
Scheme 4. P-Galactosidase-catalyzed addition of glycerol to D-galactal 16.
that the diastereofacial selectivity in protonation of D-glycals can be explained by a polarization of the R bond below the plane, with the exception of D-allal. 15.2.2 Enzyme-Catalyzed Additions to Glycals As early as 1972, Lehmann [14,151 reported that glycals could be stereospecifically converted to 2-deoxyglycosides using glycosidase enzymes. For example, D-galactal2-d 16 was transferred to glycerol using a p-galactosidase from Escherichia coli, 18 (Scheme 4). Delivery of the affording glyceryl 2-deoxy-~-~-galactopyranoside proton to C-2 of D-galactal took place from the bottom face to afford apparent trans addition. But it has been shown [ 161 that an enzymic nucleophile E-Nu is first bound on the a-face in a cis addition similar to the above acid-catalyzed process; the a-adduct 17 thus obtained is then displaced by glycerol from the B-face. Such a mechanism is the exact reversal of the enzyme's natural and stereospecific cleavage reaction. These results have been applied by Beau [17] for the synthesis of various p-2deoxyglycosides including disaccharides. With simple a-galactosides as acceptors, only p-(17-6) linkages are formed, the highest yield (50'!40) being obtained with the methyl glycoside. Limited yields are not the consequence of competing hydration or cleavage of the glycoside, but seem to be due to mutiple transfers. 15.2.3 Halogenation of Glycals Addition of halogen to double bonds occurs by a bimolecular process, with the approach of a halogen molecule perpendicularly to the n system of the enol ether from above or below the plane of the molecule. The resulting charge-transfer complex may rearrange, in the rate-limiting step, to intermediate ions (syn ion pairs, halonium ions, or open carbocations) which, according to their relative stabilities, control the stereochemical course of the reaction. Igarashi [18] reported that chlorine addition to 3,4,6-tri-O-acetyl-~-glucal10 gave the four 1,2-dichlorides 23-26 in proportions which depended on the solvent used. In nonpolar solvents, cis-addition products 24 (p-D-manno) and 25 (a-D-gluco) were predominantly obtained through ion pairs 19 and 20 in which the chloride ion is associated on the same side of the original plane from which chlorine attack first occurred. Moreover, because of the anomeric effect, ion pair 20 and therefore compound 25 were much more favored than ion pair 19 and compound 24 (Scheme 5).
311
15.2 Electrophilic Additions to Glyculs
+
ActaACO *
CI~..CI A
c AcO
Z
S
i
slow
Clp
AcO
charge-transfer complex
AcO
ACO
23
AcO
25 'I CI
24
CI
CI
AcO
CI
26
Scheme 5. Chlorine addition to 3,4,6-tri-O-acetyl-D-glucal.
In polar solvents, trans-addition products 23 (a-D-munno)and 26 (p-D-gluco)were predominantly obtained in nearly equal amounts through chloronium ions 21 and 22. The study of bromine addition to glycals is complicated by the easy anomerization of the 1,2-di-bromides. However, Boullanger and Descotes [ 191 observed that 29 in CC14 gave predominantly an a - ~ bromination of 3,4,6-tri-O-benzyl-~-glucal glum 1,2-dibromide 31, whereas bromination of 3,4,6-tri-O-acetyl-~-glucallO in the same solvent gave mainly the CL-D-WXUZY~O 1,2-dibromide 28 (Scheme 6 ) . Horton [20]
A
c
O
S
&
6r2
LOAC ei 27 cc14
10
29
AcO & *# Br
=OAC
28
-h E ! q
30
Scheme 6. Influence of protecting groups in bromine addition to D-glUCa1.
31
Br
372
15 Special Problems in Glycosylation Reactions: 2-Deoxy Sugars
demonstrated that electronic effects, rather than purely steric effects, can explain this spectacular influence of protecting groups upon the stereoselectivity of the reaction, at least in nonpolar solvents: an electron-withdrawing substituent at C-6 disfavors the formation of ion pairs or oxycarbenium ions and therefore stabilizes symmetrical bromonium ions such as 27, but has no influence upon the addition of chlorine. As seen later on for the addition of N-iodosuccinimide to glycals, preferential approach of bromine from above the plane of the acetylated glycal might result from electrostatic effects, namely orientation of the net dipole moment of the molecule. When substituents with low inductive effects (benzyl groups or 6-deoxyglycal) are present, attack from below the molecular plane leads to a syn ion pair 30 favored by the anomeric effect and readily collapsing to the a-cis isomer 31. In polar solvents, the formation of oxycarbenium ions instead of syn ion pairs will be favored and the attack by bromide ions from below the molecular plane (kinetic anomeric effect) should be easier when bromine at C-2 is above (intermediate 2 in scheme 1); higher proportions of an a-trans addition product are therefore expected whatever the protecting groups are. The results obtained in electrophilic additions of bromine (and other electro29 have been recently reexamined [21]. philes) to 3,4,6-tri-O-benzyl-~-glucal
15.2.4 Bromo- and Iodoalkoxylation of Glycals In 1977 Tatsuta et al. [22] reported that 3,4,6-tri-O-acetyl-~-glucal 10 reacted with various alcohols in the presence of N-bromosuccinimide in acetonitrile to give mainly the 2-bromo-2-deoxy-a-~-mannopyranosides in 70-90% yields. With the exception of methanol, the a-mannolP-gluco ratio was estimated to be better than 95 : 5 when the reaction was performed at 5 “C; higher temperatures increased the amount of P-glucosides. As for protonation, this procedure is limited to sugar primary alcohols such as 32 leading to an a-(1+6) disaccharide 33 in excellent yield (Scheme 7). Radical debromination was then performed with tributyltin hydride and catalytic amounts of azobis(isobutyronitri1e) (AIBN). To explain the good stereoselectivity of the glycosylation reaction, the authors postulated that the gluco bromonium ion (below-plane attack) is destabilized by the “reverse anomeric effect”. This effect, which was put forward [23] to explain the preference of anomeric cationic substituents for the equatorial position on a pyranose ring, was recently shown [241 to be not consistent with theories of molecular
NBS,CH&N % * : A AcO
+
10
% A/ AcO
OAC
32
OAc
-5”C, e 94%
AcFq A&
33 Ac*o-cAC: OAc
Scheme 7. N-bromosuccinimide-promoted glycosylation of 3,4,6-tri-O-acetyl-~-glucal.
15.2 Electrophilic Additions to Glycals
373
structures. It is more probable that in acetonitrile an intermediate oxycarbenium ion with an axial bromine atom at C-2 is preferentially attacked by an alcohol coming from the a-side (kinetic anomeric effect), as it has been postulated above for the addition of bromine. The iodoalkoxylation of acetylated glycals was initially reported by Lemieux [25] as early as 1964. As an application of well-known Prevost and Woodward methods, the reaction was promoted by a mixture of iodine, silver perchlorate and collidine (or iodonium di-collidine perchlorate [25],IDCP) in benzene. In 1978 Thiem [26] developed a very useful variation of this reaction where N-iodo-succinimide (NIS) is used as an iodonium donor. Later on, Horton [27] reported a thorough study of Thiem’s procedure where he examined the influence of the solvent, the nucleophile and the structure of the glycal in relation to the ratio of the final products. Iodonium ions being much more stable than corresponding bromonium or chloronium ions, it was not expected that the substituent at C-6 of the glycal would have a significant effect. Observing that the proportion of stereoisomers (only trans-) in acetonitrile remained the same regardless of the nucleophile used, it was concluded that in this solvent the electrophilic addition of iodine to the double bond was irreversible and the proportion of final products reflected the proportion of attack of iodine from below or above the plane of the double bond. In tetrahydrofuran (or in methanol) a complex is formed with iodine, so that the first step will be fast and reversible; then the complex collapses slowly to give the final product (Figure 1). In such a situation, the ratio of stereoisomers will be controlled by the tendency for trans-diaxial opening of the cyclic ion, and by steric factors in the glycal and the nucleophile. It appeared that in acetonitrile steric factors do not exert a major influence on formation of the iodonium ion; electrostatic efects might be responsible for the stereochemistry of the first step. 3,4-Di-O-acetyl-~-fucal34 in the favored ’H4 (L) conformation has an axial acetoxy group at C-4 and is still iodinated mainly from below the plane of the molecule. The dipole moment of the ring oxygen atom, the equatorial acetoxy group at C-3, and especially the axial acetoxy group at C-4 combine to give a net dipole moment with the negative pole below the plane, favoring electrophilic attack from this side and leading to an a-L-fucopyranoside 36 through the iodonium ion 35 (Scheme 8). 3,4-Di-O-acetyl-~-rhamnal 37 with a C-4 acetoxy group equatorial also in the preferred ’H4 (L) conformation has a smaller dipole moment and shows therefore a
3- -
R
R
\+ ,o-I
R
R
fast
0
OR’
+‘A R OH
I
Figure 1. NIS-promoted addition of an alcohol to a double bond in ether solvents.
374
15 Special Problems in Glycosylation Reactions: 2-Deoxy Sugars ROH
Me
AcO
AcO
OAc
34
I
36
35
Scheme 8. NIS-promoted glycosylation of 3,4-di-O-acetyl-~-fucal.
37
10
Figure 2. Iodonium intermediates with acetylated L-rhamnal37 and D-glucal 10.
slight decrease in reaction from below the plane of the molecule. 3,4,6-Tri-O-acetylD-glucal 10 has the same relative configuration as L-rhamnal and consequently is mainly attacked by iodine from above the plane of the molecule in its 4H5 (D) conformation (Figure 2). This interpretation could be complicated by the conformational equilibrium 4H5/ 5H4 which gives to 3,4,6-tri-O-acetyl-~-glucal10, for instance, a 40% contribution of the 5H4 (D) conformer with all its substituents axial. A vinylogous anomeric effect could explain this unusual distribution [28]. However, assuming a lower energy barrier for this conformer equilibrium in comparison to the subsequent reactions, the Curtin-Hammett principle can be applied and the product ratio will be governed by the activation energy of the electrophilic addition, rather than by the actual concentration of conformers. Thiem [29] found that methyl 3,4-di-O-acetyl-~-glucuronal 38 adopts almost exclusively an inverted 5H4 (D) half-chair conformation. A 60:40 mixture of a-D-manno-pyranoside 39 [in a flattened 1,4B(D) boat conformation] and p-Dgluco pyranoside 40 was obtained when 38 was treated with cyclohexanol and N-iodosuccinimide in acetone (Scheme 9). Other methyl glycuronals (L-ribo, D-Z~XO and L - x ~ ~ oadopt ) the usual conformation with an equatorial methoxycarbonyl
Scheme 9. NIS-Promoted glycosylation of methyl 3,4-di-O-acetyl-~-glucuronal.
15.2 Electrophilic Additions to Glycals
375
group and give almost exclusively the expected a-glycoside. A “conformational effect”, where an increased ratio of inverted half-chair conformation in the glycal shifts the a : p anomer ratio towards increased P-glycosides, was suggested. In the N-iodosuccinimide-promoted procedure the succinimide anion is frequently observed to compete with a less potent nucleophile. This leads to glycosyl succinimides which can adopt predominantly an inverted C d (D) chair conformation [30]. To this respect, nucleophilicity of the alcohol can be increased by a previous 0-tributylstannylation which allows better reactivity in NIS-promoted addition to glycals (excellent results are thus obtained with the axially configurated 4-hydroxy group of various L-fucose derivatives) [3 11. Since the stereochemistry of the iodoglycosylation reaction is most often governed by trans-diaxial addition, this methodology is quite successful for the synthesis of 2‘-deoxy-a-disaccharides. The trisaccharide sequences of dihydroaclacinomycin A, an anthracycline antibiotic, and kijanimycin, a macrolide antibiotic, containing three 2,6-dideoxysugar with a-(1-4) and a-(1-3) linkages respectively, were efficiently synthesized by Thiem using the NIS-procedure [3]. The 2-deoxy function is generated from the 2-iodo derivatives most often by radical reduction (Bu3SnH or Ph3SnH and catalytic AIBN), or sometimes by hydrogenation in the presence of palladium-charcoal and a base to neutralize HI, or with NiCl2NaBH4. Applying the “armed-disarmed’’ concept, Danishefsky [32] extended the NISprocedure to the coupling of an armed glycal donor protected by benzyl(29) or silyl ethers with one hydroxyl group of a disarmed glycal acceptor (41) otherwise protected by acyl groups. N-Iodosuccinimide or sym-collidine iodonium perchlorate are the “I+ equivalent” reagents. To reiterate the procedure, it is necessary to enhance the nucleophilicity of the obtained disaccharide glycal42 toward I+, so that it would function as a glycosyl donor to the next acceptor. For this purpose, benzoate groups are exchanged to silyl ethers. The armed disaccharide glycal43 can now be coupled with the disarmed glycal 41 to give a trisaccharide glycal 44, which can react further with a sugar alcohol to give a tetrasaccharide (Scheme 10).
’
Scheme 10. Trisaccharide assembly employing the iodoglycosylation reaction.
376
15 Special Problems in Glycosylation Reactions: 2-Deoxy Sugars
45
Scheme 11. An a-iodoacetate as a-glycosyl donor.
Using this strategy, Danishefsky [33] performed the synthesis of ciclamicin 0, an anthracycline antibiotic containing a trisaccharide with three a-(1-4) linked 2,6dideoxy sugars and an a-linkage to the daunomycin aglycone (See Figure 10 for structure of ciclamicin 0). This work and other uses of glycals for the assembly of oligosaccharides and glycoconjugates have been recently reviewed [ 341. Olivomycin A, a member of the aureolic acid family (See Figure 7 for structure), contains a 2,6-dideoxy branched sugar, olivomycose, which is a-(1-3) linked to another 2,6-dideoxy sugar. Instead of running a direct iodoglycosylation of olivomycal 45, Roush [35] preferred to convert this glycal into the iodoacetate 46 by action of acetic acid and N-iodosuccinimide at -78 "C; a 85 : 15 ratio of diaxial 46 and diequatorial isomers was thus obtained (Scheme 11). It appeared that iodoacetate 46 is an excellent glycosyl donor when activated by trimethylsilyl triflate at low temperatures; high a-stereoselectivity and excellent yields were obtained with warious acceptors including glycals such as 47 which gave a disaccharide glycal48 incorporated into the total synthesis of olivomycin A [361. At reflux the reaction of a glycal49 with acetic acid and NIS gives a 1 : 1 mixture of P-D-gluco 50 and a-D-manno 51 isomers. After separation, the manno isomer can be reduced back to the starting glycal with lithium iodide in tetrahydrofuran
PI.
Another route, completely stereospecific at C-2, to a p-D-glUC0 isomer 54 starts from a 1,6-anhydro compound 53 easily obtained [38] by iodocyclization of D-glucal 52 (Scheme 12). The 2-deoxy-2-iodo-~-glucopyranosyl acetates such as 55 appeared as excellent glycosyl donors with various sugar alcohols including glycals (56) using trimethylsilyl or tert.butyldimethylsilyl triflate as promoters. The 2'-deoxy-2'-iodo-Pdisaccharides (57) were obtained in high yield and with a stereoselectivity often higher than 9 : 1. The high levels of stereochemical control in glycosylations with either a-iodoacetates (46) or P-iodoacetates (55) are undoubtedly due to the fact that corresponding iodonium ions of type 4 or 5 respectively are configurationally stable under the glycosylation conditions (TMSOTf, low temperatures); no equilibration of these ions occurs.
15.2 Electrophilic Additions to Glycals
’
-Ro% -
49
Lil, THF, 8246%
317
50
1. (Bu,Sn),O
2.12, CHBCN
R0
OAc
80%
52
HO
H
T W O OAc
TWO
+
F
53
54
O h TMSOTf, -78°C
En0
80%
I
55
i
56
57
Scheme 12. a-Iodoacetates as p-glycosyl donors.
15.2.5 Epoxidation of Glycals
The efficient epoxidation of glycals was reported in 1989 by Danishefsky [34, 391. Since acyl protecting groups can participate in the ring opening reactions of the epoxide, they have to be replaced by ether or acetal functions. Moreover, glycal epoxides being highly sensitive to nucleophilic attack at the anomeric carbon, peracids commonly used for epoxidation of alkenes lead to products of heterolysis; the use of anhydrous potassium fluoride in the mchloroperbenzoic acid-mediated epoxidation of glycals was, however, reported [40] to be successful. 2,2-Dimethyldioxirane (DMDO) appeared as the proper reagent, since the by-product, acetone, is not expected to react with the 1,2-anhydrosugar. 3,4,6-Tri-O-benzyl-~-glucal 29 reacted smoothly with DMDO to give a 20 : 1 mixture of am-gluco 58 and p-D-manno epoxides in nearly quantitative yield (Scheme 13). Epoxidation of D-allal with an axial 3-O-substituent occurred selectively on the p-face, whereas D-gulal with hindering substituents on both faces of the double bond gave a 1 : 1 mixture of a- and 0-epoxides. Neat methanol reacted smoothly with the a-D-yluco-epoxide 58 to give quantitatively the methyl P-o-glucopyranoside. More complex acceptors, like sugar alcohols (59), required a promoter (anhydrous zinc chloride, or zinc triflate with stannylated alcohols) and gave predominantly p-Dglucopyranosides (60). Yields can be improved by constraining the C-3 and C-4 or C-4 and C-6 oxygen functions of the glycal epoxide into a cyclic motif (carbonate or acetal). The uniquely generated 2’-hydroxyl group arising from opening of the 1,2epoxide donor can therefore be exploited in the synthesis of 2-deoxy-~-glycosides [41].Deoxygenation into 62 was accomplished by free radical reduction of the 2’-
318
15 Special Problems in Glycosylation Reactions: 2-Deoxy Sugars
0
ZnCI, , THF 50% S
29
E
W
0
77%
1
R = OC(S)OPhF561
90%
K
R=H62
Scheme 13. Synthesis of a 2’-deoxy-P-disaccharide with a glycal epoxide as glycosyl donor.
pentafluorophenylthiocarbonate 61 (Scheme 13). Moreover, the glycosyl acceptor can be a glycal which will be compatible with the deoxygenation process. Glycal epoxides also react with phenols [41, 441 in the presence of potassium carbonate and a crown-ether to give (3-aryl glycosides; opening of the epoxide by phenate ions occurs according to a mechanism of S N type. ~ Deoxygenation at C-2 as above affords aryl 2-deoxy-(3-glycosides which are found in aureolic acids and other antibiotics. However, with 2,6-disubstituted aryl rings elimination of the anomeric function competes with the deoxygenation reaction and starting glycals are obtained. Under acidic conditions (ZnC12) 1,2-anhydro sugars react with phenols to give mixtures of a- and (3-glycosides; the mechanism of these glycosylations has been recently investigated [45]. In the presence of silver tetrafluoroborate stannylated primary alcohols react with the a-D-gluco-epoxide 58 to give a-glycosides [42]. When the acceptor is a glycal such as 63, an a-disaccharide glycal 64 is obtained, upon which the process can be reiterated to give a trisaccharide 65 (Scheme 14). 1. AgBF4
2. A 5
8
0
~ ~Pyr. o, 52%
*
1. 2,2-Dimethyldioxirane 2.63, AgBF4 3. Ago. Pyr.
P 51%
0-6
Scheme 14. Synthesis of an a-linked trisaccharide with glycal epoxides as glycosyl donors.
1.5.2 Electrophilic Additions to Glycals
319
4-Penten-1-01 h0 OH
ZnCI, ,63%
50% p:a6:1
66
58
67
0
-
Brio*
F
BnO
68
OH
Scheme 15. Conversion of a glycal epoxide to other p-glycosyl donors.
Reactions with secondary acceptors proceeded in lower yields and exhibited sharply diminished stereoselectivities. It should be mentioned that 1,2-epoxides can be converted [43] to other glycosylating agents, thiophenyl glycoside 66, pentenyl glycoside 67, fluoride 68, which can be helpful after acylation at C-2 for the synthesis of 2-deoxy-P-glycosides(Scheme 15). 15.2.6 Addition of Sulfur Based Electrophiles to Glycals Phenylsulfenyl chloride. Addition of phenylsulfenyl chloride, PhSCl, to 3,4,6-tetraO-benzyl-D-glucal29 was first performed by Schmidt [46]. The adduct arising from a below-plane addition was hydrolyzed with Na2C03 (80% overall yield), then converted into an w-trichloroacetimidate which in the presence of BF,.OEt2 and alcohols delivered mainly 2-deoxy-2-(phenylthio)-~-~-glucopyranosides. As for protonation, the preferred a-face selectivity in the addition of PhS+ was corroborated by theoretical studies [13]. The addition of PhSCl was thoroughly reinvestigated by Roush [47] who came to the following conclusions: the stereoselectivity of the reactions of D-glucal derivatives with PhSCl is highly dependent on the presence of an electronegative heteroatom substituent at C-6, as well as on the functionality at C-4. The C-6 substituent strongly influences the conformational preferences of the D-glucal derivatives, and greatest stereoselectivity is obtained with those glycals that exist preferentially in the inverted 5H4 (D) half-chair conformation [29]. Addition to the glycals is slow compared to 5H4/4H5 conformational interconversion, especially if the reaction is performed at low temperature. Therefore, the Curtin-Hammett principle must apply and the product distribution will be determined by the relative energies of the competing transition states and not the distribution of ground state conformers. Addition of PhSCl to the bottom face of the 4H5 (D) conformer of D-glucal 69 leads to the episulfonium ion 71 and should be favored relative to the top face addition leading to 70, owing to non-bonded interactions of the PhS- unit with the adjacent TBS ether in the latter case. This diastereofacial selectivity, however,
380
15 Special Problems in Glycosylation Reactions: 2-Deoxy Sugars
R&'
TBSO
f- TBSO
TBSO
72 a-D-Manno
S
70
O
7
GI
I
%
X
R O + ~ @
-k 'Ph 71
Ro SPh
+
*?&o PhS CI
73 a$-D-Gluco
A
Scheme 16. Stereochemistry of addition of PhSCl to D-glUCal derivatives.
should not be expected to be overwhelmingly large, according to what is observed [48] for glycals that are locked in this conformation. On the other hand, the two faces of the 5H4 (D) conformer are differentiated to a much greater extent: the bottom face is shielded only by the axial C-4 substituent. A charge-dipole interaction will stabilize the transition state when that substituent is an acyloxy group 72 > 25 : 1) was indeed obtained [27]. The best selectivity (a-D-glUCO 73/a-~-manno with bromo and chloroacetoxy substituents at C-6 and C-4 respectively (Scheme 16). This mechanistic analysis has to be, however, readjusted with glycals of other configurations, since a D-fucal derivative, which has no polar substituent at C-6, gives a bottom face addition with an excellent selectivity [49]. The 2-deoxy-2-(thiopheny1)-a,P-D-glucopyranosylchlorides such as compound 74 were hydrolyzed with Na2C03 or DBU in tetrahydrofuran-water to give lactols which can be converted [50] into aryl 2-deoxy-P-glycosides by a Mitsunobu reaction, followed by desulfurization or into a-trichloroacetimidates such as 75 which were found configurationally stable at C-2 under the TMS triflate-promoted glycosylation conditions [ 5 I]. The stereoselectivity in the glycosylation of various alcohols was found to be dependent on the substrate; the least sterically hindered alcohols gave the best selectivity for the desired P-glycosides 76 (a direct S N ~ displacement or the substitution of a tightly solvated trichloroacetamideoxycarbenium ion pair was probably occurring). The fact that the a-glycosides such as 78 comprise up to 20-50% of the product in glycosylation of hindered secondary alcohols supports the thesis that the reaction stereoselectivity is not governed by the intermediacy of episulfonium ions 77a or 77d, but rather that substitution reactions of oxycarbenium ions 77b or 77c play a dominant role (Scheme 17). The episulfonium ions have been shown [52] by theoretical methods to be less stable than the corresponding oxycarbenium ions. This subject is still a matter of high controversy [53].
15.2 Electvophilic Additions to Glycals
381
1. DBU, THF-HZO
TMSOTf, ROH CH& , -78OC
2. NaH, CC13CN
ROH Felkin-Anh control AcO
n b
SPh
PhS
nc control
76
AcO
s ' h 77d
70
OR
Scheme 17. Stereochemistry of glycosylation with a 2-thiophenyl-a-~-glucopyranosyl donor.
Desulfurization is achieved with Raney-nickel in refluxing ethanol or tetrahydrofuran at room temperature [46], or Bu3SnH-AIBN in toluene at 100°C [49]. Sulfonium salts and sulfenates. Arylbis(ary1thio)sulfonium salts were shown by Franck [48] to undergo electrophilic addition to glycals in the presence of alcohols to form principally P-glycosides. Ester-blocked glycals cannot be used since they are decomposed through a Ferrier rearrangement. The p-tolyl reagent gave the best P-selectivity, but although a major below-plane attack of sulfur onto the 3,4,6tri-0-benzyl-D-ghcal was always observed, the face selectivity was found to be highly dependent on the nature of the nucleophile. A case of double diastereodifferentiation was also uncovered with a racemic alcohol. Even 4,6-O-benzylidene or -isopropylidene D-glycals, which cannot flip to the inverted half-chair conformation, show a preference for below-plane attack. The conformational effect underlined above for iodine and PhSCl additions should therefore be discounted here. A powerful steric effect of a 3-axial alkoxy group will direct the electrophile to the opposite face. These results suggest that the reactive species associates the electrophile and the nucleophile in a dithiosulfenate structure (Figure 3). It can be seen that the aglycon and the glycal interact through space as the glycal begins to bond to the sulfur, and a face-differentiating interaction can be envisaged. The first-formed intermediate would then be a sulfurane. Franck [54] applied this methodology to the synthesis of an aureolic acid disaccharide made of two 2,6dideoxy sugars with a p-(1-3) linkage. This approach is strongly reminiscent of the reaction reported by Ito and Ogawa [55],where phenylsulfenate esters are added to
382
15 Special Problems in Glycosylation Reactions: 2-Deoxy Sugars \-
OR
SAr
Arylbis(arylthio) sulfonium
Dithiosulfenate
Figure 3. Addition of an arylbis(ary1thio)sulfonium salt and an alcohol to a glycal.
glycals in the presence of TMS triflate to give mainly P-glycosides (Figure 4). But although a similar attack of the TMS-activated sulfenate on the glycal is occurring, a close interaction between aglycon and glycal is not required here. In both cases the reaction can evolve to an episulfonium ion which is trans opened by the alcohol or a nucleophilic equivalent. 15.2.7 Addition of Selenium Based Electrophiles to Glycals
Beau and Sinay [ 561 reported the glycosyloxyselenation of glycals as an approach to 2'-deoxy-a-disaccharides. Addition of phenylselenenyl chloride, then an alcohol 79 and collidine to a protected D-glucal such as 29 in acetonitrile gave 9 : 1 mixtures of a-D-manno 80 and P-D-gluco glycosides in good yields (Scheme 18). The selective formation of a 1,2-episelenonium ion above the molecular plane was explained by complexation with benzyloxy groups. Unlike sulfur addition, the selenation is reversible and attempts to produce P-2-deoxy-glycosides in a one-step procedure were therefore investigated [57].It turned out that only trans diequatorial acetoxyselenides such as 81 can be obtained with a high selectivity (P: a 9 : 1) by adding PhSeC1, then silver acetate to the glycal in toluene. The stereochemistry of this addition was strongly influenced by the solvent polarity and the nature of the glycal protecting groups (a 4-0-benzoyl group reversed the selectivity). Acetoxy-selenides (81) were able to glycosylate even hindered secondary alcohols such as 1,2 : 5,6-di0-isopropylidene-a-D-glucofuranosein the presence of TMS triflate to give the P-disaccharide 82 in excellent yield (Scheme 18). The high P-selectivity was given
Figure 4. Addition of a phenylsulfenate ester to a glycal.
15.2 Electrophilic Additions to Glycals
383
PhSeCI, CHsCN, 0°C
%q/J then
r.t.
BnO
29
collidine -
1
79 AcHN o
h
*
80
61%
PhSeCI, Tol. then AgOAc
DlPGlcf TMSOTf
BnF* Brio
29
81%
81
SePh
a-Manno : g-Gluco 1 : 9
97%
82
a-Manno : g-G/uco I : 10
T\
Scheme 18. Stereoselective syntheses of 2/-deoxy 2'-phenylseleno-a- and 0-disaccharides.
by the efficient directing effect of the phenylselenyl group at C-2 provided that diethylether was used as a solvent. Other solvents like dichloromethane induced epimerization at C-2. Roush [47, 511 reinvestigated this two-step P-glycosylation method and came to the same conclusions as for the addition of PhSCl to glycals: the stereoselectivity of the reaction of ~-glucalderivatives with PhSeCl is highly dependent on the presence of an electronegative heteroatom substituent X at C-6 (X = Br or OTs better than X = H or OBn), as well as the functionality at C-4. A possible equilibration of episelenonium species, which does not seem to have been taken into account in other studies [ 581, makes the procedure, however, slightly less stereoselective than trichloroacetimiwith PhSCl. The 2-deoxy-2-(selenenophenyl)-a-~-glucopyranosyl dates are also highly reactive, but do not give better selectivity than the sulfur analogs. Deselenation is commonly performed with a tin hydride and AIBN in toluene at 100 "C. In a novel approach to 2'-deoxynucleosides, Castillon [59, 601 reported also a thorough study of factors controlling the stereoselectivity of the selenium-mediated N-glycosylation of pyranoid and furanoid glycals by silylated bases. The organoselenium chemistry was also applied [61] to the conversion of glycals into spiroortholactones (86), a type of 2-deoxyglycoside linkage found in orthosomycins (see Figure 8 for orthosomycin structure). Glycosyloxyselenation of 3,4,6tri-O-benzyl-D-glucal 29 by alcohol 83 was followed by unmasking of the second hydroxyl group to be glycosylated, then oxidation into a selenoxide 84. Because syn-elimination away from the two oxygen atoms is not possible for stereochemical reasons, forcing conditions provide a transient and reactive ketene acetal 85 which reacts intramolecularly and under stereoelectronic control with the hydroxyl function to give a new axial a ( l h 6 ) glycosidic linkage. The isomeric ortholactone resulting from an equatorial addition was not detected in the reaction mixture (Scheme 19).
384
15 Special Problems in Glycosylation Reactions: 2-Deoxy Sugars
29
Tol.-VinylOAc BnO
85
Scheme 19. A selenium-mediated synthesis of a 2-deoxy-glycosyl-spiroortholactone.
15.3 The Cycloaddition Way to Glycosyl Transfer Following the elegant synthesis of 2-aminosugar glycosides by [4+2] cycloaddition of azodicarboxylates to glycals [62], Franck [63, 641 developed new bicyclic donors for the synthesis of 2-deoxy-P-glycosides. A diacylthione 88 generated in situ from a phthalimidosulfenyl precursor 87 undergoes cycloaddition reactions with a variety of glycals (pyranoid 1 and furanoid); an excellent a-face selectivity leading to cycloadducts 89 was observed in all cases, except with the allal where the pseudoaxial substituent at C-3 directs the addition above the plane (Scheme 20). The resulting heterocycle 89 is a vinyl glycoside which should behave as a glycosyl donor by activation with an electrophile. The acyl side chain or the sulfur atom could provide neighboring group participation, making the glycosylation highly stereoselective. The cycloaddition appears to be a reaction with inverse electron demand, since the smallest differences in energy are between the HOMO of the glycal dienophile and the low-lying LUMO of the heterodiene. Acetates lower too much the HOMO of the glycal, so that no reaction was observed. When the reaction is performed in methanol, a 50% yield of methyl glycosides is obtained along with cycloadducts. This result was interpreted as a two-step process where episulfonium species or corresponding oxycarbenium ions are initially formed, then rearrange into cycloadducts or are intercepted by methanol.
lutidine CHzClz , r.t.
SNPhth
87
88
CHzClz, pyridine, r.t. 5 days, 80% (R = En)
Scheme 20. Cycloaddition of a diacylthione to a protected D-glucal.
15.4 Fluoroglycosylation of Glycals
385
1 . 2 R’OH, TfOH CH2CI2, - 20% RO 2. Raney nickel RO RO -OR r
liCI4, 76% 92
Scheme 21. Synthesis of 2-deoxy-P-glycosides with cycloadduct glycosyl donors.
The cycloadducts 89 cleanly opened in the presence of p-toluenesulfonic acid only with methanol. A further survey of the reaction [65] showed that the a-D-gluco cycloadduct exhibited in fact reversibility with its glycoside product, wheras the 0D-manno adduct did not. The differing anomeric effects exerted in the ground states were proposed as the force which explains the differences in behavior. As a consequence, alcohol acceptors should be added in large excess to the glum donor. The a-D-gluco 89 and D-galacto cycloadducts could be, however, activated by methylenation of the ketone function with the Nysted Reagent 90 and TiC14. affording dienes 91 which are now extended vinyl glycosides. Triflic acid in dichloromethane proved to be the promoter of choice for their conversion into P-glycosides in good yields and with excellent stereoselectivities, even with less nucleophilic secondary sugar alcohols (Scheme 2 1). The addition of tetrabutylammonium triflate suppressed the anomerization of the P-glycoside and also prevents the acidcatalyzed 0- to C-rearrangement of an aryl glycoside. Raney nickel desulfurization affords the 2-deoxy-~-glycosides92.
15.4 Fluoroglycosylation of Glycals Although 2-deoxy-2-fluoro-glycosides cannot be precursors of the corresponding 2deoxy-glycosides, they are highly appreciated for their biological activities and the stabilization of the glycosidic linkage against hydrolysis brought by the fluorine atom. In the field of electrophilic addition to glycals, it seems therefore justified to mention the recent work of Wong [66] where the electrophilic fluorinationnucleophilic addition reactions with Selectfluor-type reagents 93 upon glycals have been studied and optimized. Selectfluor 93 adds first to a glycal such as 10 predominantly from above the plane of the molecule to give a syn adduct, which is susceptible to a slow anomerization and is further displaced by the alcohol 59; heating improves yields and also affects anomeric configuration. A major 2’-deoxy-2’-fluoro-a-~-mannodisaccharide 94 is thus produced (Scheme 22). A judicious choice of protective groups was shown to improve the stereoselectivity of both fluorination and nucleophilic addition.
386
15 Special Problems in Glycosylation Reactions: 2-Deoxy Sugars
Scheme 22. Fluoroglycosylation of 3,4,6-tri-O-acetyl-~-glucal 10 with Selectfluor 93.
15.5 Glycosyl Donors with a C-2 Heteroatom Other methods than electrophilic additions to glycals can afford glycosyl donors with a C-2 heteroatom substituent that will guide the stereochemical course of the glycosylation by anchimeric participation and will be reductively removed to provide the 2-deoxyglycoside. Most often, the glycosylating intermediate (onium ion, ion pair or oxycarbenium ion) will be identical to the one obtained from a glycal. But the nature of the promoter (metal salt, Lewis acid) can strongly affect the stereoselectivity of the glycosylation. Glycosyl donors with an equatorial C-2 heteroatom should normally give access to 2-deoxy-P-glycosides.
15.5.1 2-Bromo-2-deoxyglycosyl bromides Thiem [31 introduced the use of 2-bromo-2-deoxy-a-~-glucopyranosyl bromides 96 for the p-selective glycosylation of complex aglycones involved in aureolic acid antibiotics. These bromides were prepared by the reaction of dibromomethyl methyl 95 and gave ether [67] with a methyl 2,3-O-isopropylidene-a-~-mannopyranoside predominantly (P :a 10 : 1) 2-deoxy-2-bromo-~-glucopyranosides in their silver triflate-promoted reactions with sugar alcohols. Radical debromination afforded the 2-deoxy-P-glycosides97 in excellent yields (Scheme 23). The presence of a labile fonnyl substituent at 0 - 3 of 97 is highly valuable for the
-
R O Y & ,
95
1. R"OH, AgOTf CH3N02-Tol. Ro= -78"c OCHO
BrgF
_____)
OM9
06
Br Br 2. Bu3SnH AIBN, Tol., A
OR"
07
Scheme 23. 2-Bromo-2-deoxyglucopyranosyl bromides in the synthesis of 2-deoxy-P-glycosides.
387
15.5 Glycosyl Donors with a C-2 Heteroatom
further elongation of an oligosaccharide chain at that position, a strategy that was successfully used by Thiem for the synthesis of a P-linked trisaccharide of mithramycin, an antibiotic of the aureolic acid family [68]. 15.5.2 2-Deoxy-2-(thiophenyl)-glycosylfluorides
Nicolaou [69, 701 reported that phenylthio glycosides with a trans OH group at C-2 undergo 1,2-migration of the thiophenyl group when they are treated with (diethylamino)sulfur trifluoride (DAST) at 0 "C. The glycosyl fluorides with an inverted configuration at C-2 are obtained in high yields. A starting phenylthio a-D-mannopyranoside 98 will afford the 2-deoxy-2thiophenyl-P-D-glucopyranosylfluoride 99. Activation of the fluoride with SnC12 in diethyl ether, a tin-complexing solvent, allows the glycosylation of alcohols to give P-glycosides 100 with an excellent P-stereoselectivity through a 1,2-episulfonium intermediate. In the absence of a complexing medium, a-glucosides 101 are predominantly obtained, most probably because the promoter is then coordinated to the sulfur atom and thus prevents it from participating in the coupling reaction (Scheme 24). Desulfurization with Raney nickel affords the desired 2deoxyglycosides. This strategy was applied [711 to the synthesis of the 2,2'-dideoxy-P-disaccharide B-C found in the antibiotic everninomicin, a member of the orthosomycin family (see Figure 8 for complete structure). Phenylthio a-D-mannopyranosides 102 and 104 were converted into fluoride 103 and P-methyl glycoside 105 respectively. The P-disaccharide 106 (BC), obtained by coupling of 103 and 105 in diethyl ether and subsequent protective group adjustment, was then acylated at 0-4' by fluoride 107 (A,) and a-glycosylated at 0-3' by the evernitrose fluoride 108 (A) in dichloromethane to give the AlB(A)C fragment of everninomicin (Scheme 25). Similarly, thioglycosides with a phenoxythiocarbonyl ester group at C-2 (109) undergo 1,2-migration of the anomeric phenyl- or ethylthio group when they are activated (110) by NIS/cat. TfOH. The intermediate episulfonium ion 111 is trapped by an alcohol to give P-Dglucopyranosides 112 when the starting compound is a phenyl- or ethylthio a-D-
- EtZNSF3 CHzCIz, 0°C
98
SPh
R'OH, SnCI, EtpO, - 15°C + : R Ro
RO
Ro *R
99
I
PhS
OR'
loo
PhS
101
PhS OR'
R'OH. SnCI, CHzCI2,-15"C RO !!
Scheme 24. Stereospecific syntheses of 2-deoxy-2-thiophenyl-P- and a-glycosides via 1,2-migration of the PhS group.
388
15 Special Problems in Glycosylation Reactions: 2-Deoxy Sugars
Twkq 1. DAST, CH2C12
Me
~o~~~
2. MeOH. SnCI, , EbO
104
F
Sph 3. BUdNF, THF
105
95%
SPh
1. SnCI, , Et,O. -15"C, 78% 2. Raney Ni, MeOH, 65OC 3. BnBr, NaH. Bu4NI, DMF, 0°C 4. BUdNF, THF, 25°C
Me
BnO
C Me0
109
2. DDQ 3.BF3 OEt, , CH,CI,,
NO2
95% (a-glycosylation)
Scheme 25. Synthesis of an everninomicin fragment.
mannopyranoside 109 (Scheme 26) [72]. 2'-Ethylthio disaccharides are, however, desulfurized by Raney nickel much more sluggishly than the corresponding 2'phenylthio compounds. 15.5.3 2,6-Anhydro-2-Thio-Glycosyl Donors Tatsuta and Toshima [73] designed conformationally rigid 2,6-anhydro-2-thio sugars for the stereocontrolled synthesis of 2,6-dideoxy-a- and -P-glycosides which are very commonly found in bioactive natural products. These new donors have a very rigid structure (boat conformation) and the stereoselectivity of the glycosylation should not be affected by the anomeric effect in the same manner as the more usual chair conformers.
S B
n
?
!
BnO
109
R = Ph or Et
- - -
OPhNIS
TtOH
SR
~
n
Bno
110
OAOPh ?
t
SR
q
B
n
?
!
Bno
R'OH
B n E o w Bno
111
:+
SR
112
R
Scheme 26. 1,2-Ethyl (or phenyl) thio group migration for the stereospecific synthesis of 2-deoxy-Pglycosides.
15.5 Glycosyl Donors with a C-2 Heteroatom
389
The required methyl 2,6-anhydro-2-thio-u-~-altropyranoside 113 was prepared according to known procedures [74, 751, then converted into a phenylthio glycoside 114,a fluoride 115 or a p-acetate 116.Activation of the thioglycoside 114 with Nbromosuccinimide or methyl triflate, and of the fluoride 115 with various Lewis acids (SnC12, SnC12-AgC104, SnCI2-ZnClz, Cp2 HfC12-AgC104, CpzZrC12-AgBFq, TMSOTf) allowed the glycosylation of various alcohols to give a-glycosides 117 in high yields and nearly complete stereoselectivity . Strikingly, that selectivity was independent of the solvent, the anomeric configuration of the donor and the nature of the promoter. In a drastic contrast, the P-glycosides 118 were almost exclusively obtained when the p-acetate donor 116 was activated by various Lewis acids (TMSOTf, Tf20, SnC14, TrC104); only in diethyl ether and tetrahydrofuran was the selectivity reversed, because of deactivation of the promoter. Anomerization of ainto p-glycosides by the Lewis acid alone prooved that a thermodynamic control is responsible for the high P-stereoselectivity of the glycosylation reaction (Scheme 27). Methanolysis, then hydrogenolysis with Raney nickel or radical reduction with Bu&H afforded the 2,6-dideoxy-a-(119) and ~-(120)-~-ribo-hexopyranosides. Moreover, 2,6-anhydro-2-thiomannopyranosyldonors 121 (Scheme 28) having an equatorial 3-0-substituent can be obtained by oxidation of the 3-hydroxyl group
OAc
4
OAc ‘ I ”
I
”*
OH
120
-& hF :$=
NBS.DAST
____)
AcO
AcO
OM
OAc
AcO
SPh
OAc
113
OAc
114 E2, ROH
1 . NaOMe 2.[HI E2 = SnClz or SnC12-AgCIC or SnCi2-ZnCI, OR
119
OAc
117
OR
or Cp2HfCI2-AgCIO, or Cp2ZrCI2-AgBF4 or TMSOTf E, = TMSOTf or Tf20 or SnCi, or TrCIO,
Scheme 27. 2,6-Anhydro-2-thio sugars as glycosyl donors for the stereospecific synthesis of 2deoxy-a- and P-glycosides.
390
A+4- %& 15 Special Problems in Glycosylution Reactions: 2-Deoxy Sugars
TPSO
AcO
NIS,TMSOTf
TPSO
121
SPh
Ho
122
TPSO
SPh
AcO
TPSO
123
0
SPh
1. LAH. THF 2. AQO, Pyr. 3. NBS, ROH
Scheme 28. Armed-disarmed glycosylation with 2,6-anhydro-2-thio sugars.
into a ketone, then stereoselective reduction with diisobutylaluminum hydride; they all give 2,6-dideoxy-a-~-arabino-hexopyranosides. Since the configuration of the glycosidic bond is completely independent of the configuration at the anomeric center of the glycosyl donor, these glycosylation reactions must involve an oxycarbenium ion as an intermediate. Under kinetic conditions, a repulsive electronic interaction between the sulfur atom of the 2,6bridge and the alcohol approaching from the p-face impedes the reaction and aglycosides are exclusively obtained. In contrast, under thermodynamic conditions, an a-glycoside with an axial 3-0-substituent will anomerize to the more stable pglycoside (Figure 5). When the 3-0-substituent is equatorial, no 1,3-diaxial interaction is present and the a-glycoside is thermodynamically stable. The high reactivity of 2,6-anhydro-2-thioglycosyldonors results from the electron-donating nature of the bridging sulfur atom. Indeed, the derived sulfoxides 122 and sulfones have no glycosylating power and can thus be implied in block syntheses exploiting the “armed-disarmed effect” (Scheme 28). Coupling of the armed phenylthio glycoside 121 with the disarmed acceptor 122 gives the a-disaccharide 123 in 89% yield. Activation of 123 by lithium aluminohydride (LAH) reduction of the sulfoxide group and subsequent reacetylation allows to glycosylate an alcohol in an a-selective procedure (124 is obtained in 98% yield when R = cyclohexyl). I
H
0-R
&-
AcO OAc
EZ‘
Figure 5. Repulsive interactions in glycosylation with a 2,6-anhydro-2-thio glycosyl donor.
15.5 Glycosyl Donors with a C-2 Heteroatom
HO
391
OH
OM W
O
H Cladinose
Me
SPh
Me
Erythromycin A
S
Figure 6. 2,6-Anhydro-2-thio-cladinose as a glycosyl donor in erythromycin A synthesis.
This highly stereocontrolled method of glycosylation was applied to the synthesis of erythromycin A and the C-D-E trisaccharide of olivomycin A [76]. The 3hydroxyl group of erythromycin A aglycone is sterically hindered and forms a hydrogen bond with the C-1 carbonyl group. Its glycosylation by a L-cladinose donor is a difficult task, as noticed by Woodward in his total synthesis of erythromycin A in 1981. The use of a phenyl2,6-anhydro-2-thio-~-cladinose thioglycoside activated by N-iodosuccinimide and triflic acid allowed the stereocontrolled synthesis of the desired a-glycoside in 90% yield (Figure 6). The olivomycin A trisaccharide (under the form of a P-cyclohexyl glycoside) was assembled through three 2,6-anhydro-2-thio sugars. The P-glycosidic bonds of units C and D were made (90% yields, no a-anomer) with a p-acetate donor which has an axial 3-O-substituent, the conversion to the reauired equatorial configuration being made after each coupling. The a-glycosidic bond of unit E was built in 89% yield and a fully stereoselective glycosylation reaction from a phenylthio glycoside derived [77] from olivomycose (Figure 7).
HO &eM
OAc
?Me OH
DEIPSO
D ‘PrOCO
HO E
Olivomycin A
Figure 7. Synthesis of the CDE trisaccharide of olivomycin A with 2,6-anhydro-2-thio sugars as glycosyl donors.
392
15 Special Problems in Glycosylation Reactions: 2-Deoxy Sugars
15.5.4 1,2-Di-O-Acetyl-fi-Hexopyranoses and N-Formylglucosamine Derivatives An efficient, general and stereospecific preparation of 2'-deoxy-P-disaccharides reported by Sinay [78, 791 makes use of a 2-0-acetyl substituent as a stereodirecting 126 (and -D-galacto-)pyranoses group. 1,2-Di-0-acetyl-3,4,6-tri-O-benzyl-P-~-glucowere obtained from the known corresponding orthoesters 125 (they could have been also prepared from glycal epoxides). They reacted with various sugar alcohols in the presence of TMS triflate at -20 "C to give P-disaccharides in high yields (75-90%) and a total stereoselectivity attributed to the efficient anchimeric assistance of the 2-0-acetyl group. After removal of this group, the Barton-McCombie procedure allowed deoxygenation at C-2' to give the 2-deoxy-P-glycosides 127 in 70-95% yields (Scheme 29). This method could be extended to 6-deoxysugars giving thus a straightforward access to 2,6-dideoxy-P-glycosides. If necessary, a 2-0-acylpyranosyl bromide can be used in place of the 1,Zdiacetate; Konigs-Knorr conditions are then applied to obtain the P-disaccharide [80]. The only limitation lies in the impossibility of using acylated glycosyl acceptors, inasmuch as regiospecific deoxygenation at C-2' would be problematic. This can be circumvented, however, by the following procedure [78, 791. 1,3,4,6-Tetra-0-acetyl-2-deoxy-2-fo~amido-~-~-glucopyranose 128, which is readily obtained from glucosamine, can be converted to an a-chloride 129 or an atrichloroacetimidate 130. These three glycosyl donors, when properly activated at room temperature (TMS triflate for the acetate 128 and the imidate 130, silver tri131 (Scheme flate for the chloride 129),give 2'-deoxy-2'-formamido-~-disaccharides 29). The excellent stereocontrol of the glycosylation reaction results from the participating property of the N-formyl group (oxazolinium ion).
1. ROH, TMSOTf, - 20°C 2. NaOMe, MeOH 3. NaH, irn., CSz, Me1 4. Bu,SnH. AlBN OR
126
127
Me ROH 1. POC13, NEt3 2. Bu,SnH, AlBN
TMSoTf A AcO c ;
A-li
a
O
A
r.t.
c
~
AcO
128 NHCHo 13' HCONH
OR
AcO
NHCHO
t
132
1ax=cI ROH, AgOTf, r.t. 130 X = OC: NHCCI, ROH, TMSOTf, r.t.
Scheme 29. fi-stereocontrol with 2-0-acetyl or 2-N-formyl groups in the synthesis of 2-deoxy-Pglycosides.
15.6 2-Deoxyglycosyl Donors
393
Q Figure 8. Synthesis of the BCDE tetrasaccharide fragment found in orthosomycins.
When the donor is correctly adjusted to the acceptor, yields are high (64-97%). However, acidic conditions of the glycosylation reaction may induce partial anomerization at the reducing end of the obtained disaccharide. In that case, more reactive N-phthaloylglucosamine donors can be used; a sequential treatment converts then the N-phthaloyl group into the required N-formyl substituent. Radical deamination at C-2' through an isonitrile delivered 2/-deoxy-P-disaccharides 132 in 65-89% overall yields (Scheme 29). This methodology was applied to the synthesis of the B-C-D-E tetrasaccharide fragment found in orthosomycins (Figure 8) [82].* The interglycoside spiroortholactone junction between disaccharide B-C and D-E fragments was constructed by glycosyloxyselenation of the glycal, oxidation at selenium, and syn-elimination of the resulting selenoxide, according to the procedure already described [611.
15.6 2-Deoxyglycosyl Donors In the absence of an heteroatom at C-2 a pyranosyl donor is expected, when activated by a promoter, to give an oxycarbenium intermediate which will be attacked
*After completion of this chapter, this methodology has been used as a key step for the total synthesis of everninomicin 13,384-1 by Nicolaou's group (Angew. Chem. Int. Ed. 1999,38, 3334-3350).
394
15 Special Problems in Glycosylation Reactions: 2-Deoxy Sugars
-
Montrnorillonite K-10 ~d-0
CH&,
25’C
Me
TBSO
67%
133
134
om
135 @a 69:31
OMe
Montrnorillonite K-10
CHPCI~, 25°C 136
137a:P 89:19
OMe
Scheme 30. Montmorillonite-promoted glycosylations of olivose.
by an alcohol preferentially on its a-face (kinetic anomeric effect) if no impeding steric interaction is present. However, in a few cases (insoluble silver silicate catalyst, anomeric phosphites), a reasonable 0-stereoselectivity can be obtained. 15.6.1 2-Deoxy-Hexopyranoses
It has been recently reported [ 831 that the stereocontrolled glycosylation of olivose (2,6-dideoxy-~-arabino-hexose) by a primary alcohol such as 134, using montmorillonite K-10 as a solid acid promoter as well as a dehydrating agent, leads predominantly to p-glycosides (135) when a 4-0-acetyl group is present (133); a participating effect of that group was postulated [ 851. With a 4-0-tert-butyldimethylsilyl substituent like in 136, a major a-glycosylation leading to 137 is observed as expected from the kinetic anomeric effect (Scheme 30). In his total synthesis of the macrolide avermectin Bla, Ley 1841 reported the in situ activation of an oleandrose unit (2,6-dideoxy-3-0-methyl-~-arabino-hexose) by carbonyldiimidazole or thiocarbonyl-diimidazole to give imidazolylcarbonyl and imidazolyl-thiocarbonyl glycosides respectively which undergo predominant aglycosylation in the presence of silver perchlorate in tetrahydrofuran. 15.6.2 Tert-Butyldimethylsilyl 2-Deoxyglycosides
Tert-Butyldimethylsilyl 2-deoxy-p-glycosides have been used for the glycosylation of primary [86]and secondary [ 111 sugar alcohols in the presence of TMS triflate at low temperatures. 2’-Deoxy-a-disaccharides are obtained in excellent yields. 15.6.3 1-0-Acyl- and Acetimidyl-2-Deoxy-Hexopyranoses
Remarkable selectivity for an attack from the a-side was observed [87a] when the anomeric p-nitro-benzoate of evernitrose 138 was reacted with the sugar alcohol
15.6 2-Deoxyglycosyl Donors
395
Scheme 31. Stereoselective a-glycosylation of the branched-chain nitro sugar evernitrose.
139 in the presence of TMS triflate at low temperature. In spite of a repulsive 1,3syn-axial interaction between the 3’-methyl branch and the anomeric substituent, a 2’-deoxy-a-disaccharide 140 (AB fragment of everninomicin) was exclusively obtained in a 64% yield (Scheme 31). As early as 1979 and as an application of the “imidate procedure”, Sinay [87b] reported the use of anomeric (N-methy1)acetimidates of 2-deoxysugars for the highly stereoselective synthesis of 2’-deoxy-a-disaccharides in excellent yields. 15.6.4 2-Deoxyglycosyl Bromides and Fluorides ~ of an Insoluble silver salts such as silver silicate favor direct S N displacement anomeric bromide by alcohols [ 881. When applied to 2-deoxy-pyranosyl bromides, this procedure can lead to 2‘-deoxy-P-disaccharideswith a good stereoselectivity. For instance (Scheme 32), bromide 141, where an ester group at 0-4 can also favor [85] P-glycoside formation, reacted with the sugar alcohol 142 in the presence of silver silicate to give the disaccharide 143 in 85% yield (P:a 5.4: 1) [89]. 2-Deoxy-a- and P-pyranosyl fluorides react with sugar alcohols in diethyl ether in the presence of TiF4 to give predominantly glycosides of the same anomeric configuration; a fluoride/catalyst/solvent complex is postulated in a “double s~~displacement”. A non-nucleophilic solvent such as hexane can neither form a complex nor stabilize an oxycarbenium ion and, therefore, in such solvents major S N ~ reactions are observed [90]. 2-Deoxyglycosyl fluorides have also been activated in neutral conditions by LiC104-diethyl ether mixtures 1911. Wiesner [92] introduced the concept of 1,3-participation of an axial 3-0substituent (p-methoxy-benzoate or N-methylcarbamate) for the stereoselective A 1,3-acyloxonium or a P-glycosylation of digitoxose, a 2,6-dideoxy-~-riho-hexose. 1,3-bridging iminium ion was proposed as an intermediate species where the a-face of the donor would be blocked during the glycosylation step (Figure 9).
+
++ozB BnO
;horn + / ; p & Silver silicate
~
BZO
BnO
05%
141
Br
142
Scheme 32. Silver silicate-promoted P-glycosylation.
143 @:a5.4:l
O M
396
R
15 Special Problems in Glycosylation Reactions: 2-Deoxy Sugars
O
or
W
R
O
R"OH w
W
0.
R
O
w
o
R
.
R'COO
R' = pmethoxyphenyl
+o
or NHMe
+ OMe Figure 9. Hypothetical 1,3-~articipationof a p-methoxybenzoate or a carbamate in glycosylation.
However, several attempts [93, 941 to apply this concept with digitoxosyl halides under silver triflate activation (or thioalkyl digitoxosides under mercury salt activation) showed no reliability in enhancing the P :a ratio of 2-deoxyglycosides. 15.6.5 S-(2-Deoxyglycosyl)phosphorodithioates
Michalska [95, 961 introduced S-(2-deoxyglycosyl)phosphorodithioatesfor the synthesis of 2-deoxy-glycosides. 0,O-Dialkylphosphorodithioic acids add to acylated (144) or benzylated glycals in benzene (Scheme 33) to give predominantly crystalline a-adducts (145). Protonation at C-2 occurs from the a-face as it has been seen with Ph3P.HBr, and overall the addition is cis [ 121. Simple alkoxides react upon these a-adducts to give 2-deoxy-P-glycosides ac~ [97]. Activation by thiophilic promoters such as silver cording to a S N mechanism perchlorate in acetonitrile [96], or NIS in dichloromethane, or IDCP in acetonitrile [ 981 leads with alcohols mainly to 2-deoxy-a-glycosides. 2'-Deoxy-a-disaccharides (147) can thus be prepared in good yields, but with a : P ratios better than 9 : 1 only in a few cases (sugar alcohol 146). Anomeric a$-S-phosphorodithioates can also be obtained [99] from glycal epoxide 58. After pivaloylation at 0-2, they can be activated by methyl triflate to give with sugar alcohols exclusively 0-disaccharides in good yields. Selective deoxyge-
-
vc S
S
II
HS P(OEt);!
PhH, r.t.
BZO W
144
Bzd
OEt OEt
$ BzO
s
'
IDCP, CHSCN, 0°C
80%, a:t3 9:l
145
BZO BZO
Scheme 33. 0,O-Diethylphosphorodithioic acid-mediated a-glycosylation of a glycal.
147
15.6 2-Deoxyglycosyl Donors
391
Scheme 34. Generation of anomeric 2-deoxy-a-glycosyl phosphate donors by a radical rearrangement.
nation at 0-2’ as already described [41, 78, 791 would then afford 2’-deoxy-pdisaccharides. 15.6.6 2-Deoxyglycosyl Phosphates, Phosphoramidites and Phosphites An anomeric 2-deoxy-a-glycosyl phosphate 150 is generated by a radical 2-1 migration of the phosphate ester group, which occurs when bromide 148 is irradiated in the presence of tributyltin hydride [loo]. The intermediate anomeric radical 149 (in a boat conformation) rearranges into a C-2 radical in an usual 4C1 (D) chair conformation; an hydrogen atom is then trapped mostly from the less shielded above face (Scheme 34). The unstable phosphate 150 reacts with alcohols in the presence of magnesium perchlorate to give mainly 2-deoxy-a-glycosides 132 in good yields (a: p ratio 2.34.5 : 1). 3,4,6-Tri-O-benzyl-2-deoxy-~-arabino-hexopyranose 151, easily obtained [ 101 from 3,4,6-tri-O-benzyl-~-glucal 29, was converted into an anomeric N,Ndiisopropyl phosphoramidite 152 which, under activation by TMS triflate at low temperatures, reacted with sugar alcohols to give 2’-deoxy disaccharides 153 in high yields and excellent a-stereoselectivity (Scheme 35) [ 1011. 1,2-Cis-2-(p-methoxypheny1thio)-a-D-glycopyranosylphosphoroamidate donors, with an equatorial 2-SAr participating group and the anomeric OP:O(NMe*)Z
EtOP(NiPr,),, CH2C12 HNiPr, - Tetrazole
YEt
BnO
&lo
OH
151
o4
p .
152 a:p1.6:1
OH
BnO NiPr,
- fi 153
CIP(0Et)z
151
ROH, CHzCIZ TMSOTf, -78°C
OR
ROH. Tol. TMSOTf, -94OC
90%
OR
154 a : p 9 4 : 6
OP(OEt)p
127
Scheme 35. 2-Deoxyglycosyl phosphoramidites and diethyl phosphites for respective a- and glycosylations.
p-
398
15 Special Problems in Glycosylation Reactions: 2-Deoxy Sugars
Scheme 36. 2-Deoxy (2-pyridy1)thioglycosides as a-glycosyl donors
leaving group, have been previously [ 1021 used for the P-glycosylation of alcohols. This variety of anomeric phosphates was claimed [ 1031 to be more stable than other phosphates such as 152. Diethyl phosphite 154, when activated in the same conditions (except toluene as solvent) as 152, gave predominantly 2-deoxy-~-glycosides127 (Scheme 35) [ 1041. The highest P-stereoselectivity was observed with primary alcohols (P :a 9 : 1). The glycosylation reaction was assumed to proceed through the intermediacy of a thermodynamically more stable 2-deoxy-a-~-glucopyranosyl triflate (or its tight a-ion pair). Anomeric a,P-bis(trichloroethy1) phosphites undergo mainly a-glycosylation when activated by boron trifluoride etherate or tin (11) triflate at room temperature [105]. 15.6.7 2-Deoxy Thioglycosides
2-Deoxy-(2-pyridyl)thioglycoside 155, when activated by methyl iodide, reacts smoothly with sugar alcohol 156 to give 2'-deoxy-a-disaccharide 157 in good yield and high stereoselectivity (Scheme 36) [ 1061. Tatsuta [ 1071 reported that highly a-stereoselective glycosylation of phenyl 2deoxy-3,4-0-isopropylidene- 1-thio-D-fucopyranoside 158 could be accomplished because of its rigid and unusual boat conformation. 2-Deoxy-a-glycosides (161 versus 159) were obtained with a much lower selectivity from 2-deoxyfucosyl donors 160 in an usual 4C1 (D) chair conformation (Scheme 37). It was also shown that
0
%
" QNBS
0
T SPh
99% 0 -
158
159 a:P>99:1
A d
A m ! & ,
160
97 idem %
SPh
AcO
AcO&
161 a$ 11:l
\
I
-o
0
Scheme 37. Highly stereoselective glycosylation by conformational assistance.
15.6 d-Deo,~yglycosylDonors
399
selectivity came from conjormational ussistunce, not from a 1,3-diaxial interaction due to the configuration of the 3,4-O-isopropylidene group, and was highly independent of alcohol acceptors. This effect was largely exploited in the 2,6-anhydro-2thio-glycosyl donors as already seen [73, 761. 15.6.8 2-Deoxyglycosyl Sulfoxides The “armed/disarmed concept” takes into account that the electron-donating/ withdrawing power of substituents in carbohydrates has a profound impact on the reactivity of the anomeric center. It can therefore be exploited to construct tri- and larger oligosaccharides in a one-step reaction. In 1993 Kahne [lo81 reported the synthesis of the trisaccharide of ciclamycin 0, an anthracycline antibiotic, in 25% yield by a “polymerization reaction” using two 2-deoxyglycosyl sulfoxides B and C and a phenyl2-deoxy-thioglycoside A as monomers (Figure 10). The reactivities of the sulfoxide donors and nucleophiles were tuned such that the linkage between A and B would occur first (a p-methoxybenzyl sulfoxide B is more reactive than an unsubstituted phenyl sulfoxide C), followed by the linkage to C (a silyl ether AB is a good acceptor when triflic acid is used as a promoter, but reacts more slowly than an unprotected alcohol). Methyl propiolate is used as a sulfenic acid scavenger. The trisaccharide has an anomeric phenyl sulfide on the A ring and can be readily oxidized into a sulfoxide, allowing an iterative strategy for oligosaccharide synthesis. The use of p-methoxybenzyl (PMB) groups in place of benzyl ethers was required for final deprotection of ciclamycin 0, but was found to decrease the yield of the one-step coupling reaction. Therefore, a stepwise approach to the trisaccharide was finally performed with noticeable improvements in glycosylation steps [ 1091. Activation of an anomeric sulfoxide with triflic anhydride releases phenylsulfenyl tri-
TfOH, Et20-CHZCIZ HC&CO,Me -70to-70°C 25%
c
1I
Ciclarnycin 0 b
p
C
0 ’
Figure 10. One-step synthesis of the trisaccharide of ciclamycin 0.
SPh
Me& A
15 Special Problems in Glycosylation Reactions: 2-Deoxy Sugars
400
Me$sph
TO’
kSPh
-&+Me?g-7 2 ‘:SPh
HO OPMB
AcO
AcO
PhSOTf
- q m l V e n g m J
AcO
OPMB
PhSSPh
Me0 -
m
;
P
Me$sph
h
Me0
b@o OPMB
b&o AcO
-
Me@
OPMB
Me#o AcO
OPMB
OPMB
Figure 11. Formation of a trisaccharide by-product by competitive PhSOTf activation of a phenylthio glycoside.
flate, PhSOTf, which causes problems by activating thiophenyl glycosides even at low temperatures; a trisaccharide resulting from such an activation was identified among by-products (Figure 1 1). It was found that 4-allyl-1,2-dimethoxybenzene could trap efficiently the phenylsulfenyl triflate, although some trisaccharide was still present. A second strategy was then examined: a hindered sulfide (2,6-dichlorophenyl) with decreased nucleophilicity would react more slowly with PhSOTf, providing a better opportunity for the scavenger to operate more efficiently. Finally, an “inverse addition” procedure (the sulfoxide is slowly added to the mixture of acceptor and promoter) minimizes the formation of anomeric sulfenates. With these modifications disaccharide A-B, then trisaccharide A-B-C could be obtained in 82 and 68% yields respectively. Oxidation of trisaccharide A-B-C with 2,2-dimethyldioxirane gave a sulfoxide (900/,)which was coupled to the aglycone of ciclamycin 0 under the modified conditions in 75% yield (Figure 10). After removal of the p-methoxybenzyl ethers with DDQ the ciclamycin 0 was obtained in a 17% overall yield and with only six steps from the starting monomers.
15.7 Other Approaches to 2-Deoxyglycosides All the approaches which have been described so far involved glycosylation reactions, that is to say the creation of an exocyclic C-1-0 bond on the pyranose ring.
15.7 Other Approaches to 2-Deoxyglycosides
162
I
1. PhSeCl 2. Bu3SnH
401
%& OR
127
Scheme 38. Electrophile-mediated intramolecular cyclization of an acyclic enol ether.
However, it should be also possible to construct a 2-deoxy-glycoside by formation of the endocyclic C-1-0 bond from an acyclic sugar, or the C-1-H-1 bond from an anomeric radical. 15.7.1 Cyclization of Acyclic Sugars Acyclic sugars obtained by Wittig reactions. Acyclic enol ethers 163 obtained by Wittig reaction upon 2,3,5-tri-O-benzyl- D-arabinofuranose 162 undergo intramolecular cyclizations when they are treated by an electrophile. N-Iodosuccinimide gives predominantly 2-deoxy-2-iodo-a-~-mannopyranosides which can be deiodinated to give 2-deoxy-a-glycosides 153. The reaction of phenylselenenyl chloride with 163, followed by radical deselenation, gives mainly the 2-deoxy-P-glycosides 127 (Scheme 38) [110, 1111. Redox glycosylation via thionoester intermediates. Barrett [ 112, 1131 introduced the concept of “redox glycosylation”, where the usual glycosylation reaction is replaced by the esterification of an aldonic acid 164, followed by reduction to a reactive intermediate (enol ether from an ester or 0,s-acetal 166 from the thionoester 165 obtained by treatment of the ester by the Lawesson’s reagent). Cyclization of 166 by silver tetrafluoroborate gives the 2-deoxyglycoside 167 in good yield (Scheme 39).
1. ROH, DCC 2. Lawesson’s reagent
165
OL.OR S
OMe 167
Scheme 39. Redox glycosylation via thionoester intermediates.
166
II
A m 4
collidine
402
15 Special Problems in Glycosylation Reactions: 2-Deoxy Sugars 1. Lawesson's reagent 2.286-Di-t-butyl-4-methy~En~+ Mel. MeOH, 40°C
Eno+
pyridine
sMe-
BnO 168
169
' 0
81 %
En0
om
OMf
170 p:a6:1
Scheme 40. Alkoxy-substituted anomeric radicals for the construction of 2-deoxy-P-glycosides.
15.7.2 Use of Alkoxy-Substituted Anomeric Radicals The hemithio orthoester 169 was prepared from lactone 168 by treatment with Lawesson's reagent, then reaction with methyl iodide and methanol. Radical desulfurization of 169 gives predominantly a 2-deoxy-P-glycoside 170 by transfer of hydrogen to an intermediate methoxy-substituted anomeric radical preferentially from below (Scheme 40). P-Selectivity is, however, enhanced when an alkoxy substituent is present at C-2 [ 1141. Crich [ 115-1 171 generated anomeric alkoxy-substituted radicals by irradiation of "Barton esters" (0-acyl thiohydroxamate 176) in the presence of a thiol. Conversion of a protected D-galactall71 into the anomeric sulfone 172, then into the thiophenyl glycoside of a methyl 2-ulosonate 173, was followed by a classical 0-glycosylation (alcohols or phenols). The carboxylic acid 174 was coupled with the salt 175 to give the 0-acyl thiohydroxamate 176. Its photolysis in the presence of a thiol generated an anomeric radical by decarboxylation; hydrogen transfer took place stereoselec177 were tively from below as for compound 169 and 2-deoxy-~-galactopyranosides predominantly produced (Scheme 41).
RO
- -
1. HCI, tol. 2. PhSH, Et(iPr),N OR 3. MCPBA. NaHC03
1 . Buti, O:C(OMe),
2.U Naphth., (PhS)2
RO
171
172
RO
OR'
+-
177
R = tBuMe,Si
Scheme 41. Reductive decarboxylation of 3-deoxy-ulosonic acid glycosides
I I SPh
179
1 . R'OH, HgCI, or NBS
R'SH CHZC12 hv, 5°C
RO
CO2Me
RO
References
403
References 1. A.C. Weymouth-Wilson, Nut. Prod. Rep., 1997, 14, 99-110. 2. R.W. Franck, S.M. Weinreb in Studies in Natural Products Chemistry (Ed.: Atta-UrRahman), Elsevier, Amsterdam, 1989, p. 173. 3. J. Thiem, W. Klaffke, Top. Curr. Chem., 1990, 154, 285-332. 4. K. Toshima, K. Tatsuta, Chem. Reo., 1993, 93, 1503-1531. 5. A. Kirschning, A.F.-W. Bechtold, J. Rohr, Top. Curr. Chem., 1997, 188, 1-84. 6. A.J. Kirby, The Anomeric Effect and Related Stereoelectronic Effects at Oxygen, Springer Verlag, New York, 1983. 7. P. Deslongchamps, Stereoelectronic Effects in Organic Chemistry, Pergamon Press, Oxford, 1983. 8. R.W. Franck in Conformational Analysis and Stereochemistry of Six-membered Rings (Ed.: E. Juaristi), VCH Publishers, Inc., New York, 1995, Chapter 5 , p. 157-198. 9. V. Bolitt, C. Mioskowski, S.-G. Lee, J.R. Falck, J. Org. Chem., 1990, 55, 5812-5813. 10. S. Sabesan, S. Neira, J. Org. Chem., 1991, 56, 5468-5472. 11. C. Kolar, G. Kneissl, Angew. Chem. Znt. Ed. Engl., 1990, 29, 809-81 1. 12. N. Kaila, M. Blumenstein, H. Bielawska, R.W. Franck, J. Org. Chem., 1992, 57, 4576-4578. 13. R.W. Franck, N. Kaila, M. Blumenstein, A. Geer, X.L. Huang, J.J. Dannenberg, J. Org. Chem., 1993,58, 5335-5337. 14. J. Lehmann, E. Schroter, Carbohydr. Res., 1972, 23, 359-368. 15. J. Lehmann, B. Zieger, Carbohydr. Rex, 1977, 58, 73-78. 16. O.M. Viratelle, J. Yon, Biochemistry, 1980, 19, 4143-4149. 17. J.-M. Petit, F. Paquet, J.-M. Beau, Tetrahedron Lett., 1991, 32, 6125-6128. 18. K. Igarashi, T. Honma, T. Imagawa, J. Org. Chem., 1970,35, 610-616. 19. P. Boullanger, G. Descotes, Carbohydr. Res., 1976, 51, 55-63. 20. D. Horton, W. Priebe, 0. Varela, J. Org. Chem., 1986, 51, 3479-3485. 21. G. Bellucci, C. Chiappe, F. D’Andrea, G. Lo Moro, Tetrahedron, 1997, 53, 3417-3424. 22. K. Tatsuta, K. Fujimoto, M. Kinoshita, Carbohydr. Res., 1977, 54, 85-104. 23. R.U. Lemieux, A.R. Morgan, Can. J. Chem., 1965,43,2205-2213. 24. C.L. Perrin, M.A. Fabian, J. Brunckova, B.K. Ohta, J. Am. Chem. Soc., 1999, 121, 69116918 and references therein. 25. a) R.U. Lemieux, S. Levine, Can. J. Chem., 1964, 42, 1473-1480; b) R.U. Lemieux, A.R. Morgan, Can. J. Chem., 1965,43, 2190-2198. 26. J. Thiem, H. Karl, J. Schwentner, Synthesis, 1978, 696-698. 27. D. Horton, W. Priebe, M. Sznaidman, Carbohydr. Rex, 1990, 205, 71-86. 28. D.P. Curran, Y.G. Suh, Carbohydr. Rex, 1987, 171, 161-191. 29. J. Thiem, P. Ossowski, J. Carbohydr. Chem., 1984, 3, 287-313. 30. J. Thiem, S. Kopper, J. Schwentner, Liebigs Ann. Chem., 1985, 2135-2150. 31. J. Thiem, W. Klaffke, J. Org. Chem., 1989, 54, 2006-2009. 32. R.W. Friesen, S.J. Danishefsky, J. Am. Chem. Soc., 1989, 111, 6656-6660. 33. K. Suzuki, G.A. Sulikowski, R.W. Friesen, S.J. Danishefsky, J. Am. Chem. Soc., 1990, 112, 8895-8902. 34. S.J. Danishefsky, M.T. Bilodeau, Angew. Chem. Int. Ed. Engl., 1996,35, 1380-1419. 35. W.R. Roush, K. Briner, D.P. Sebesta, Synlett, 1993, 264-266. 36. W.R. Roush, R.A. Hartz, D.J. Gustin, J. Am. Chem. Soc., 1999, 121, 1990-1991. 31. W.R. Roush, C.E. Bennett, J. Am. Chem. Soc., 1999,121, 3541-3542. 38. D. Tailler, J.-C. Jacquinet, A.-M. Noirot, J.-M. Beau, J. Chem. Soc., Perkin Trans. I , 1992, 3 163-3 164. 39. R.L. Halcomb, S.J. Danishefsky, J. Am. Chem. Soc., 1989, 111, 6661-6666. 40. G. Bellucci, G. Catelani, C. Chiappe, F. D’Andrea, Tetrahedron Lett., 1994, 35, 8433-8436. 41. J. Gervay, S.J. Danishefsky, J. Org. Chem., 1991, 56, 5448-5451. 42. K.K.-C. Liu, S.J. Danishefsky, J. Org. Chem., 1994, 59, 1895-1897. 43. D.M. Gordon, S.J. Danishefsky, Carbohydr. Rex, 1990,206, 361-366. 44. R.G. Dushin, S.J. Danishefsky, J. Am. Chem. Soc., 1992, 114, 3472-3475.
404
15 Special Problems in Glycosylution Reactions: 2-Deoxy Sugars
C. Chiappe, G. Lo Moro, P. Munforte, Tetrahedron, 1997, 53, 10471-10478. R. Preuss, R.R. Schmidt, Synthesis, 1988, 694-697. W.R. Roush, D.P. Sebesta, C.E. Bennett, Tetrahedron, 1997,53, 882558836, G. Grewal, N. Kaila, R.W. Franck, J. Org. Chem., 1992, 57, 2084-2092. W.R. Roush, X.-F. Lin, J. Am. Chem. Soc., 1995, 117, 2236-2250. W.R. Roush, X.-F. Lin, J. Org. Chem., 1991, 56, 5740-5742. W.R. Roush, D.P. Sebesta, R.A. James, Tetrahedron, 1997, 53, 8837-8852. D.K. Jones, D.C. Liotta, Tetrahedron Lett., 1993, 34, 7209-7212. T.J. Dudley, I.P. Smoliakova, M.R. Hoffmann, J. Org. Chem., 1999, 64, 1247-1253. R.W. Franck, N. Kaila, Carbohydr. Rex, 1993,239, 71-83. Y. Ito, T. Ogawa, Tetrahedron Lett., 1987, 28, 2723-2726. G. Jaurand, J.-M. Beau, P. Sinay, J. Chem. SOC.,Chem. Commun., 1981, 572-573. M. Perez, J.-M. Beau, Tetrahedron Lett., 1989, 30, 75-78. A. Kaye, S. Neidle, C.B. Reese, Tetrahedron Lett., 1988,29, 2711-2714. A. El-Laghdach, M.I. Matheu, S. Castillon, Tetrahedron, 1994, 50, 12219-12234. Y. Diaz, A. El-Laghdach, S. Castillon, Tetrahedron, 1997,53, 10921-10938. G. Jaurand, J.-M. Beau, P. Sinay, J. Chem. Soc., Chem. Commun., 1982, 701-703. Y. Leblanc, B.J. Fitzsimmons, J.P. Springer, J. Rokach, J. Am. Chem. Soc., 1989, I l l , 29953000. 63. R. W. Franck, C.H. Marzabadi, J. Org. Chem., 1998, 63, 2197-2208 and references therein. 64. A. Dios, A. Geer, C.H. Marzabadi, R.W. Franck, J. Org. Chem., 1998,63, 6673-6679. 65. A. Dios, C. Nativi, G. Capozzi, R.W. Franck, Eur. J. Org. Chem., 1999, 1869-1874. 66. S.P. Vincent, M.D. Burkart, C.-Y. Tsai, Z. Zhang, C.-H. Wong, J. Org. Chem., 1999, 64: 5264-5279, and references therein. 67. K. Bock, C. Pedersen, J. Thiem, Carbohydr. Rex, 1979, 73, 85-91. 68. J. Thiem, M. Gerken, B. Schottmer, J. Weigand, Carbohydr. Res., 1987, 164, 327-341. 69. K.C. Nicolaou, T. Ladduwahetty, J.L. Randall, A. Chucholowski, J. Am. Chem. Soc., 1986, 108, 2466-2467. 70. K.C. Nicolaou, C.W. Hummel, N.J. Bockovich, C.-H. Wong, J. Chem. Soc., Chem. Commun., 1991, 870- 872. 71. K.C. Nicolaou, R.M. Rodriguez, H.J. Mitchell, F.L. van Delft, Angew. Chem. Znt. Ed., 1998, 37, 1874-1876. 72. H.M. Zuurmond, P.A.M. van der Klein, G.A. van der Marel, J.H. van Boom, Tetrahedron, 1993,49, 6501-6514. 73. K. Toshima, S. Mukaiyama, Y. Nozaki, H. Inokuchi, M. Nakata, K. Tatsuta, J. Am. Chem. SOC.,1994, 116, 9042-9051 and references therein. 74. J. Kocourek, Carbohydr. Rex, 1967, 3, 502-505. 75. A.B. Foster, J.M. Duxbury, T.D. Inch, J.M. Webber, Chem. Commun., 1967, 881-883. 76. K. Toshima, Y. Nozaki, S. Mukaiyama, T. Tamai, M. Nakata, K. Tatsuta, M. Kinoshita, J. Am. Chem. Soc., 1995,117, 3717-3727. 77. K. Toshima, T. Yoshida, S. Mukaiyama, K. Tatsuta, Carbohydr. Res., 1991, 222, 173188. 78. M. Trumtel, A. Veyrieres, P. Sinay, Tetrahedron Lett., 1989, 30, 2529-2532. 79. M. Trumtel, P. Tavecchia, A. Veyrieres, P. Sinay, Carbohydr. Rex, 1989, 191, 29-52. 80. C. Zagar, H.-D. Scharf, Carbohydr. Rex, 1993, 248, 107-118. 81. P. Tavecchia, M. Trumtel, A. Veyrieres, P. Sinay, Tetrahedron Lett., 1989, 30, 2533-2536. 82. M. Trumtel, P. Tavecchia, A. Veyrieres, P. Sinay, Carbohydr. Rex, 1990,202, 257-275. 83. T. Jyojima, N. Miyamoto, Y. Ogawa, S. Matsumura, K. Toshima, Tetrahedron Lett., 1999, 40, 5023-5026. 84. S.V. Ley, A. Armstrong, D. Diez-Martin, M. J. Ford, P. Grice, J.G. Knight, H.C. Kolb, A. Madin, C.A. Marby, S. Mukherjee, A.N. Shaw, A.M.Z. Slawin, S. Vile, A.D. White, D.J. Williams, M. Woods, J. Chem. Soc., Perkin Trans. I , 1991, 667-692. 85. C.A.A. van Boeckel, T. Beetz, S.F. van Aelst, Tetrahedron, 1984, 40, 4097-4107. 86. E. Petrakova, C.P.J. Glaudemans, Carbohydr. Res., 1995, 268, 35-46. 87a. P. Jutten, H.-D. Scharf, G. Raabe, J. Org. Chem., 1991, 56, 7144-7149. 87b. J. Szymoniak, P. Sinay, Tetrahedron Lett., 1979, 6, 545-548. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62.
References
405
88. a) H. Paulsen, W. Kutschker, 0. Lockhoff, Chem. Ber., 1981, 114, 3233-3241; b) H. Paulsen, 0. Lockhoff, Chem. Ber., 1981, 114, 3102-3114. 89. a) R.W. Binkley, D. J. Koholic, J. Org. Chem. 1989, 54, 3577-3581; b) R.W. Binkley, M.R. Sivik, J. Carbohydr. Chem., 1991, 10, 399 -416. 90. J. Jiinnemann, I. Lundt, J. Thiem, Liebiys Ann. Chem., 1991, 759-764. 91. H. Schene, H. Waldmann, Synthesis, 1999, 1411-1422. 92. K. Wiesner, T.Y.R. Tsai, H. Jin, Helu. Chzm. Acta, 1985, 68, 300-314. 93. R.W. Binkley, D.J. Koholic, J. Carhohydr. Chem., 1988, 7, 487-499. 94. J. Thiem, S. Kopper, Tetrahedron, 1990, 46, 113-138. 95. J. Borowiecka, P. Lipka, M. Michalska, Tetrahedron, 1988, 44, 2067-2076. 96. H. Bielawska, M. Michalska, Tetrahedron Lett., 1998, 39, 9761-9764. 97. M. Michalska, J. Borowiecka, J. Carhohydr. Chem., 1983, 2, 99-103. 98. L. Laupichler, H. Sajus, J. Thiem, Synthesis, 1992, 1133-1136. 99. O.J. Plante, P.H. Seeberger, J. Org. Chem., 1998, 63, 9150-9151. 100. A. Koch, C. Lamberth, F. Wetterich, B. Giese, J. Org. Chem. 1993, 58, 1083-1089. 101. H. Li, M. Chen, K. Zhao, Tetrahedron Lett., 1997,38, 6143-6144. 102. S. Hashimoto, Y. Yanagiya, T. Honda, S. Ikegami, Chem. Lett., 1992, 1511-1514. 103. S. Hashimoto, Y. Yanagiya, T. Honda, H. Harada, S. Ikegami, Tetrahedron Lett., 1992, 33, 3523-3526. 104. S. Hashimoto, A. Sano, H. Sakamoto, M. Nakdjima, Y. Yanagiya, S. Ikegami, Synlett, 1995, I27 1-1273. 105. T. MuIler, R. Schneider, R.R. Schmidt, Tetrahedron Lett., 1994, 35, 4763-4766. 106. D. Ravi, V.R. Kulkarni, H.B. Mereyala, Tetrahedron Lett., 1989, 30, 4287-4290. 107. K. Toshima, Y. Nozaki, K. Tatsuta, Tetruhedron Lett., 1991, 32, 6887-6890. 108. S. Raghavan, D. Kahne, J. Am. Chem. Soc., 1993, 115, 1580-1581. 109. J. Gildersleeve, A. Smith, K. Sakurai, S. Raghavan, D. Kahne, J. Am. Chem. Soc., 1999,121, 6176-61 82. 110. K. Suzuki, T. Mukaiyama, Chem. Lett., 1982,683-686. 111. K. Suzuki, T. Mukaiyama, Chem. Lett., 1982, 1525-1528. 112. A.G.M. Barrett, B.C.B. Bezuidenhoudt, A.R. Howell, A.C. Lee, M.A. Russell, J. Org. Chem., 1989,54, 2275- 2277. 113. A.G.M. Barrett, B.C.B. Bezuidenhoudt, L.M. Melcher, J. Org. Chem., 1990,55, 5196-5197. 114. D. Kahne, D. Yang, J.J. Lim, R. Miller, E. Paguaga, J. Am. Chem. Soc., 1988, 110, 87168717. 115. D. Crich, T.J. Ritchie, J. Chem. Soc., Chem. Commun., 1988, 1461-1463. 116. D. Crich, T.J. Ritchie, Carbohydr. Rex, 1989, 190, C3-C6. 117. D. Crich, T.J. Ritchie, J. Chem. Soc., Perkin Trans. I , 1990, 945-954.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
16 Orthogonal Strategy in Oligosaccharide Synthesis Osamu Kan ie
In the pursuit of efficient chemical synthesis of oligosaccharides, important progress has been made. Landmarks include chemoselective glycosylations and polymersupported oligosaccharide synthesis. Although polymer-supported synthesis is advantageous in respect of the isolation process, it needs to be tuned to be a more realistic prospect, especially in oligosaccharide synthesis. Also, because the final product is released along with deletion byproducts, delicate purification manipulations, e.g. by HPLC must usually be performed at the final stage. The advantage of the chemoselective activation strategy is recognized as its potential that no manipulations are required before the coupling reactions because the glycosyl acceptor has a pre-installed leaving group which also protects the anomeric center. The number of available protecting groups and chemoselective glycosylation conditions, however, limit the chemoselective sequential glycosylation strategy. This chapter addresses the problem of the chemoselective activation strategy and describes the successful introduction of a novel coupling strategy-an orthogonal glycosylation strategy in which two chemically distinct glycosylation reactions are utilized-and its application to polymer-supported oligosaccharide synthesis.
16.1 Introduction Much effort has been devoted to identification of the functions of the oligosaccharides found on the cell surface, where they covalently attached to a variety of organic molecules such as proteins and lipids. Because of the diversity of oligosaccharide structures, they are often described as the ‘finger print’ of a certain cell. The possibility that the complex structures are directly related to their functions has prompted many synthetic chemists to synthesize glycoconjugates. During the last two decades the technology for the synthesis of these structures has, therefore, improved dramatically. The key reaction in the construction of oligo-
408
16 Orthogonal Strategy in Oligosaccharide Synthesis
saccharides is, of course, the glycosylation reaction, in which stereo- and regiospecificities must be carefully controlled. There are several ways of controlling the stereochemistry of glycosidic bond formation: e.g.: 1) 1,Ztrans glycoside formation by means of a C-2 participating group [ l , 21; 2) axial glycoside formation resulting from the anomeric effect [3]; 3) equatorial glycoside formation using solvation of the carbonium cation by acetonitrile [4]; 4) axial glycoside formation by SN2 displacement of P-halides formed by halide equilibration in the presence of quaternary ammonium halide [ 51; 5) P-mannosylation by means of insoluble silver salts [6]; and 6) P-mannosylation by intramolecular aglycone delivery [71. The generalized scheme for the synthesis of oligosaccharides is shown in Figure 1 as an example of complex oligosaccharide synthesis. One must decide which method (1-6) should be applied for each coupling step before the synthesis. Also, a protecting group must be removed after each glycosylation step to release the hydroxyl function required for the next glycosylation reaction.
16.2 Analysis of the Strategic Aspects of Oligosaccharide Synthesis 16.2.1 General Aspects
To achieve efficient synthesis, both stepwise and convergent methods must be employed. A Stepwise method in which synthesis starts from the reducing end has classically been used for oligosaccharide synthesis because of the difficulty of transforming an anomeric protecting group into a leaving group. The recent development of anomeric protecting groups has enabled alternative synthesis which starts from the non-reducing end and a convergent synthesis (Figure 2). These anomeric protecting groups include the allyl, methoxyphenyl (MP), npentenyl, 2-trimethylsilylethyl (SE), t-butyldimethylsilyl, and t-butyldiphenylsilyl groups. Removal of this class of protecting groups releases the anomeric hydroxyl function to be converted into leaving groups [8].
16.2.2 The Pursuit of Efficiency in Oligosaccharide Synthesis
The other significant contribution in oligosaccharide synthesis (see Figure 3) was the introduction of ‘stable’ leaving groups which can be used as protecting groups until exposed to certain activation conditions. The thioglycosides are typical examples of this class of leaving group. Thioglycosides can be activated under alkylating
16.2 Analysis of the Strategic Aspects of Oligosaccharide Synthesis
409
Suitably protected sugar [A]
1
Anomeric deprotection
1
GI~COSYI
Activation
acceptor
Glvcosvl donor
p2d (NP) Sugar [A-B]
J Figure 1. Generalized oligosaccharide synthesis. Iterative reactions consisting of protection, deprotection, and glycosylation reaction are performed to obtain oligosaccharides. L = leaving group; P = protecting group.
or oxidative conditions but are stable under the traditional glycosylation conditions such as the Koenigs-Knorr method, so that halides can be chemoselectively activated without affecting them (Figure 3C). In addition, they can be transformed into halides or other leaving groups (Figure 3B). For this reason, thioglycosides are ideal intermediates in a flexible synthetic strategy [9]. The possibility of chemoselective activation of anomeric leaving groups has thus emerged [9-111. The armed and disarmed concept (Figure 3C), which employs a single potential leaving group, n-pentenyl glycosides, for both the donor and acceptor has been developed on the basis of the observation that the reactivity of glycosyl donors is
410
16 Orthogonal Strategy in Oligosaccharide Synthesis B
A
reducing end
m]
C
non-reducingend
product
: glyoosyl donor
Figure 2. Two stepwise syntheses (A and C ) and block condensation (B). One of the stepwise methods in which synthesis starts from the reducing end (A) has classically been used for oligosaccharide synthesis. The recent development of anomeric protecting groups has enabled alternative synthesis starting from the non-reducing end (C) and a convergent one (B).
affected by the protecting groups (i.e. ether or ester) especially at 0-2 [12a]. The utility of n-pentenyl glycosides is obvious, because small fragments of oligosaccharides can be synthesized in a very efficient manner and the n-pentenyl group can be temporarily protected as the dibromide to be used as the acceptor for block condensations. The armed and disarmed concept has proven to be applicable also to glycals [ 12b], thioglycosides [ 12~1,selenyl glycosides [ 12d], and glycosyl phosphoroamidates [ 11, 12el.
16.3 The Introduction of the Orthogonal Glycosylation Strategy 16.3.1 Limitation of Current Concepts
The advantage of the chemoselective activation strategy lies in its efficiency, because the glycosyl acceptor has a potential leaving group which also temporarily protects the anomeric center. Therefore, in principle, there is no need for deprotection or protecting group manipulations before the coupling reactions. Because the methodology relies on the relative reactivities of donor molecules, and these are controlled by the protecting groups and/or different leaving groups available, one must very carefully select the combination of leaving groups to be used, and thus this naturally imposes a limitation on this chemoselective sequential glycosylation strategy. Therefore, a different methodology which does not rely on the different reactivities of molecules is needed to reduce the complexity of the coupling process. An idea of such methodology can be derived by comparing the reactivities of glycosy1 halides and thioglycosides as an example (Figure 4).Although their reactivities are different in each group (A),there is a possibility of selective activation of halides and thioglycosides (B) in both directions, which might open a new coupling strategy.
16.3 The Introduction of the Orthogonal Glycosylution Strategy
A '$-L
41 1
B
, H O e P
O
Reactivity of leaving groups L' > L2> L3> L"
L
H O O P
Reactivity of leaving groups L1> L2(conditions A) L2> L1(conditions B)
P O G L H O G P
F
t P P O G L HO-P
O
*
G
P
t e
L
2T M S O G P
pod.,, Figure 3. Schematic representation of stepwise synthesis. (For A-D the direction of elongation is non-reducing end + reducing end and for E and F it is reducing end 3 non-reducing end. A: Most traditional method in this series (A-D) involving glycosylation reaction, deprotection of anomeric protecting group, and activation of the anomeric hydroxyl group into a donor. B: This method eliminates a deprotection step but involves direct transformation of the anomeric protecting group into a leaving group. C: This method utilizes a leaving group as the protecting group and so the number of reaction steps is minimized. This group includes the armed-disarmed and chemoselective glycosylations. D: This method also features the minimum number of steps in the synthetic scheme. It does not, however, rely on the hierarchy of the reactivities but on orthogonality and, therefore, hypothetically, eliminates the limitation of the number of coupling reactions. E: Most traditional method involving glycosylation reaction and deprotection to release the hydroxyl group for the next coupling reaction. F: This method utilizes a carefully selected combination of leaving groups and hydroxyl protecting groups, which enables the shortest, one-pot, synthetic scheme.
412
16 Orthogonal Strategy in Oligosaccharide Synthesis
labile
4
stable
silver salts
Br
l
CI
x n
5 oddative m conditions
'-Me
'\Ph
Figure 4. Comparison of the reactivities of glycosyl halides and thioglycosides as examples. Although their reactivities are different in each group (A) there is a possibility of selective activation of halides and thioglycosides (B) in both directions, which might lead to a new coupling strategy.
16.3.2 The Orthogonal Coupling Concept
The minimum requirement for this scheme would be to combine two chemically distinct glycosylation reactions in which one of the leaving groups is activated while the other behaves as a protecting group, and vice uersa (Figures 3D, 4 and 5). To fulfil the requirement for this orthogonal system, each selected leaving group should be unaffected under the conditions used to activate the other. Also, both leaving groups should tolerate routine manipulations of temporary protecting groups. For this orthogonal strategy, a set of leaving groups and activation conditions for each group-L', phenylthio group and L2, fluoride, and (a), NIS-TfOH (or AgOTf) [ 131 and (b),Cp2HfClz-AgC104 [ 141-are selected as candidates.
Figure 5. A scheme for orthogonal glycosylation. The combined use of two chemically distinct glycosylation reactions, where one of the leaving groups is activated while the other behaves as a protecting group and vice versa, enables continuous coupling without any transformations. L = leaving group; P = protecting group; a = activation condition for L1; b = activation condition for L2. N.B. L2 is stable under condition a and L' is stable under condition b.
16.3 The Introduction o j the Orthogonal Glycosylation Strategy
41 3
Figure 6. Manipulations of orthogonal protecting groups. Orthogonal protecting groups are “a set of completely independent classes of protection groups, such that each class can be removed in any order and in the presence of all other classes” as defined by Baranay and Merrifield. X, Y, Z = functional groups; A, B, C = orthogonal protecting groups; a, b, c = orthogonal deprotection conditions.
16.3.3 What is Orthogonality Anyway? The term ‘orthogonality’ is frequently used in a generalization of its mathematical meaning to describe sets of primitives or capabilities that span the entire ‘capability space’ of the system and are non-overlapping or mutually independent. The term ‘orthogonal system’ in chemistry was first defined by Baranay and Merrifield as “a set of completely independent classes of protection groups, such that each class can be removed in any order and in the presence of all other classes” (Figure 6) [ 151. Manipulations of orthogonal protecting groups are widely accepted in peptide chemistry and can also be seen in other fields [16]. Despite the importance of the idea in the synthesis of polymeric materials such as peptides, oligonucleotides, and oligosaccharides, the term is used to describe the independence of a set of protecting groups during deprotection, and is not used to describe condensation reactions. The synthesis does, therefore, require multiple steps before the coupling reactions. To reduce the number of synthetic steps, the challenge is to apply the idea of orthogonality to the reactive site of coupling. In this system, each synthon which is involved in the coupling reactions must have a functional group with orthogonal behavior in the coupling reactions, e.g. inertness and reactiveness under the given conditions. The basis of the orthogonal condensation system is the combined use of individual reactions which enable the synthesis of polymeric compounds without deprotection or activation steps, as explained above. It should, furthermore, be
414
16 Orthogonal Strategy in Oligosaccharide Synthesis
noted that the number of conditions to be employed is not limited in a system as long as the conditions are orthogonal to each other. 16.3.4 Orthogonal Glycosylation and Solid-Phase Oligosaccharide Synthesis Such systems are effectively suited to solid-phase oligosaccharide synthesis [ 171. Although the main advantage of solid-phase synthesis is the elimination of tedious purification processes, one drawback of this advantage is that each reaction conducted on the support must give nearly quantitative yield or the unreacted reaction center must be capped (again, the capping must be quantitative). These are required to ensure elimination of the possibility that there are deletion compounds in the mixture of compounds obtained. When a mixture that consists only of product and precursors for each step is given after several reaction cycles on a solid support, they can be separated by conventional purification protocols such as HPLC. If, on the other hand, the final mixture contains compounds in which some of the synthons are randomly deleted, isolation will not be a practical proposition. To avoid this problem, we usually give the above two answers; when, however, the orthogonal glycosylation strategy is applied to the solid-phase synthesis, these problems can be solved simultaneously (Figure 7), because (i) the hydrolyzed byproduct cannot be a candidate for the glycosyl donor in the next coupling cycle [17d], and (ii) the activation step is no longer necessary because the leaving group is incorporated as the protecting group for the previous reaction. Thus, in this system, one of the leaving groups is activated in the presence of hemiacetals and the other leaving group. There is, therefore, no need for capping. High yielding coupling reactions are, of course, needed but it is not necessary if one can efficiently extract the desired product from a product mixture. A tag might be incorporated at the end of the coupling reactions, because the only compound that has leaving group is the desired compound and others are presumably hydrolysis or elimination by-products formed at some stage in the orthogonal glycosylation strategy. Such tags include hydrophobic aglycones; these have proved to be extremely useful for the rapid solid-phase extraction of oligosaccharides in the assay system known as the SepPak assay [18].
16.4 The Orthogonal Glycosylation Strategy 16.4.1 Orthogonal Chain Elongation of Homo-Oligosaccharides: Synthesis of Chito-Oligosaccharides 1191 To assess the orthogonality of the above-mentioned combination of reactions, the interconversion of the two anomeric potential leaving groups phenylthio and fluoro group was performed. Thiophenyl glycoside 1 was treated under Nicolaou’s conditions [20] (u) to yield the glycosyl fluoride 2 quantitatively. The fluoride 2 could,
16.4 The Orthogonal Glycosylution Strutegy
415
Orthogonal condensation
PO Sugar [2] Polymer
-
PO Sugar [ l ] PO Sugar [3]
&O
Polymer-
Activate Y
PO
Sugar [l+]-Tag deprotection
L L O HOW*
Ho
H0-1-
L o
Ho
OX/* HO
HO
O
L o O HO w* HO
Sugar [l-n]-Tag HO etc
Figure 7. The overall idea of solid-phase oligosaccharide synthesis on the basis of the orthogonal glycosylation strategy. The orthogonal glycosylation strategy can be used in solid-phase oligosaccharide synthesis and can accelerate the process by dramatically reducing the number of reaction steps. A hydrophobic tag can be incorporated at the end of the synthesis and this is used to extract the desired compound from the accumulated by-products.
16 Orthogonal Strategy in Oligosaccharide Synthesis
416
-
OAc
.OAc
a quant
AAGO c O s S pSPh h NPhth 1
F NPhth
A&& AcO* AcO
b
2
93%
Scheme 1. An interconversion of potential donors. Reagents and conditions: (a) SnC12-AgC104PhSH in CH2C12, room temp.; (b) NBS-DAST in CHZC12, room temp.
on the other hand, be activated under Mukaiyama's conditions [21] (b)in the presence of thiophenol to give the thioglycoside 1 in 93% yield (Scheme 1). This successful interconversion reaction prompted us to investigate further. Using this orthogonal set of reactions, a set of glycosylation reactions was investigated using 1 and 2 as donors and 3 and 4 as acceptors. It should be noted here that because the selected anomeric functional groups are orthogonal, all the compounds used were synthesized from a single compound. The thioglycoside 1 was reacted with the acceptor 3 under conditions (a)to afford disaccharide 5 (90%). The fluoride 2 was also successfully activated under conditions (b) and reacted with 4, without affecting the thioglycosidic linkage, to give disaccharide 6 (78%) (Scheme 2). Self-condensed product was not detected in either sequence. These results indicate that less reactive acyl protected donors were activated in preference to the potentially more reactive ether-protected acceptors; the reactions are therefore clearly distinguishable from armed-disarmed reactions. The chosen set of reactions is therefore shown to be orthogonal; chemoselective in both directions. To show the applicability of this strategy to the synthesis of longer-chain oligosaccharides, iterative couplings were further examined by constructing the heptasaccharide 12 in which ail synthons were derived from 1. Firstly, thioglycoside donor 7 was coupled with acceptor fluoride 3 under conditions (a) to give the disaccharide fluoride 8 (85%) which was then reacted with the acceptor 4 to produce 9
Ester protected donors Ether protected acceptors
AcO AcO&Sph
NPhth
+
1
i$&&F
NPhth
8_ 90%
Ap*CO-og BnO NPhth
3
NPhth F
5
NPhth
AcO 2
+
! f o a SNPhth p h 4
&
A
NPhth BnO
NPhth ~ SPh
6
Scheme 2. A set of orthogonal reactions showing complete distinctness of the reactions used. Reagents and conditions: (a) NIS (1.3 equiv.)-AgOTf (0.1 equiv.) in CH2C12, -50 "C + room temp.; (b) Cp2HfClz (1.3 equiv.)-AgC104 (2.6 equiv.) in CH2C12, -78 "C .+ room temp.; (c) NaOMe.
16.4 The Orthogonal Glycosylution Strategy
417
[conditions (b), 72%]. Subsequent reaction of 9 with 3 [conditions (a), 65%] gave tetrasaccharide 10. Having accomplished the synthesis of a tetrasaccharide by the orthogonal strategy, we examined a block condensation approach. Tetrasaccharide acceptor 11, prepared by Zemplen deacetylation of 10, was coupled with its precursor 9 to give compound 12 [conditions (a), 670/0],which is again ready for further use as a oligosaccharide donor. The orthogonal glycosylation reactions were put together with the block condensation to show its efficiency and flexibility (Scheme 3 ) .
BnO
-+&+ -
4%
“
4
w
SNPhtP
h
-
a
65%
9
10 R = A c ‘(11 R = H
NPht
-$&++-F
12
Scheme 3. An example of the orthogonal glycosylation strategy showing only the continuous coupling reactions required to obtain the oligomer. Reagents and conditions: (a) NIS (1.3 equiv.)AgOTf (0.1 equiv.) in CH2C12, -50°C 4room temp.; (b) CpzHfClz (1.3 equiv.)-AgClOa (2.6 equiv.) in CHIC12, -78 “C + room temp.; (c) NaOMe, 91%.
4 18
16 Orthogonal Strategy in Oligosaccharide Synthesis
fj-D-GalNAc-(1 -3)-a-D-Gal-( 1-3)-p-D-Gal-( 1-3)-p-D-GalNAc-( 1-4)-fj-D-Gal-( 1-4)-fj-D-Gle( 1-)-Cer a-L-FuC(1-2)
J
13
14
Figure 8. The structure of a novel extended blood-type B glycosphingolipid (13)and the target structure (14).
16.4.2 Orthogonal Coupling for Hetero-Oligomer Synthesis [22] To verify generality of the aforementioned orthogonal system, a branched tetrasaccharide 14, a partial structure of a novel blood-type related glycosphingolipid 13 (Figure 8), was chosen as a target molecule. Four monosaccharide units 15, 16, 18, and 22 were synthesized as either glycosyl fluorides or thioglycosides to fulfil the criteria of the orthogonal coupling concept. All necessary selective protections and anomeric transformations were completed at the monosaccharide stage, thereby minimizing manipulations during elongation of the sugar chain. The protecting groups especially at C-2 position of each synthon were carefully chosen for the stereoselective glycosylation reactions (Scheme 4). The synthesis was started from the suitably protected galactosyl fluoride 15 (a : P = 1.7 : 1) which was glycosylated with GalNAc precursor 16 under conditions (a) to afford 17. The stereochemical assignment was confirmed by 'H NMR; J1,,2, % 8.5 Hz for H-1' indicated the P-D-configuration for the GalNAc residue. No anomerization of the reducing-end fluoride was observed during the glycosylation reaction, which eliminated the potential ambiguity of this strategy. The disaccharide 17 was used directly as the donor for the next glycosylation reaction with the phenylthioglycoside 18. Glycosylation under conditions (b) afforded the desired trisaccharide 19. The a configuration of the newly formed glycosidic linkage was evident from the coupling constant of the anomeric proton at 64.77 ( J = 3.6 Hz). Glycosylation of 19 with octanol was then conducted under conditions (u) to yield the octyl glycoside 20. The applicability of the current strategy was proven by showing direct orthogonal coupling reactions three times for introduction of various forms of interglycosidic linkage. Deprotection of the levulinoyl group on trisaccharide 20 by use of hydrazine acetate [23] afforded 21 (85.4%) which was then used as an acceptor for the a-
16.4 The Orthogonal Glycosylution Strutegy
419
OBn I .-Oh
AcO
SPh 18
AcO
73%
OBn 17
Octyl alcohol
19
OLev
SPh % +
Scheme 4. Synthesis of blood-type-related oligosaccharides by the orthogonal strategy. Reagents and conditions: (a) *NIS-AgOTf in CH2C12, -50 + -20°C; **NIS-AgOTf in CH2C12, 10°C; (b) Cp2HfC12-AgC104 in -20°C + room temp.; *** DMTST in benzene-CH2Cl2, 0 CH2C12-Et20, -78 "C ---t room temp.; (c) NHzNH2.AcOH; (d) (i) H2NCH2CH2NH2-EtOH, 90 "C; (ii) Ac20-MeOH-Et3N (cat.), room temp.; (e) Hz-Pd/C-MeOH, H+. ---f
420
16 Orthogonal Strategy in Oligosaccharide Synthesis
fucosylation reaction. Thus the levulinoyl group was used not only as a participating group to yield the P-glycoside but also as a selectively removable protecting group. The coupling reaction of 21 using methylthio glycoside 22 [24] gave the desired tetrasaccharide (88%). Ethylenediamine [25]treatment of the tetrasaccharide then selective acetylation of the resulting amine yielded 14 (85%) after hydrogenolysis under acidic conditions. In the same manner, compound 21 was deprotected to afford 25. We have demonstrated the applicability of the orthogonal glycosylation strategy to the synthesis of a branched oligosaccharide with four different types of glycosidic linkage. The synthesis of the target tetrasaccharide 14 was thus achieved in seven steps (38%) overall yield) from protected monosaccharide units. With the trisaccharide 25 and the known blood-type B trisaccharide available it might be possible to utilize these oligosaccharides as probes to investigate an unknown biosynthetic pathway. 16.4.3 Application to Polymer-Supported Synthesis 1261 The elongation of the sugar chain from the non-reducing end in polymer-supported oligosaccharide synthesis is less straightforward, mainly for two reasons: 1) accumulation of by-products on the support together with the desired compound; and 2) a transformation reaction must be performed to obtain the glycosyl donor. When, however, the orthogonal glycosylation strategy is applied to polymersupported synthesis, an important improvement, namely pre-installation of a potential leaving group, is achieved. This has potential to make the methodology more advantageous-because the leaving group is attached, there will be no need for the activation reaction and no need to protect the anomeric hydroxyl group produced by hydrolysis. As a result, again only one reaction step is required per cycle; this is the same as was shown for solution synthesis. In addition, because only the last introduced sugar unit has a leaving group, one can introduce a tag to ease isolation of the final product after deprotection. The recoverability of excess glycosyl acceptor used in the coupling reaction should be noted in addition to the above advantages as a common feature of polymer-supported synthesis starting from non-reducing end terminus. To test this hypothesis, the first attempt was made using ‘soluble’ poly(ethy1ene glycol) (PEG) as the polymer [17b]; the trimannoside portion (27) of the highmannose type glycoprotein (26) was chosen as the target. In addition, the 2-(trimethylsily1)ethyl (SE) group was used as a hydrophobic tag [27] in this case in addition to the temporary protecting group [8d,e] (Figure 9). Initial coupling of the polymer, PEG monomethyl ether (average MW = 5000)supported methylthiomannoside 28 with the fluoride 29 was performed in the presence of DMTST, prepared in situ from MeOSOzCF3 and MeSSMe [conditions (a)], to afford the disaccharide-PEG conjugate 30 in 89% yield. Subsequent coupling with the reducing-end mannoside was performed by using SE-glycoside 31 as aglycone under conditions (b)to afford the trisaccharide-PEG conjugates 32.
16.5 Conclusions and Prospects a-0-Man-(1-2))-a-D-Man-(l-6)
42 1
1
a-D-Man-(1-6) a-D-Man-(l-2)-a-D-Man-(l-6) p-D-Man-(l-.+p-D-GicNAc-(
1
1-.4)-B-D-GlcNAc-Asn-Protein
a-D-Man-(l-2)-a-D-Man-(l-+2)-a-D-Man-( 1 ’ 3 ) -1 26
I
27
OSE
Figure 9. The structures of the high mannose-type N-linked oligosaccharide 26 and the target tri. mannoside 27.
Having shown the construction of a trisaccharide sequence, PEG was cleaved off under basic conditions and the crude trisaccharide was hydrogenated over Pd(OH)*. The resulting mixture was then applied to a short column of CISreversed-phase silica gel, which was first eluted with water to elute all by-products. Subsequent elution with 50% MeOH gave a mixture of trisaccharides from which 27 was isolated by eluting with a linear gradient from water to 50% MeOH (Scheme 5). This strategy, featuring a novel combination of polymer-support chemistry, the concept of orthogonal glycosylation, and simple isolation by incorporating a hydrophobic tag has proven successful. It should also be noted that the aglycone in 27, which is used as a hydrophobic tag, can be selectively cleaved if one wants to conjugate the formed oligosaccharide with other structures. One expansion of this strategy can be seen in connection with the intramolecular aglycone delivery approach, where a polymer-substituted benzyl group was used as both the linker and the tether [28] (Scheme 6 ) . Thus, methylthiomannoside 33 was coupled with a fluorinated GlcNAc derivative 3 to yield only P-linked mannoside which can be used as an acceptor itself or as a donor after protection of released 2OH. The advantage of this particular use is that the only compound to be released from the polymer is the desired product.
16.5 Conclusions and Prospects An orthogonal glycosylation strategy has been developed by combined use of phenylthioglycosides and glycosyl fluorides both as donors and acceptors. By use of this
422
16 Orthogonal Strategy in Oligosaccharide Synthesis
BnO BnO BnO BnO
30
SEO :
BnO BnO
'siWo 'I
32
OSE
H O T OH
HO HO
HO
OH
HO
HO *H r HO
Wafer eluent
27
OSE
Organic (alcohollc) eluent Scheme 5. Polymer-supported synthesis of trimannoside on the basis of the orthogonal strategy. Reagents and conditions: (a) DMTST in CHzC12, room temp., (b) CpzHfClz-AgOTf in CH2CI2, 0 "C + room temp., (c) (i) NaOMe; (ii) Hz-Pd(OH)z in MeOH-EtOAc. *Yields were calculated on a weight basis.
16.5 Conclusions and Prospects
423
SMle
34
,OBn
lb TBDPSO
BnO
NPhth
36
NPhth
d 85%
35 pTBDPSO hTo0?~*+;& NPhth
N3 NPMh
37
Scheme 6. Combination of polymer-supported chemistry, P-mannosylation, and orthogonal strategy. Reagents and conditions: (a) DDQ, 4 A mol. sieves in CH2C12, room temp., (b) AczO-Pyr., 48% from 3, (c) Cp*HfClz-AgOTf in ClCH2CH2C1, -15°C + room temp.
strategy, syntheses of several oligosaccharides have been achieved. As was clearly demonstrated in the synthesis of the chito-oligosaccharide, the novel strategy is free from the effects of the protecting groups, unlike the armed-disarmed concept. The usefulness of the strategy is obvious, because by use of this method an oligosaccharide can be synthesized highly efficiently with the lowest number of reactions. The syntheses of homo- and hetero-oligosaccharides has been demonstrated successfully. Further, the strategy was also examined for polymer-supported synthesis; this showed the potential of the methodology in solid-phase oligosaccharide synthesis. A hydrophobic tag was incorporated at the end of the synthesis to demonstrate the idea of simple isolation of the final product and/or the possibility of direct use of the isolated material for biological evaluations. The orthogonal coupling strategy can be applied to the synthesis of other polymeric compounds. Several approaches have been reported for construction of polymeric and dendritic structures on the basis of the orthogonal coupling strategy [29] (Figure 10).
424
16 Orthogonal Strategy in Oligosaccharide Synthesis
conditions a
PPhB - DEAD
b
Pd(PPh,)$l2
-
- CUI
Pd(0Ac)z - P(o-tOlyl), NBu3
Pd2(dba)3- DPPF - NaOtBu
NaH
Et3N DMAP
-
Figure 10. Some examples of the orthogonal coupling strategy in the synthesis of polymers and dendnmers.
Acknowledgments
The author thanks Professor Tomoya Ogawa and Dr. Yukishige Ito for their continuous encouragement and suggestions. Professors Hans Paulsen, Ole Hindsgaul, Robert Field, Chi-Huey Wong, Makoto Kiso, Tadahiro Takeda, and Dr. Frank Barresi are also acknowledged for their encouragement. Most of all, the author would like to express his sincere respect and gratitude to the late Professor Akira Hasegawa. References 1. (a) Igarashi, K. The Koenigs-Knorr reactions. In: Adu. Curbohydr. Chem. Biochem., 1977, 34, pp. 243-283. (b) Paulsen, H. Angew. Chem. Znt. Ed. Enyl., 1982, 21, 155-175. 2. (a) Lemieux, R. U., Takeda, T., Chung, B. Y. A. C.S. Symp. Ser., 1976, 4, 90-1 15. (b) Ito, Y., Ogawa, T. Tetrahedron. 1990, 46, 89-102. (c) Okamoto, K., Goto, T. Tetrahedron. 1990, 46,
5835-5857. 3. Lemieux, R. U. In: Molecular Rearrangements, de Mayo, P. Ed., Interscience, New York, 1964, p. 709. 4. Brccini, I., Derouet, C., Esnault, J., Heme du Penhoat, C., Mallet, J.-M., Michon, V., Sinay, P. Carbohydr. Res., 1993, 246, 23-41. and references cited therein. 5. Lemieux, R. U., Hendriks, K. B., Stick, R. V., James, K. J. Am. Chem. Soc., 1975, 97,40564062. 6. (a) Paulsen, H., Lockhoff, 0. Chem. Ber., 1981,114, 3102-3114. (b) Garegg, P. J., Ossowski, P. Acta Chem. Scand., 1983, B37, 249-250. (c) Van Boeckel, C. A. A., Beetz, T., van Aelst, S. F. Tetrahedron, 1984, 40, 4097-4107. 7. (a) Barresi, F., Hindsgaul, 0. J. Am. Chem. Soc., 1991, 113, 9376-9377. (b) Stork, G., Kim, G. J. Am. Chem. Soc., 1992, 114, 1087-1088. (c) Barresi, F., Hindsgaul, 0. Synlett, 1992, 759761. (d) Bols, M. Tetrahedron, 1993, 49, 10049-10060. (e) Barresi, F., Hindsgaul, 0. Can. J. Chem., 1994, 72, 1447-1465. (f) Ito, Y., Ogawa, T. Angew. Chem. Int. Ed. Engl., 1994, 33, 116551767.
References
425
8. (a) Sugimoto, M., Ogawa, T. Glycoconjugate J., 1985, 2, 5-9. (b) Murakata, C., Ogawa, T. Carbohydr. Rex; 1992, 235, 95-1 14. (c) Fraser-Reid, B., Udodong, U. E., Wu, Z.: Ottosson, H., Merritt, J. R., Rao, C. S., Roberts, C., Madsen, R. SYNLETT., 1992, 927-942. (d) Jansson, K., Frejd, T., Kihlberg, J., Magnusson, G. Tetrahedron Lett., 1988, 29, 361-362. (e) Jansson, K., Ahlfors, S., Frejd, T., Kihlberg, J., Magnusson, G. J. Org. Chem., 1988, 53, 56295647. (f) Murase, T., Kameyama, A,, Kartha, K. P. R., Ishida, H., Kiso, M., Hasegawa, A. J. Carbohydr. Chem., 1989, 8, 265-283. (g) Kanie, O., Takeda, T., Ogihara, Y. Carbohydr. Res., 1989,190, 53-64. (h) Kinzy, W., Schmidt, R. R. Liebigs Ann. Chem., 1985, 1537-1545. (i) Nakahara, Y., Ogawa, T. Tetrahedron Lett., 1987,28, 2731-2734. 9. (a) Koto, S., Uchida, T., Zen, S. Bull. Chem. Soc. Jpn., 1973, 46, 2520-2523. (b) Nicolaou, K. C., Dolle, R. E., Papahatjis, D. P., Randall, J. L. J. Am. Chem. Soc., 1984, 106, 4189-4192. (c) Lonn, H. Carbohydr. Rex, 1985, 139, 105-113. (d) Fiigedi, P., Garegg, P. J., Lonn, H., Norberg, T. GlycoconjugateJ., 1987, 4, 97-108. ( e ) Nilsson, M., Norberg, E. Carhohydr. Rex, 1988, 183, 71-82. 10. Chemoselective glycosylation reaction in general: (a) Paulsen, H.; Tietz, H. Carbohydr. Rex, 1985,144, 205-229. (b) Friesen, R. W., Danishefsky, S. J. J. Am. Chem. Soc., 1989,111, 66566660. (c) Trumtel, M., Veyrieres, A., Sinay, P. Tetrahedron Lett., 1989, 30, 2529-2532. (d) Veeneman, G. H., van Boom, J. H. Tetrahedron Lett., 1990,31,275-278. (e) Veeneman, G. H., van Leeuwen, S. H., Zuurmond, H., van Boom, J. H. J. Carbohydr. Chem., 1990, 9, 783-796. (f) Mori, M., Ito, Y., Uzawa, J., Ogawa, T. Tetrahedron Lett., 1990, 31, 3191-3194. (g) Mehta, S., Pinto, B. M. Tetrahedron Lett., 1991, 32, 4435 4438. (h) Marra, A,, Gauffeny, F., Sinay, P. Tetrahedron, 1991,47, 5149-5160. (i) Zegelaar-Jaarsveld, K., van der Marel, G. A,, van Boom, J. H. Tetrahedron, 1992, 48, 10133-10148. (j) Jain, R. K., Matta, K. L. Carbohydr. Rex, 1992, 226, 91-100. (k) Marra, A., Esnault, J., Veyrieres, A., Sinay, P. J. Am. Chem. Soc., 1992, 114, 6354-6360. (1) Hashimoto, S., Yanagiya, Y., Honda, T., Kobayashi, H., Ikegami, S. Presented at 18th Symposium on Progress in Organic Reactions and Syntheses-Applications in the Life Sciences, Sapporo, Japan, October 1992, Abstr. pp 276-280. (m) Raghavan, S., Kahne, D. J. Am. Chem. Soc., 1993, 115, 1580-1581. (n) Mehta, S., Pinto, B. M. J. Org. Chem., 1993, 58, 3269-3276. ( 0 ) Sliedregt, L. A. J. M., Zegelaar-Jaarsveld, K., van der Marel, G. A., van Boom, J. H. SYNLETT, 1993, 335-337. (p) Yamada, H., Harada, T., Miyazaki, H., Takahashi, T. Tetrahedron Lett., 1994, 35, 3979-3982. (4) Yamada, H., Harada, T., Takahashi, T. J. Am. Chem. Soc., 1994, 116, 7919-7920. (r) Chenault, H. K., Castro, A. Tetrahedron Lett., 1994, 35, 9 145-9148. 11. Latent-active strategy: (a) Roy, R., Andersson, F. O., Letellier, M. Tetrahedron Lett., 1992, 33, 6053-6056. (b) Boons, G.-J., Isles, S. Tetrahedron Lett., 1994, 35, 3593-3596. 12. Armed-disarmed strategy: (a) Mootoo, D. R., Konradsson, P., Udodong, U., Fraser-Reid, B. J. Am. Chem. Soc., 1988, 110, 5583-5584. (b) Friesen, R. W., Danishefsky, S. J. J. Am. Chem. Soc., 1989: I l l , 6656-6666. (c) Veeneman, G. H., van Leeuwen, S. H., van Boom, J. H. Tetrahedron Lett., 1990, 31, 1331-1334. (d) Grice, P., Ley, S. V., Pietruszka, J., Priepke, H. W. M., Walther, E. P. E. Synlett, 1995, 781-784. (e) Hashimoto, S., Sakamoto, H., Honda, T., Abe, H., Nakamura, S., Ikegami, S. Tetrahedron Lett., 1997, 38, 8969-8972. 13. (a) Konradsson, P., Mootoo, D. R., McDevitt, R. E., Fraser-Reid, B. J. Chem. Soc., Chem. Commun., 1990, 270-272. (b) Veeneman, G. H., van Leeuwen, S. H., van Boom, J. H. Tetrahedron Lett., 1990,31, 1331-1334. (c) Konradsson, P., Udodong, U. E., Fraser-Reid, B. Tetrahedron Lett., 1990,31,4313-43 16. 14. (a) Suzuki, K., Maeta,; H. Matsumoto, T., late Tsuchihashi, G. Tetrahedron Lett., 1988, 29, 3571-3574. (b) Suzuki, K., Maeta, H., Matsumoto, T. Tetrahedron Lett., 1989,30, 4853-4856. 15. (a) Barany, G., Merrifield, R. B. J. Am. Chern. Soc., 1977, 99, 7363-7365. (b) Barany, G., Merrifield, R. B. J. Am. Chem. Soc., 1980, 102, 3084-3095. 16. Schelhaas, M.: Waldmann, H. Angeiv. Chem. Znt. Ed. Engl., 1996, 35, 2056-2083. 17. For the recent advances of the solid-phase ohgosaccharide synthesis, see: (a) Yan, L., Taylor, C. M., Goodnow Jr., R., Kahne, D. J. Am. Chem. Soc. 1994,116, 6953-6954. (b) Douglas, S. P., Whitfield, D., Krepinsky, J. J. J. Am. Cliem. Soc. 1994, 117, 2116-2117. (c) Schuster, M., Wang, P., Paulson, J. C., Wong, C.-H. J. Am. Chem. Soc. 1994, 116, 1135-1136. (d) Randolph, J. T., McClure, K. F., Danishefsky, S. J. J. Am. Chem. Soc. 1995, 117, 5712-5719. (e) Rademann, J., Schumidt, R. R. Tetrahedron Lett. 1996, 37, 3989-3990. (f ) Shimizu, H., Ito, Y . ,
426
18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
16 Orthogonal Strategy in Oligosaccharide Synthesis
Kanie, O., Ogawa, T. Bioorg. Med. Chem. Lett. 1996, 6, 2841-2846. (8) Nicolaou, K. C., Winssinger, N., Pastor, J., DeRoose, F. J. Am. Chem. Soc. 1997, 119, 449-450. Palcic, M. M., Heerze, L. D., Pierce, M., Hindsgaul 0. GlycoconjugateJ. 1988, 5, 49-63. Kanie, O., Ito, Y., Ogawa, T. J. Am. Chem. Soc., 1994, 116, 12073-12074. Nicolaou, K. C., Caulfield, T. J., Kataoka, H., Stylianides, N. A. J. Am. Chem. Soc., 1990, 112, 3693-3695. Mukaiyama, T., Murai, Y., Shoda, S. Chem. Lett., 1981, 431-432. Kanie, O., Ito, Y., Ogawa, T. Tetrahedron Letters, 1996, 37, 4551-4554. Koeners, H. J., Verhoeven, J., van Boom, J. H. Rec. Trav. Chim. Pays-Bas., 1981, 100, 65-72. Sato, S., Ito, Y., Nukada, T., Nakahara, Y., Ogawa, T. Carbohydr. Rex, 1987, 167, 197-210. Kanie, O., Crawley, S. C., Palcic, M. M., Hindsgaul, 0. Carbohydr. Res., 1993, 243, 139-164. Ito, Y., Kanie, O., Ogawa, T. Angew. Chem. Int. Ed. Engl., 1996, 35, 2510-2512. Ito, Y., Paulson, J. C. J. Am. Chem. Soc. 1993, 115, 1603-1604. Ito, Y., Ogawa, T. J. Am. Chem. Soc. 1997, 119, 5562-5566. (a) Zeng, F., Zimmermann, S. C. J. Am. Chem. Soc. 1996,118,5326-5327. (b) Maddux, T., Li, W., Yu, L. J. Am. Chem. Soc. 1997, 119, 844-845. (c) Deb, S. K., Maddux, T. M., Yu, L. J. Am. Chem. Soc. 1997, 119, 9079-9080. (d) Frost, C. G., Mendonp, P. Chem. Lett. 1997, 1 159-1 160.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
17 Protecting Groups: Effects on Reactivity, Glycosylation Stereoselectivity, and Coupling Efficiency Luke G. Green and Steven V. Ley
17.1 Introduction In general, protecting groups are seen as an expedient, performing the uninteresting task of masking functionality that would otherwise interfere with a carefully planned strategy. A protecting group or protecting group strategy is usually chosen on the basis of its compatibility (in protection, deprotection and lability to other transformations), selectivity (in protection) and sequence (in deprotection when multiple protecting groups are used) [I]. Ultimately the same criteria apply in oligosaccharide assembly where the protecting group pattern is principally dictated by the target structure. Even within these constraints there are, however, options which can mean the difference between success and failure, efficiency and tedium. Protecting groups are not innocent bystanders in carbohydrate chemistry and to relegate them in the synthesis design is foolhardy. It is not the purpose of this chapter to review methods for installing and removing carbohydrate protecting groups, because this has already been covered by other texts [2]. Rather, it is intended to outline the mechanisms by which they effect glycosylations thereby enabling readers to decide which protecting group strategy best serves their purpose. Before investigating the roles various protecting groups can play it is pertinent to analyze briefly the glycosidic mechanism, because this is central to oligosaccharide assembly processes. There have been many detailed studies of the processes that occur during a glycosylation [ 3 ] but much of the evidence used to substantiate proposed inter-glycosyl coupling mechanisms is anecdotal or circumstantial. Monitoring actual glycosylation reactions is complex and often not very illuminating, given the number of factors involved and the instability of the intermediates. The mechanism presented here is not intended to be definitive but serves well as a working model.
428
I7 Protecting Groups
17.2 Glycosidic Mechanism The first rule is that glycosylations do not, under most reaction conditions, proceed through a free oxonium ion in an s N 1 manner [4]. Jencks has estimated the lifetime s, a time too short to of a glycosyl oxonium ion in water to be approximately enable solvent equilibration. It therefore seems unlikely that in typical, low-polarity glycosylation solvents the ion-pairs can be separated sufficiently to generate the free glycosyl oxonium ion as an intermediate. Hence, glycosylations can be considered as proceeding through an 'exploded' transition state in which the incipient oxacarbenium moiety is stabilized electrostatically by both entering and leaving groups [ 5 ] , as in Capand CBataken from Lemieux's mechanism (Figure 1) [6].
R.
*.
!?
X
Aa
Ba
R.
Figure 1. Lemieux's glycosidic mechanism.
17.2 Glycosidic Mechanism
429
Lemieux's mechanism (based on earlier work by Rhind-Tutt and Vernon [7], and Ishikawa and Fletcher [S]) was put forward to explain the high u-selectivity observed on alcoholysis of anomeric bromides with an ether-type protecting group on C-2. The first step involves dissociation of the glycosyl bromides to tight ion-pairs (B) [9]. This process is promoted by donation from the oxygen lone pair (n) into the carbon oxygen antibonding orbital (o*), requiring the two orbitals to be appropriately aligned [ 101. For the u-halide, with the required orbitals naturally in an antiperiplanar arrangement, this interaction only results in flattening of the pyran ring as the oxygen takes on more sp2 character. For the (3-halide, however, the sugar must reorganize to a boat conformation (AD) to place the orbitals anti-periplanar (there is mounting evidence that the reaction probably proceeds through a synperiplanar arrangement via the lower energy twist-boat formation) [ 111. The resulting ion pairs are then intercepted by the acceptor alcohol (SOH) with inversion, in the rate limiting step, leading to the protonated glycosides (E). The product from the a-halide pathway proceeds through a boat-like intermediate (Ep, whereas the (3-halide leads to a lower energy chair-like intermediate (Ea). This smaller activation barrier, coupled with the higher ground-state energy of the (3-halide (relative to the anomerically stabilized u anomer), more than compensate the energy penalty incurred in aligning the oxygen lone pair with the antibonding orbital (CurtinHammet principle) [ 121. Hence the p-bromide is more reactive than the u-bromide. Even though the u-bromide is predominant (as it is the more thermodynamically stabilized species) interconversion between u- and (3-bromides (C ) is considerably faster than glycosylation (especially when lowly nucleophilic alcohols such as glycosy1 acceptors are used and a source of bromide ion is present-% situ anomerization protocol') [6] and glycosylation occurs preferentially on the more reactive (3bromide, resulting in the u-glycoside as the major product. This is one of the earliest examples of a dynamic kinetic resolution. This mechanism is readily translated to other glycosylation systems if an activation step is added (Figure 2). The leaving group (X) is initially coordinated by the activating group (M+Y,-) to form a catalyst-coordinated ion-pair. Although this pair can be attacked by the alcohol directly, affording products with inverted stereochemistry of the initial ROU
Figure 2. Generalized glycosylation process.
430
17 Protecting Groups
leaving group, in general [ 131, this does not occur and anion exchange occurs before alcohol attack. Schuerch first demonstrated that silver triflate activation of glycosyl halides generates glycosyl triflates [ 141 and it has been shown recently that anion exchange occurs in a variety of other systems [15]. As with the bromide, any functionality sufficiently reactive to act as a leaving group will not be configurationally stable and the same mechanism for glycoside formation applies. The a-selectivity of these reactions is not, however, usually as consistent as for the 'in situ anomerization protocol' of anomeric bromides.
17.3 Electronic and Torsional Effects The first step of any glycosylation involves dissociation into ion-pairs, a process which results in reorganization of the glycoside and the development of a positive charge on the ring oxygen. Clearly, any factors that influence this process will have profound consequences on the outcome of a glycosylation. Riiber et al. noticed that the removal of the oxygen at C2 of glycosides greatly increased the rate at which the methyl glycosides were hydrolysed (Scheme 1) [ 161. Overend et al. [17], amongst others [18], further investigated the effects of deoxygenation at other positions on the rates of hydrolysis of methyl glycosides (Table 1). Richards proposed the explanation that the oxygen atoms, owing to their electronegative nature, inductively destabilize the developing positive charge required for the formation of the ion-pairs (B) [ 181. Hence, their removal results in an increase in the rate of hydrolysis, with the C2 oxygen, because it is nearest the reacting center, having the greatest effect. Even the C6 can, however, affect the reaction, as is evi-
HO
H+
I
*
&HH
OMe
OH
Scheme 1. Reagents and conditions: HCl(aql.
Table 1. Rates of hydrolysis of methyl glucopyranosides at 58 "C in 0.01 M HCI. Glucopyranoside
Relative rate
Methyl-a-D-glucopyranoside Methyl-2-deoxy-a-~-glucopyranoside Methyl-3-deoxy-a-~-glucopyranoside Methyl-4-deoxy-a-~-glucopyranoside
1 2090 20 40
17.4 Influence o j Protecting Group on Donor Reactivity
43 1
Table 2. Comparison of the rates of hydrolysis of methyl glycosides and their 6-deoxy analogs. GIycoside
Rate relative to Me-a-D-glucopyranoside
Methyl-a-D-mannopyranoside Methyl-a-L-rhamnopyranoside Methyl-a-D-galactopyranoside Methyl-6-deoxy-a-~-galactopyranoside
2.4 8.3 5.2 28.2
Figure 3. Torsional strain in the incipient oxonium ion
dent from comparing the rates for methyl-a-D-mannose and methyl-a-L-rhamnose [19], or for methyl-a-D-galactose and its 6-deoxy analog [17] (Table 2). The enhanced lability of the methyl-6-deoxygalactopyranosideis mirrored by the acid-sensitivity of a-fucosyl linkages in general [20]. Another pattern that emerges from these data is the relative reactivities of the differing hexoses-galactosides > mannosides > glucosides. Although there are many factors responsible for this sequence (ground state energies in particular) one contributing factor, as Edwards suggested [2I], arises from the eclipsing interactions that develop between the C2-C3 and C4-C5 substituents, disfavoring formation of the ion-pairs (Figure 3 ) . An axial substituent will interact less with its neighbor and therefore provide less resistance to the conformational reorganization. Overend et al. later argued that the larger substituent on C5 results in the C4-C5 interaction being more influential than the C2ZC3 interaction [ 171, purportedly explaining why galactosides were more reactive than mannosides. Although this explanation is incomplete in rationalizing the complex processes involved in glycoside activation, it was the first recognition that torsional effects could be a controlling influence in a glycosylation; an idea that did not resurface for many years [22]. Inductive effects, however, were all too evident to early researchers; Fletcher et al. demonstrated that increasing numbers of ester protecting groups reduced the reactivity of glycosyl halides under methanolysis conditions (Table 3 ) [23].
17.4 Influence of Protecting Group on Donor Reactivity The origin of t h s effect lies in the destabilizing effect of the exocyclic ring oxygens on ion-pair formation. An ester protecting group, owing to its electron-withdrawing
432
17 Protecting Groups
BnO
1
3
2
Table 3. Influence of protecting group on the rates of methanolysis of arabinosyl chlorides. Substrate
Relative rate of methanolysis
1
106
2 3
13 1
nature, increases the electronegativity of the oxygen it is protecting thereby increasing the deactivating effect of that oxygen. This deactivation effect translates to increased resistance to incipient oxonium ion formation i. e. the leaving group is stabilized. In contrast, a benzyl group does not disturb the oxygen in the same manner (not being electron withdrawing) and thus the fugacity of the leaving group is untempered. Ley et al. have investigated this effect in detail, quantifying the individual contributions of a variety of protecting groups to the different hydroxyl positions of rhamnosides and mannosides [24]. Two differently protected glycosyl donors were made to compete for a standard glycosyl acceptor (Scheme 2). A carbohydrate
Donor A 2 equiv.
Donor 0 2 equiv.
Disaccharide AC
Acceptor C (R'OH) 1 equiv.
Disaccharide BC
Scheme 2. Reagents and conditions: NIS (N-Iodosuccinimide), TfOH (cat.), 4 (CH2C1)2-Et20.
A molecular sieves,
17.4 Influence of Protecting Group on Donor Reactivity
433
Table 4. The possible protecting group patterns of ethyl 1-thio-a-L-rhamnoside. R'
R2
R3
Bn BZ Bn Bn Bz BZ Bn BZ Bn
Bn Bn BZ Bn BZ Bn BZ BZ CDA
Bn Bn Bn BZ Bn BZ BZ BZ
acceptor was specifically chosen to simulate actual glycosylation conditions because the steric profile of the alcohol can influence reaction kinetics through variations in its nucleophilicity [26]. All possible combinations of benzyl and benzoyl protecting group patterns were prepared (Table 4). The torsionally deactivating protecting group cyclohexane-1,2diacetal (CDA), introduced for the selective protection of trans diequatorial 1,2diols (Scheme 3) [25], was also included in the survey of donor activity. The rigidity imposed by the fused cyclic acetals resists the conformational changes required for glycosylation, hence the reactivity of the sugar is diminished. The ratio of the product disaccharides AC and BC was determined by 'H NMR (each disaccharide had already been prepared discretely) thus providing a quantitative analysis of reactivity of the various protecting group patterns (Table 5). Not surprisingly the amount of electronic deactivation arising from a benzoyl protecting group varies with proximity to the ring oxygen/reacting center in a manner similar to that in the deoxygenation experiments of Overend et al. (cf. Table 1); i.e. the order of importance is C2 > C4 > C3 [27]. The torsional deactivation effect from the CDA group is marked, being slightly greater than a benzoate at C2 and slightly less than the 3,4-dibenzoyl compound. As a simplifying approximation, the individual contributions from each protecting group can be treated as being independent of any other protecting groups in the system. Although the validity of this approximation is questionable [28] it does
Me0 i
55% UH
--
D
OH -. .
M e0-
Scheme 3. Reagents and conditions: 1,1,2,2-tetramethoxycyclohexane,CSA (1 0-Camphorsulfonic acid), CH(OMe)3, MeOH.
434
I7 Protecting Groups
Table 5. Selected results from competition experiments. Donor A
Donor B
Quotient AC/BC
TriBn TriBn TriBn 2,4-DiBz 2,3-DiBz 3,4-DiBz 3,4-DiBz 3,4-CDA
3-BZ 4-BZ 2-BZ TriBz TriBz TriBz 3,4-CDA 2-BZ
3.1 8.9 26.6 2.5 13.0 24.2 1.7 1.7
enable semi-quantitative prediction of donor reactivities by multiplication of the quotients or deactivation factors (DFs) for each position. For example: reactivity of 2,4-DiBz (Di-benzoyl) = 8.9 x 26.6 = 236.7 reactivity of 2,3,4-TriBz (Tri-benzoyl) = 3.1 x 8.9 x 26.6 = 733.9 therefore ratio of 2,4-DiBz/TriBz = 733.9/236.7 = 3.1 observed ratio = 2.5 That the predicted result is of similar magnitude to the observed value is indicative of the usefulness of this approximation; the difference cannot, however, be explained away merely as statistical error and should in part be attributed to the failure of the approximation. A similar set of competition experiments was performed for thiomannosides which included the second generation of diacetal protecting groups; butane diacetal (BDA) [29] (Table 6). The relative importance of the various positions is unchanged but C6 emerges as the position of most significance after C2, which could be further tuned by varying the electronics of the benzoate. Making the benzoate more electron rich with a donating p-methoxy group reduced the deactivation; conversely the
Table 6. Standardized DFs for non-Bn protecting groups on thiomannosides. Position of non-Bn group
DF
3-BZ 4-BZ 6-(p-MeOBz) 6-BZ 6-(p-N02Bz) 3,4-CDA 3,4-BDA 2-BZ
1.1 5.0 6.6 8.2 18.9 13.9 16.5 33.6
17.4 Influence o j Protecting Group on Donor Reactivity
BzO BzQ
BzO Bz&
BZO
1.o
To’ BZO
BzO Bz&
2.3
435
To1 HO
3.1
BzO
5.1
11.8
Figure 4. Relative reactivities of selected thiogalactosides.
more electron deficient p-nitro group served to increase the deactivation. Again the torsional effects of the diacetal protecting groups are amply demonstrated, reducing the reactivity of the donor by a factor of around 15. Wong et al. have investigated protecting group effects on p-methylphenyl thiogalactosides and p-methylphenyl thioglucosamines employing a similar competition strategy except with the significant difference of using methanol as the acceptor alcohol [28]. This facilitated examination of effects of hydroxyl groups on donor reactivity by suppressing homocoupling that might otherwise occur if a less nucleophilic acceptor (e.g. carbohydrate alcohol) was used. Curiously, the order of importance of the positions seems to be dramatically altered with C2 and C6 being demoted in favor of C4 and C3 (Figure 4).This result seems to be at odds with the pattern so far established in this chapter, even allowing for the variations that might be expected from changes in stereochemistry of the hydroxyl and leaving group (axial uersus equatorial). Whether this is particular to galactose [30] or is the result of comparing a benzoyl group with a hydroxyl function whose ability to hydrogen bond (providing some torsional resistance) will vary with position and adjacent protecting groups, has yet to be determined. It is, however, clear that a hydroxyl group can be considered as more activating than a benzyl protecting group (Figure 5).
pP
pP Bnb
1.o
2.8
Figure 5. Relative reactivities of a benzylated and an unprotected thiogalactoside.
436
17 Protecting Groups
Wong et al. also demonstrated that the deactivating power of the different electron withdrawing groups investigated was -=Nj> -0AcCl > -NPhth > -0Bz > NHTroc (Troc = 2,2,2-trichloroethurycurbonyl> -0Bn. It should now be clear that protecting groups profoundly affect the reactivities of carbohydrates through torsional and electronic effects. The size of this effect varies with the position and nature of the protecting group and for multiple protecting groups the effects are additive. The reactivity difference can be significant enough to enable successful competitive coupling of two sugars containing identical leaving groups (see Chapters 6(?) and 19(?)).This reactivity tuning can also influence the stereoselectivity and yield of a glycosylation.
17.5 Stereoselectivity 17.5.1 Neighboring-Group Participation The most obvious way in which a protecting group can influence the stereoselectivity of a glycosylation is through anchimeric assistance (neighboring-group participation) [31]. When there is a carbonyl functionality present at C2 [32] (e.g. ester, carbonate, phthalimide, etc.) it can attack the incipient oxonium ion to form the thermodynamically more stable dioxonium species 5 (Figure 6). The position of this equilibrium will depend upon the nucleophilicity of the carbony1 oxygen which varies with the electronics of substituent R. A trichloroacetate, for example, cannot participate whereas a rnonochloroacetate does, but with lower efficiency than a simple acetate [33]. Attack by an alcohol on 5 occurs anti to the carbonyl bond resulting in exclusive generation of the trans glycoside [34]. Hence, donors bearing a 2-acyl protecting group react to give predominantly trans glycosides as they react through the lower energy dioxonium species. If, however, there are severe steric clashes (vide infra Section 17.7) between donor and acceptor that disfavor reaction on the dioxonium species, reaction will instead occur on the incipient oxonium ion with concomitant loss of stereoselectivity [35]. This problem can sometimes be resolved by deactivating the donor; stabilizing the sugar makes intermolecular attack on the incipient
*x
?R
-
-% Lo
4
Figure 6. Anchimeric stabilization.
x-
R
5
437
17.5 Stereoselectivity
Ac
AcA&B & :sph AcO
Ac
P hthN
6
Bn
+
BnO BnO
i
*
BnO
86%
Bn BnO
Me
p:a 15:l 7
OMe
* 9
74% +
I
10
7
OMe
P:a30:l
Scheme 4. Reagents and conditions: (i) NIS, AgOTf, 4 A molecular sieve, CH2C12-toluene, -50 "C to -30 "C; (ii) AgOTf, 2,6-lutidine, 4 molecular sieve, CHzCl2-toluene, -50 " C .
A
oxonium ion a less favorable process, forcing the reaction to proceed through the dioxonium species. Such a situation arose during the synthesis of the glycans of glycodelin (Scheme 4) [36].Coupling of lactosamine donor 6 to trimannoside acceptor 7 provided pentasaccharide 8 as a 15: 1 mixture of anomers (p : a). The stereoselectivity of the coupling was improved to 30 : 1 (p : a) if the glucosamine residue was protected with ester groups instead of benzyl groups. Although it is possible that the observed improvement in stereoselectivity originates from potential alteration of the steric profile of the lactosamine donor as a result of changing the protecting groups, a completely acetylated lactosamine donor [37] gave identical selectivity to 9. 17.5.2 Reactivity Control
If, however, the C2 is functionalized by a group that cannot participate (e.g. azide, benzyl ether, etc. ) the stereoselectivity of a glycosylation is more unpredictable. The two competing pathways, reaction on the a-leaving group and reaction on the pleaving group, are of more similar activation energy than the incipient oxonium ion and the dioxonium species. The choice of protecting group can, however, still effect
438
17 Protecting Groups
Bn BnO Br
12
11
Nc%
BnoBr
14
13
02%
15
BnO
,
16
Table 7. Stereoselectivity of methanolysis of glucosyl halides (molar ratio of methanol to monomer -390: 1). Substrate
a : p Ratio
11 12
7:93 16:84 16:84 37 : 63 92:s 16:84
13 14 15 16
the stereochemical outcome electronically, as was noted by Frechet and Schuerch (Table 7) [38]. They investigated the effect of different protecting groups at C6 on the stereoselectivity of methanolysis of glucosyl halides (building on earlier work by Ishikawa and Fletcher) [ 81. A clear pattern emerged-electron-rich benzoates promoted pselectivity and the more electron-withdrawing the benzoate became (p-nitro being the most deactivating) the greater the proportion of a-glycoside formed. This can be rationalized in terms of the reactivities of the donors; from the data in Table 6 it is clear that the more electron-deficient the benzoyl, the more the reactivity of the donor is diminished (the greater the DF) owing to the inductive effects highlighted above (Section 17.3). This results in an increase in the activation energies for glycosylation of both the a- and P-bromide. The a-anomer, with its lower ground-state energy (anomerically stabilized), becomes too unreactive to glycosylate methanol at a rate that is competitive with the P-bromide pathway (4). Interconversion of the ato P-bromide is fast and the reaction therefore (for 15) generates the a-glycoside as the major product (Figure 7). If, however, the reactivity of the donor is reduced too much, as in 16, in which the leaving group is changed to the chloride (some 20-30 times less reactive), the rate of
17.5 Stereoselectivity U-D-GIY-1-X
a-D-Gly+, x-
p -~-Gly-l-X
p - ~ - G l y +X,
439
(3) MeOH
(4) M eOH
Methyl 0-D-glycopyranoside
Methyl a-D-glycopyranoside
Figure 7. Methanolysis of glycopyranosides.
interconversion of a- to P-chloride (2) is reduced (relative to glycosylation). Because the a-chloride is present at a higher concentration (because it is the thermodynamically favored arrangement) than the p-chloride, reaction proceeds through the achloride pathway (3) and the p-glycoside is the major product. Alternatively, if the donor is made sufficiently reactive (as in ll), the a-bromide is reactive enough to glycosylate methanol at a rate competitive with the lower energy p-pathway, aided by its increased concentration, such that the P-glycoside predominates. Unfortunately these results were not reproduced when carbohydrate acceptors were used; only a-glucosides were obtained, irrespective of the protecting group on C6 [39]. This is a reflection of the sensitivity of the kinetics of glycosylation-a carbohydrate alcohol is considerably less nucleophilic than methanol for reasons both of size and electronics. This reduced nucleophilicity ensures that the a- to phalide equilibration process is faster than the glycosylation and hence reaction proceeds through path (4) [40]. The reduced concentration of alcohol relative to donor (ratios of 390: 1 are not practical for synthesis of oligosaccharides) would also ensure that equilibration was faster than glycosylation, thus favoring formation of the a-glycoside [41]. The underlying principles, however, are still valid and can be successfully applied in many systems; in general, deactivation of the donor improves the a-selectivity of a glycosylation if the donor species can still anomerize. Such an occasion arose during various groups’ syntheses of glycosyl phosphatidyl inositol membrane anchors [42], in which an oligosaccharide is linked to an inositol moiety via an a-glucosamine residue. a-Linkages in gluco-configured sugars are particularly troublesome because, unlike manno-configured sugars whose axial C2 hydroxyl serves to stereoelectronically promote a-selectivity [43], the equatorial C2 imparts no particular bias. Ley’s initial approach employed glycosyl donor 17 but even under optimized conditions [44] the best selectivities obtained consisted of a 3 : 1 mixture (a : p) (Scheme 5). The in situ-generated glycosyl triflate/perchlorate was too reactive for the two glycosylation pathways ((3) and (4) in Figure 7) to be cleanly differentiated. Ley’s solution was to change the donor species to the less reactive iodide (as opposed to the triflate/perchlorate), this deactivation afforded the desired disaccharide 19 with near complete a-selectivity [42a]. Schmidt’s solution was also to deactivate the donor but instead of deactivating the active leaving group (triflate), he deactivated the donor by employing acetyl protecting groups
440
T Bn B
17 Protecting Groups
S
O
~
F
N3 I
17
+
b
60% a:p3:1
19
18
T Bn B s
o
20
G
ii
Br
+ 18
b
65%
TBs4& Bn
Al I
Bn
a$ >30:1 19
Scheme 5. Reagents and conditions: (i) Yb(OTf)Z, CaC03, Ba(C104)2,Et20, room temp; (ii) TBAI (tetra-N-butyl-ammonium iodide), CH2C12, room temp; (iii) TMSOTf, Et20, room temp. Mnt = menthyl.
(21), affording the same result [42c].The different protecting groups on the acceptor alcohol 22 may also have contributed to this improvement in selectivity (uideinfru). A similar problem was encountered during the synthesis of the glycans of glycodellin-S [36]. The synthesis of LewisY epitope 26 required bis-a-fucosylation of lactosamine moiety 24 (Scheme 6). The tri-0-benzylated fucosyl donor 25 provided the tetrasaccharide 26 with acceptable selectivity but unfortunately as an inseparable mixture. Recourse to a bromide donor under halide ion catalysis (as in the previous example) was not successful because the deactivation was so great it made glycosylation of the hindered hydroxyls prohibitively slow. Deactivating the donor species by acetylating the 3- and 4-positions (27) [45], however, increased the a-selectivity to the point that the other anomer was unobservable. There are other ways to influence the stereochemical outcome by affecting the
441
17.6 Injluence of the Protecting Group on the Acceptor
i OH
PhthN
24
*
57%
++% PhthN
+ P Bn
OBn Bn
BnO
25
i OH
PhthN
24 +
26
a$ 15:l
*
SEt
47%
F' AcO
OBn AcO
24
28
Scheme 6. Reagents and conditions: (i) AgC104, 2,6-lutidine, Et,O, THF, -35 "C.
anomerization equilibrium; lower temperatures 'freeze out' the a-donor species promoting p-selectivity, especially when the donor is deactivated [38, 461 (see Chapter 16(?) on p-mannose synthesis) and the polarity and donating ability of the solvent can alter the equilibrium [47] (see chapter on solvent effects). As has already been mentioned, the nucleophilicity of the acceptor alcohol can also effect the stereoselectivity of glycosylation. The more nucleophilic alcohols can react on the a-ion pair whereas less nucleophilic alcohols glycosylate more slowly, allowing the donor species to equilibrate to the more reactive p-ion pair, generating the a-glycoside. Hence glycosylation of the primary C6 is usually less a-selective than any of the secondary positions. The choice of protecting group can also electronically influence the nucleophilicity of the acceptor alcohol.
17.6 Influence of the Protecting Group on the Acceptor In the same way that protecting groups influence the electron density on the anomeric carbon, tuning the reactivity, they also effect the electron density of the hydroxyl groups within the sugar, adjusting their nucleophilicity. Sinay noted in the synthesis of lactosamine derivative 32 that the protecting group at C3 of the glucosamine had a dramatic effect on the glycosylation yield with galactosyl bromide 29 (Scheme 7) [48]. Although the acetylated acceptor 30 was such a poor nucleophile that the coupling with 29 only produced a 5% yield of the desired
442
I 7 Protecting Groups
Scheme 7. Reagents and conditions: (i) HgBr2, (CH2C1)2, room temp.
i A
* Ac
Ac H OBn
Br
AcO
OBn
R= Ac, 30 R= Bn,31
29 Acceptor
Yield (%)
30 31
5 87
32
product, benzyl protection increased the nucleophilicity of acceptor 31 affording 32 in 87% yield [49]. The protecting group at the anomeric position also influences hydroxyl reactivity; thioglycosides tend to be less nucleophilic than the equivalent 0-glycoside [ 501. This can result in reduced glycosylation yields for difficult couplings as was the case in the synthesis of disaccharide 36, taken from van Boom's synthesis of a pentasaccharide from Crytococcus neoformans (Scheme 8) [ ~ O C ] .Coupling of xyloside 33 with thiomannoside 34 provided disaccharide 36 in a disappointing 38% yield, methyl mannopyranoside 35, however, coupled with 33 in 68% yield. The final mechanism by which protecting groups can influence the outcome of a glycosylation is through the way they alter the steric profile of the donor and acceptor.
Scheme 8. Reagents and conditions: (i) TMSOTf, (CH2C1)2, room temp
A%*
i
AcOO
-
;&-% A
AcO
Bn
33
BnO Bn
R=SEt, 34 R=OMe, 35 Acceptor
Yield ('%)
34
38 68
35
R
36
17.7 Steric EfSects on Glycosylution
443
17.7 Steric Effects on Glycosylation Of all the ways in which protecting groups can influence a glycosylation, steric effects are perhaps the least quantifiable and yet ultimately, the most influential. Although the rate-limiting step for most glycosylations is the activation of the leaving group [51] the product determining step is the approach of the acceptor to the donor (step C in Figure 1) [52]. The different steric interactions in the two transition states CaPand CPaaffect the activation energies of each pathway, which determine the selectivity (and yield) of a glycosylation [53]. This is obvious when the protecting group is close to the reacting center, as in Scheme 9 [54].Attack by the rhamnoside 40 on the torsionally deactivated mannosyl a-triflate, preformed from 37 at low temperature by activation with triflic anhydride, proceeded only with modest P-selectivity when the C2 of mannosyl donor was protected with a bulky tert-butyldimethylsilyl (TBDMS) group. Replacement of this group with the smaller trimethylsilyl (TMS) or benzyl group greatly improved the P-selectivity (for optimized conditions see Chapter 16 (?) on p-mannose). Even remote positions, the C6 for example, have, however, been reported to have an effect on the stereoselectivity of glycosylation [55]. The issue is unfortunately clouded by the change in electronic properties which frequently accompanies a change in protecting groups. Perhaps the most dramatic example of a protecting group affecting the outcome of a glycosylation comes from a synthesis of a glycan from laminin by Zhu et al. (Scheme 10) [56].Attempted coupling of lactose donor 42 with P-methylglycoside 43p failed to produce any of desired hexasaccharide 44p. When coupling was attempted on the a-methyl glycoside 43a under identical conditions, however, this
Scheme 9. Reagents and conditions: (i) Tf20, DTBMP, Et,O, -78 "C. Me P
BnQ
i
q
+
Bn
VEt R=TBDMS,37 0 R=TMS, 38 R=Bn, 39
41
40
Donor
Selectivity (j3 : a )
37 38 39
1.6: 1
19
5.1 : 1 6.1: 1
91
Yield (%)
85
\
444
17 Protecting Groups
Scheme 10. Reagents and conditions: (i) BF3 . OEtz, CH2C12,-10°C to room temp.
Ac
AcO
Ac i
I
Ac BnO
*
Ac
44
AcO
OH
T&
BnO BnO
B -nBn
R
43 BnO
R’
A 43a 43P
R
R’
Yield (%)
Me0 H
H Me0
65 0
single stereochemical alteration was sufficient to rectify the situation, furnishing hexasaccharide 44u in 65% yield. Rationalization of this reversal of fortune lies in the different conformations of 43u and 43p. Molecular modeling demonstrated that the steric hindrance about the hydroxyl group of 43p was greater than that for 43u, making the hydroxyl less amenable to glycosylation. Add to the equation the steric hindrance about the donor site and the net ‘steric mismatch’ is enough to prevent coupling between donor 42 and 43p. It is unusual for such a controlling element to be so readily identified or modified and steric mismatch, whose likelihood increases with increasing size of coupling partners, can be the Nemesis of block coupling-based strategies [ 571.
17.8 Conclusions In the last 25 years much progress has been made in the development of glycosyl donors and activating systems [58],with few corners of the periodic table remaining untouched in the search for an ever-better system. With few exceptions [59], however, they all depend on the displacement of an anomeric leaving group (frequently a triflate) in a bimolecular fashion and are thus all subject to the influences outlined hitherto. That different donors provide varying results for the same glycosylation
445
17.8 Conclusions
i
BnO
30%
45
-
All
-
‘&OAIl
47
PhthN
46
a:P 23:l
Scheme 11. Reagents and conditions: (i) 0.07 M LiC104, CHZC12, room temp.
can be ascribed to their differing rates of activation under the conditions used. This was encountered by Waldmann and Schmid in their investigations into the synthesis of blood group determinants (Scheme 1 1) [60]. Coupling of the a-fucosyl fluoride 45 with the acceptor 46 proceeded with good stereocontrol to afford the a-( 1,4)-linked disaccharide 47 albeit in poor yield. When, however, the more reactive P-fluoride was employed under identical conditions, the desired disaccharide was obtained in better yield (68%) but with worse stereoselectivity ( a : P 4: 1). In this example even though the donors are of the same type, the difference in stereochemistry was enough to affect the course of the reaction. The reaction mechanism has not changed, merely the positions of the various equilibria. This result is not unique to a certain leaving group but to the entire system-the activator, the solvent, the temperature, the donor, the acceptor, the protecting groups, and the leaving group-potentially requiring that each individual reaction be optimized. This predicament prompted carbohydrate chemists, as early as 1973, to bemoan “unfortunately the reactivity of C1 and the stereoselectivity of its reaction depend in a complex way on the leaving group, the blocking groups, the configuration of the sugar and the structure of the molecule bearing the entering hydroxyl function” [611. Although the glycosylation reaction is by no means tamed, owing to the unpredictable interplay of these factors (steric effects in particular), its behavior has mostly been characterized, even if not entirely understood. Protecting groups, as first summarized by Paulsen in his seminal review [62], can alter the course of a reaction in a predictable manner; providing the shrewd chemist with some control over the reaction. To gain complete control of a glycosylation will require the development of a coupling strategy which evades these influences, one that does not depend on a bimolecular collision as its key step [63]. Acknowledgments
We thank Professor A. J. Kirby and Dr S. L. Warriner for helpful discussion in the preparation of this manuscript.
446
I 7 Protecting Groups
References 1. P. J. Kocienski Protecting Groups;Georg Thieme Verlag: Stuttgart: 1994. 2. (a) T. W. Greene, P. G. M. Wuts Protective Groups in Organic Synthesis, Wiley: New York: 1991 (b) ref [ I ] (c) M. Bols Carbohydrate Building Blocks, Wiley: New York: 1996 (d) S. Hanessian Preparative Carbohydrate Chemistry, Marcel-Dekker: New York: 1997 (e) G.-J. Boons Carbohydrate Chemistry, Blackie Academic & Professional: London: 1998. 3. B. Capon Chem. Rev. 1969, 69, 407. 4. M. L. Sinott, W. P. J. Jencks J. Am. Chem. Soc. 1980, 102, 2056. 5. (a) T. L. Amyes, W. P. J. Jencks J. Am. Chem. SOC.1989, 111, 7888 (b) M. L. Sinott Chem. Reu. 1990, 90, 1171. 6. R. Lemieux, K. B. Hendriks, R. V. Stick, K. James J. Am. Chem. SOC.1975,97, 4056. 7. A. J. Rhind-Tutt, C. A. Vernon J. Chem. Soc. 1960, 4637. 8. T. Ishikawa, H. G. Fletcher J. Org. Chem. 1969, 34, 563. 9. R. U. Lemieux, G. Huber Can. J. Chem. 1955,33, 128. 10. (a) P. Delongchamps, C. Moreau, D. FrCchet, P. Atlani Can. J. Chem. 1972, 50, 3402 (b) P. Deslongchamps ACS Symposium series: The Anomeric Effect and Associated Stereoelectronic effects; G. R. J. Thatcher Ed.; American Chemical Society: Washington, D. C., 1993; Vol. 549. 11. (a) A. J. Ratcliffe, D. R. Mootoo, C. Webster Andrews, B. Fraser-Reid J. Am. Chem. SOC. 1989, 111, 7661 (b) P. Deslongchamps, P. G. Jones, S. Li, A. J. Kirby, S. Kusela, Y. Ma J. Chem. SOC.,Perkin Trans. I 1997, 12, 2621. 12. D. Y. Curtin Rec. Chem. Proc., 1954, 15, 111. 13. There are of course exceptions: (a) where there is no available counter-ion e.g. BF30Et2 activation of anomeric fluorides and trichloroacetimidates but N.B. the catalyst coordinated ionpair is also not configurationally stable either. Some silver salts e.g. AgZC03, AgzO, and Ag silicate also operate via a ‘push-pull’ mechanism--(;. Wulff, G. Rohle, U. Schmidt Chem. Ber. 1972, 105, 111-however, in this case the initial bromides are not usually configurationally stable. ZnC12 mediated concerted nucleophilic opening of deactivated glycal epoxides relies on direct attack for selectivity. (b) In cases of solvolysis, as the nucleophile is present in the solvent cage, attack immediately follows activation of the leaving group, leading to product with inversion of the leaving group stereochemistry. A similar special case includes the reaction of trichloroacetimidates with carboxylic acids-R. R. Schmidt, J. Michel Angew. Chem. Znt. Ed. Engl. 1980,19,731-and dibenzyl phosphate-R. R. Schmidt, M. Strumpp Liebig Ann. Chem. 1984, 680-where the nucleophile is again present in the solvent cage at the point of activation of the leaving group. 14. (a) V. Marousek, T. H. Lucas, P. E. Wheat, C. Schuerch Carbohydr. Res. 1978, 60, 85. Anomeric perchlorates had been reported previously-K. Igarashi, T. Honma, J. Irisawa Curbohydr. Res. 1970, 15, 329. 15. D. Crich, S. X. Sun Tetrahedron 1998, 54, 8321. 16. C. N. Riiber, N. A. Ssrensen Kgl. Norske. Videnskab. Selskabs Skrifter 1938, I , 1. 17. W. G. Overend, C. W. Rees, J. S. Sequeria J. Chem. SOC.1962, 3429. 18. G. N. Richards Chem. Znd. 1955, 228. 19. M. S. Feather, J. F. Harris J. Org. Chem., 1965, 30, 153. 20. E. A. Kabat, H. Baer, A. E. Bezer, V. Knaub J. Exp. Med., 1948, 88, 43. 17. J. T. Edward Chem. Znd. 1955, 1102. 22. B. Fraser-Reid, A. Wu, C. Webster Andrew, E. Skowronski J. Am. Chem. SOC.1991, 113, 1434. 23. C. P. J. Glaudemans, H. G. Fletcher J. Am. Chem. SOC.1965, 87,4636. 24. N. L. Douglas, S. V. Ley, U. Lucking, S. L. Warriner J. Chem. SOC.,Perkin Trans. I , 1998, 51. 25. L. R. Schroeder, J. W. Green, D. C. Johnson J. Chem. SOC.B, 1966, 447. 26. (a) S. V. Ley, H. W. M. Priepke, S. L. Warriner Angew. Chem. Znt. Ed. Engl. 1994, 33, 2290 (b) P. Grice, S. V. Ley, J. Pietruszka, H. W. M. Priepke, S. L. Warriner, J. Chem. SOC. Perkin Trans. I , 1997, 351. 27. A plausible explanation for the increased importance of the C4 relative to the C3 based upon hyperconjugation with the endocyclic oxygen lone-pairs has been put forward-C. A. A. van Boeckel, T. Beeta, S. F. van Aelst Tetrahedron, 1984, 40, 4097.
References
447
28. Z. Zhang, I. R. Ollmann, X.-S. Ye, R. Wischnat, T. Baasov, C.-H. Wong J. Am. Chem. Soc. 1999,121, 734. 29. (a) N. L. Douglas, S. V. Ley, H. M. I. Osborn, D. R. Owen, H. W. M. Priepke, S. L. Warriner Synlett, 1996, 793 (b) U. Berens, D. Leckel, S. C. Oepen J. Org. Chem. 1995, 60, 8204 (c) J.-L. Montchamp, F. Tian, M. E. Hart, J. W. Frost J Org. Chem. 1996, 61, 3897. 30. That the C4 of galactose has a particular bearing has been noted by others (a) M. Miljkovic, D. Yeagley, P. Deslongchamps, Y. L. Dory J. Org. Chem. 1997, 62, 7597 (b) 0. Kwon, S. J. Danishefsky J. Am. Chem. Soc. 1998, 120, 1588. 31. R. U. Lemieux Adu. Carbohydr. Chem. 1954, 9, I . 32. Although participation from other positions has been postulated by many early investigators, there is little evidence to substantiate that this is possible. 33. R. U. Lemieux, C. Brice, G. Huber Can. J. Chem. 1955, 33, 134. 34. In truth this is a simplification; although attack at C1 of 6 leads to the thermodynamic product, kinetic attack occurs first at the carbonyl carbon leading to an orthoester product. If the reaction is performed under acidic conditions, the acid sensitive orthoester rearranges to form the trans glycoside. However if the donor is very deactivated the orthoester may require a higher acid concentration to induce rearrangement which can result in transesterification (to the acceptor alcohol) predominating, especially if an acetate is used. Destabilizing the orthoester by activating the donor or using benzoyl or pivaloyl protecting groups can resolve this problem. 35. This problem has been reported by many other authors (a) S. Oscarsson Topics in Current Chemistry: Glycoscience; H. Driguuez, J. Thiem Ed.; Springer: Berlin Heidelberg, 1997; Vol. 186 (b) A. V. Nikolaev, T. J. Rutherford, M. A. Ferguson, J. S. Brimacornbe Bioorg. Med. Chem. Lett. 1994, 4, 785 (c) T. Ziegler, B. Adams, P. Kovac, C. P. J. Glaudemans Carbohydr. Chem. 1990, 9, 135 (d) H. Jiao, 0. Hindsgaul Angew. Chem. Int. Ed. Engl. 1999, 38, 346 (e) N. Kochetkov The Synthesis of Polysaccharides in The Total Synthesis of Natural Products; J. ApSimon Ed.; Wiley-Interscience: New York, 1992; Vol. 8, p 245. 36. D. Depre, A. Duffels, L. G. Green, R. Lenz, S. V. Ley, C.-H. Wong Chem. Eur. J. 1999, 5, 3326. 37. J. Arnop, M. Haraldsson, J. Lonngren J. Chem. Soc., Perkin Trans. 1 1982, 1841. 38. J. M. Frechkt, C. Schuerch J. Am. Chem. Soc. 1972, 94, 604. 39. J. M. Frechkt, C. Schuerch Polymer Supported Reactions in Organic Synthesis; P. Hodge Ed.; Wiley: Chichester, 1980. 40. Schroeder et al. demonstrated that a-selectivity increased with increasing size of the alcoholL. R. Schroeder, J. W. Green, D. C. Johnson J. Chem. SOC.B 1966, 447. 41. J. E. Wallace, L. R. Schroeder J. Chem. Soc., Perkin Trans. 1 1976, 1938. 42. (a) D. K. Baeschlin, A. R. Chaperon, V. Charbonneau, L. G. Green, S. V. Ley, U. Lucking, E. Walter Angew. Chem. Int. Ed. Engl. 1998,37, 3423 (b) A. S. Campbell, B. Fraser-Reid J. Am. Chem. Soc. 1995,117, 10387 (c) T. G. Mayer, B. Krazer, R. R. Schmidt Angew. Chem. Znt. Ed. Engl. 1994, 33, 2177 (d) C. Murakata, T. Ogawa Curbohydr. Res. 1992,235, 95. 43. The A2 effect-R. E. Reeves Adu. Carbohydr. Chem. 1960, 15, 11-the axial oxygen at C2 serves to both to reinforce the anomeric effect, further lowering the ground state energy of the a-leaving group relative to the P-leaving group and sterically shields the p-face thus promoting a-glycosidation. 44. W. S. Kim, S. Hosono, H. Sasai, M. Shibisaki Heterocycles 1996, 42, 795-perchlorate counter-ions have been proposed to provide higher a-selectivities than triflates owing to their slightly reduced fugacity and ether type solvents at room temperature also serve to promote aselectivity (see chapter on solvent effects). 45. Others have used deactivated fucosyl donors-(a) M. Djeter, H. M. Flowers Carbohydr. Res. 1972, 23, 41 (b) V. Behar, S. J. Danishefsky, Angew. Chem. Int. Ed. Engl. 1994, 33, 1468 (c) R. Windmuller, R. R . Schmidt, Tetrahedron Lett. 1994, 65, 7927 (d) T. Kiyoi, Y. Nakai, H. Kondo, H. Ishida, M. Kiso, A. Hasegawa, Biocirg. Med. Chem. 1996, 4, 1167. 46, J. E. Wallace, L. R. Schroeder J. Chem. Soc., Perkin Trans. I1976, 1633. 47. R. Eby, C. Schuerch Carbohydr. Res. 1974, 34, 79. 48. P. Sinay Pure Appl. Chem. 1978, 50, 1437. 49. Boons and Zhu have exploited this effect for the synthesis of small oligosaccharides-G.-J. Boons, T. Zhu Synlett 1997, 809.
448
17 Protecting Groups
50. (a) V. Pozsgay Carbohydrates: Synthetic Methods and Applications in Medicinal Chemistry: Synthesis of Oligosaccharides Related to Plant, Vertebrate, and Bacterial Cell-wall Glycans: H. Ogura, A. Hasegawa, T. Suami Ed.; Kodansha, VCH: Tokyo, New York, 1992, p 188 (b) H. Paulsen, W. Rauwald, U. Weichert Liebig. Ann. Chem. 1988, 75 (c) K. Jaarsveld Zegelaar, S. A. W. Smits, G. A. van der Marcel, J. H. van Boom Bioorg. Med. Chem. 1996,4, 1819. 51. e.g. for sulfoxides D. Kahne, S. Raghavan J. Am. Chem. SOC.1993, 114, 1580. 52. For glycosyl bromides under halide catalysis this is the rate determining step [6]. 53. N. M. Spijker, C. A. Boeckel Angew. Chem. Int. Ed. Engl. 1991,30, 180. 54. D. Crich, S. X. SunJ. Org. Chem. 1997,62, 1198. 55. inter alia (a) M. Wakao, Y. Nakai, K. Fukase, S. Kusumoto Chem. Lett. 1999, 27 (b) R. Rodebaugh, S. Joshi, B. Fraser-Reid, H. M. Geysen J. Org. Chem. 1997, 62, 5660. 56. X.-X. Zhu, M . 3 . Cai, R.-L. Zhou Carbohydr. Res. 1997, 303, 261. 51, (a) Y.-M. Zhang, A. Brodzky, P. Sinay, G. Saint-Marcroux, B. Perly Tetrahedron: Asymmetry 1995, 6, 1195 (b) V. Pozsgay, E. P. Dubois, L. J. Pannell J. Org. Chem. 1997, 62, 2832. 58. K. Toshima, K. Tatsuta Chem. Rev. 1993, 93, 1503. 59. These include: (a) direct alkylation of the anomeric position-R. R. Schmidt, Angew. Chem. Int. Ed. Engl. 1986, 25, 212 (b) attempts at SNi based strategies-@) M. E. Behendt, R. R. Schmidt Tetrahedron Lett. 1993, 34, 10733; (ii) T. Iimori, T. Shibizaki, S. Ikegami Tetrahedron Lett. 1996, 37, 2267 iii) G. Scheffler, R. R. Schmidt Tetrahedron Lett. 1997, 38, 2943 iv) G. Scheffler, R. R. Schmidt J. Org. Chem. 1999, 64, 1319 (c) tethered strategies (see Chapter 16 (?) on 0-mannose) (d) redox glycosylation-A. G. M. Barrett, B. C. B. Bezuidenhoudt, A. F. Gasiecki, A. R. Howell, M. A. Russell J. Am. Chem. SOC.1989, 111, 1392 (e) acetal reduction-H. Ohtake, T. Iimori, S. Ikegami Tetrahedron Lett. 1997,38, 3413. 60. H. Waldmann, U. Schmid Chem. Eur. J. 1998,3, 494. 61. C. Schuerch Acc. Chem. Res. 1973, 6, 184. 62. H. Paulsen Angew. Chem. Int. Ed. Engl. 1982, 21, 155. 63. F. Baressi, 0. Hindsgaul Modern Synthetic Methods: Glycosylation Methods in Oligosaccharide Synthesis, B. Ernst, C. Leuman, Ed.; VCH, Basel, 1995.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
18 Intramolecular Glycosidation Reactions Jacob Mudsen and Mikael Bols
18.1 Introduction Carbohydrate derivatives found in nature are almost always linked to each other by glycosidic bonds. There has been increasing interest in the synthesis of biologically active oligo- and polysaccharides and reliable methods for the formation of glycosidic linkages have been the subject of intensive study. Two problems are usually encountered in the synthesis of a glycosidic bondunreactive hydroxyl groups and lack of diastereoselectivity (i.e. formation of a and epimers). To overcome these problems in the last decade chemists have explored a new approach-intramolecular glycosidation reactions. In this review we will define the term intramolecular glycosidation reaction as a type of reaction in which the glycosyl donor (the glycone) is linked to the glycosyl acceptor (the aglycone) by means of a tether producing a 0-glycosidic bond (Scheme 1). The tether can be either a temporary tether which is removed during the glycosidation reaction or it can be a stable tether which is cleaved after the glycosidation procedure. This chapter is divided into two main parts: 1) reactions in which the tether participate in the reaction; and 2) reactions in which the tether does not participate in the reaction.
Non-tethered intramolecular glycosidations which produce spiroketals [ 11 or natural products [2, 31 have been reported; these will not be discussed here.
18 Intramolecular Glycosidation Reactions
450
GIycosyl
X
donor
7d@& Tetherin procedure
L
Glycosyl acceptor
Ho przdure GI sidationb L Tethered disacchari d
RoL*
X
Scheme 1. Outline of tethered intramolecular glycosidation reactions. (T1 = temporary tether, T2 = stable tether)
18.2 Reactions in which the Tether Participates in the Reaction 18.2.1 Tethering to the Glycosyl Donor Carbon Tethers
In 1991 Barresi and Hindsgaul reported the first example of an intramolecular glycosidation procedure and introduced the expression ‘intramolecular aglycone delivery’. The idea was to use this approach in syntheses of 1,2-cis glycosidic linkages in the difficult mannose case [4]. The set-up for this reaction was thioglycoside 1, a glycosyl donor with a 2-0-acetate group (Scheme 2). The acetate was converted to methylene derivative 2 with the Tebbe reagent. Treatment of 2 with the alcohols a, b or c in the presence of a catalytic amount of acid formed the key acetals 3a-3c in moderate yield. Activation of 3a-3c with 5 equiv. N-iodosuccinimide (NIS) resulted in selective intramolecular glycosidation at the p face via intermediates 4a-4c, which on aqueous work-up gave the desired pmannopyranosides 5a-5c. In a later paper an improved method, in which the base 2,6-di-t-butyl-4methylpyridine (DBMP) was added in the activation step, was reported [5].This increased the yields of 5b and 5c significantly. Further investigation, by means of competition experiments, showed that the reaction was intramolecular, but also revealed some limitations of the method. The mixed acetals 3a-3d were relatively unstable and had to be prepared carefully to avoid hydrolysis to the corresponding monosaccharides or dimerization. Furthermore the yield dropped drastically when trisaccharides, e. g. 5d, or tetrasaccharides were synthesized [6]. A similar approach was taken by Ito and Ogawa who introducedp-methoxybenzyl (PMB) as carbon tether [7]. Alcohols protected with PMB, e.g. 6, can be oxidatively deprotected with DDQ [8] or CAN [9] presumably via the oxocarbenium intermediate 7 (Scheme 3 ) .
451
18.2 Reactions in which the Tether Participates in the Reaction
~~7% A
- Bny%FiR
reagent, Tebbe
BnO
ROH
BnO
cat. TsOH
2
SEt
SEt
BnO
4"C,20min
1:z
SEt
NIS (and base)
R- OH
BnO BnO O *"
Mixed
P-glycoside 5 without basel with base
51%
61%
-
45%
42%
17%
r
-I
Brio OMe
a
(OBn HO%
BnO Brio OMe b
OBn BnO BnO
Hosoe C
38%
BnO
NPhthBno d
-
21%
O(CHz)&H3 NF'hth
Scheme 2. Isopropylidene-tethered glycosidation.
r
6
L
+ l
7
J
CHO
Scheme 3. Protection and deprotection of PMB ethers.
OR
5
452
18 Intramolecular Glycosidation Reactions
OMe
I
Q Brio\
OPMB R-OH
BnO BnO
8
F
'
/
BnO
-
L
R- OH
~
P-glycosides
9
F
yield[%]*'
OH
Brio* BnO
OBn OBn
10a
61-14'
BnO BnO
a
OR
10
OBn
OBn
Ho?&& BnO
OBn NPhth
1oc
40
P
* Variations in different solvents(CH,CI,, ** Total yields for two reactions.
Et,O, MeCN).
Scheme 4. PMB-tethered glycosidation in the synthesis of P-mannopyranosides.
The basic idea was to trap intermediate 7 under anhydrous conditions and react it with another alcohol producing the corresponding mixed acetal. The authors showed that treatment of mannosyl donor 8 with DDQ and alcohols a-c gave the mixed acetals 9a-9c (Scheme 4). Without purification, 9a-9c were activated with AgOTf and SnC12 in the presence of DBMP. Aqueous work-up gave the expected P-mannopyranosides lOa-lOc in good overall yields. By use of this strategy the core penta- and hexasaccharides found in asparaginelinked oligosaccharides were synthesized in good yield [ 10- 121. It has recently been reported that the choice of protection groups also plays an mannose derivative important role. Thus using a 4,6-O-cyclohexylidene-protected as glycosyl donor gave the desired P-mannopyranosides in high yields [13]. Ito and Ogawa have also reported the synthesis of P-mannopyranoside linkages by use of the 'intramolecular aglycone delivery' strategy with PMB as tethering group linked to a solid phase (Scheme 5) [ 141. It is notable that the products 13a-13d contain free hydroxyl groups. By this
18.2 Reactions in which the Tether Participates in the Reaction
453
R-OH
DDQ,CH2C12 room temp.
TBSO $3hp
ph3%+oR TBSO
1
12 SMe
11 sMe
MeOTf
%&H2Cl
P-rnannopyranoside
R- OH
Yield[%]
-.aoBn 139
BnO
50
%"P
OBn
P
TBSO
6
BnO BnO
OBn
b
13b
43
13c
37
13d
54
13
OR
OBn
Ho&F BnO NPhth C
QBn HO-,H,
N3 d
Scheme 5. Solid-phase version of PMB-tethered glycosidation.
means the P-mannopyranoside derivatives will be the only compounds not connected to the polymer phase after the glycosidation reaction. Unreacted starting material, hydrolysed mixed acetals, or 1,2 elimination products will stay bound to the polymer phase. This means that the disaccharides should be easily isolated. A variation of the PMB strategy was used by Krog-Jensen and Oscarson to produce (3-D-fructofuranoside linkages (Scheme 6) [ 15, 161. Reaction of the a-Dfructofuranoside derivative 14 with mannose derivatives 15 or 16 and DDQ gave the corresponding mixed acetals 17 which were not characterized. Immediately after work-up the acetals were treated with dimethyl(methylthi0)sulfonium trifluoromethanesulfonate (DMTST) in CHZC12 to give P-D-fructofuranoside derivatives 19, both in 77% yield.
454
18 Intramolecular Glycosidation Reactions
DMTST
Bnow
____)
CHZCIZ, 71%
OBn
OBn
-
1
17
19
Scheme 6. PMB-tethered glycosidation in the synthesis of P-fructofuranosides
Other promoters (MeOTf, NIS, iodonium dicollidine perchlorate (IDCP), and iodonium dicollidine triflate (IDCT)) have also been investigated, but DMTST usually seems to be the right choice [ 161. Silicon Tethers The use of silicon as a temporary tether in intramolecular reactions has found widespread application in organic chemistry [ 171. This concept was first described by Stork et al. in 1992 for intramolecular stereocontrolled glycosidation reactions. With a silyl group connected to the 0-2 position of a P-mannosyl donor it was demonstrated that the glycosyl acceptors could contain primary and secondary hydroxyl groups [18, 191. Treatment of sulfoxide 20 with MezSiClz and alcohols a-f gave the mixed silyl acetals 21a-21f in good yields (Scheme 7). Activation of the sulfoxide with trifluoromethane sulfonic acid anhydride (Tf2O) and base, presumably resulting in the five-membered intermediates 22a-22f responsible for exclusive glycosidation at the [3 face, gave 0-mannopyranosides 23a-23f. Special interest was now devoted to glycoside 23d because it was produced in 12% yield only. It was found that compound 24, containing a silicon bridge, could be isolated as the major product in 82% yield (Scheme 8). A similar observation was made when silyl acetal 21f was activated, producing the expected glycoside 23f in 54% yield and 41% of a debenzylated compound [ 191. A similar approach was simultaneously investigated by Bols et al. Initially thioglycoside 25 was reacted with Me2SiC12 and primary, secondary, or tertiary aliphatic alcohols or phenol in the presence of pyridine to produce the corresponding silyl acetals 26a-26d in good yields (Scheme 9). Activation of 26a-26d with NIS and a catalytic amount of TfOH resulted in exclusive formation of a-glucopyranosides 28a-28d [20].
455
18.2 Reactions in which the Tether Participates in the Reaction
+B ;n
? '
. l
Me$iC12/imidazole
* : nB
BnO
ROWTHF
Hh
BnO
CH2CI2, Et2O
. l
BnO BnO
21 OGs'Ph
20 OGS'Ph
R- OH
DTBP/Tf20
BnO
'x,,
silyl a d s 21
P-mannopyranosides23
89%
92%
84%
65%
88%
82%
Brio OMe a
BnO, Bno*OMe BnO OH b
&:nB HO Brio OMe
BnO BnO
OR
23
98%
12%
60%
48%
d
HBnO O% Brio OMe e
BnO.
54%
+ @-O-debenzylated product.(as 24) 41%
Scheme 7. Dimethylsilyl-tethered glycosidation in the synthesis of fi-mannopyranosides.
It is worthy of note that only small differences were found between the reactivity of primary and tertiary alcohols a-c in the above glycosidation reactions. The study was therefore extended to include carbohydrate derivatives e, f, and g, with one unprotected hydroxyl group as glycosyl acceptor. It was found that no acid catalyst was needed when the solvent was changed from CH2C12 to MeN02. Thus activation of silyl acetals 26e-26g with NIS in MeN02 gave the desired a-glucopyranosides 28e-28g in good yield. No P-glucopyranosides were detected [21, 221. In addition
456
18 Intramolecular Glycosidation Reactions
"*,.sil BnO BnO B+ O \*OBn
OBn
BnO
'... 1 Tf20, DTBP
OMe
BnO
' g o - O B n
Et20, CH2C12,829:'
OMe
Brio BnO
o/'i'ph 24
2ld
Scheme 8. Side-reaction in dimethylsilyl-tethered glycosidation.
+
AcO AcO
2 ROSiMe&I
-
OAc
Aco%sph
Pyridine
THF, 2h, 25'C
AcO
25
..
Activation of 2694: 2.5 eq.NIS cat. TmH, CH2C12, 10 min.
I
Si
silyl acetals 26
a-glumsides28
15%
59%
Cyclohexyl b
14%
62%
terr-Butyl
16%
61%
54%
12%
R
n-Octyl
'
'OR 26
Activation of 26e-g: 2.5 eq. NIS, MeNO,
a
C
Phenyl d
I
(OBn
66%
BnO* BiO
74%
I
OMe AcO% AcO
O @ OfF
12%
85%
Ho OR
28
39%
Scheme 9. Dimethylsilyl-tethered glycosidation in the synthesis of a-glucopyranosides,
18.2 Reactions in which the Tether Participates in the Reaction
451
AcO MezSiCl2,pyridine
AcO “&SPh
alcohole,73%
*
OH
29
5
OMe
30
I) NIS, MeN02 NIS, MeNOz
OAc
31(31%)
+
AcO
OMe
0 OMe
31 R = Bn (32%) 32 R = H (49%)
Scheme 10. Debenzylation observed in dimethylsilyl-tethered glycosidation.
the intramolecular nature of the glycosidation reaction was proven by means of a competition experiment [ 231. Treatment of thiogalactopyranoside 29 with MezSiCl2 and glucose derivative e gave silyl acetal30 in 73% yield (Scheme 10). Activation of 30 with NIS in MeN02 produced the desired a-galactopyranoside 31 in 320/0yield and, highly unexpectedly, another a-galactopyranoside 32 (identified by careful spectroscopic and chemical studies) which had lost a benzyl group, in 49% yield. Compound 33 could, furthermore, be isolated when the reaction mixture was not quenched with acid, so it was proposed that debenzylation occurred via an intramolecular pathway involving silicon [22]. It has recently been demonstrated that treatment of disaccharide 31 with NIS produces the debenzylated compound 32, probably via a benzylidene intermediate and without participation of silicon [24]. In the glycosidation reactions described above the silyl groups are attached to the 0-2 positions of the glycosyl donors. Bols and Hansen investigated the effect of having the silyl group attached to other positions of the donor [25].This could be a useful strategy because a free hydroxyl group, which could be a target for further glycosidation, is obtained in this type of glycosidation reaction. Thioglucopyranoside 34 was silylated to give 35 in 77% yield. Activation of 35 with NIS in MeN02 gave a 1 : 4 mixture of glucopyranosides 36 in 22% yield (Scheme 11). Higher yields and better selectivity were obtained when the silyl group was anchored to the 0-4 position of thioglucoside derivative 37, giving exclusively the a-
458
I8 Intramolecular Glycosidation Reactions OBn
CISiMe20C8HI,, pyridine SMe HO
* C*H,,OMqSiO BnO-&&
SMe
77%
OBn
OBn
35
34
BnO& HO
0 OBn
36 a$
1:4
Scheme 11. Dimethylsilyl-tethered glycosidation with tethering to the 3 position of the donor.
glucopyranoside 38 in 45% yield (Scheme 12). Silylation of 39 at the 0 - 6 position gave P-glucopyranoside 40 and tribenzyl levoglucosan 41 in 2 :7 ratio. Finally, silylated ribose derivative 42 was treated with NIS in MeN02 to give P-riboside 43 in 63% yield. That complete stereocontrol is observed in the glycosidation of 37, 39, and 42 indicates that this approach could lead to new strategies for oligosaccharide synthesis. Kojitriose has been prepared with complete stereocontrol by use of silicon tethers at two points in the synthesis (Figure 1) [26].
6
C,H,,OMqSiO BnO
NIS, MeNO, >
SMe
--.S;i BnO
45%
BnO
OBn
37
38
OBn
arn
39
40
yJal\sph 63% YYo-
OSiMqOC,H,,
.
.
O 42 2
OBn
41 211
OH
NIS, MeNO,
.
.
O 43 X
Scheme 12. Dimethylsilyl-tethered glycosidation with tethering to the 4, 5 and 6 positions of the donor.
18.3 Reuctions in which the Tether does not Participate in the Reaction
459
HO HO
HO
HO
Figure 1. Kojitriose.
HO
Kojitriose
18.2.2 Tethering to the Leaving Group
In contrast with the above strategies, Schmidt and Behrendt investigated another approach in which the glycosyl acceptor was attached directly to the leaving group [27]. Treatment of 44 with LDA and benzaldehyde resulted in 45 as a 1 : 1 mixture of diastereomers in 90% yield (Scheme 13). Without separation of the diastereomers 45 was reacted with triflates a-c, in the presence of NaH and 15-crown-5 as base, to give predominant P-glucopyranosides 48a-48c and p-lactone 49 in good yields. It was not possible to obtain glycopyranosides 48a-48c directly from 44 and no alkylation on the hydroxyl group in 45 was observed when 45 was treated with triflates a-c, apparently because of steric hindrance. In support of this idea some alkylation was found when the a isomer of 45, which has a less hindered hydroxyl group, was treated with triflate c. It was, therefore, proposed that alkylation occurred at the oxide of the carbonyl group via intermediate 46 and orthoester 47 to produce 48a-48c and 49. A similar approach was taken by Hanaoka et al., who used CO~(CO)S as complexing agent in the glycosidation step [28]. A variation of the above strategy has been applied by Ikegami et al. Decarboxylation of a carbonate tether under acid conditions was claimed to produce intramolecular glycosidic bonds, even though anomeric selectivity was not very high [29]. Schmidt et al. have re-investigated this decarboxylative glycosidation reaction and by means of a competition experiment have found evidence that the mechanism is intermolecular rather than intramolecular [30].
18.3 Reactions in which the Tether does not Participate in the Reaction Ziegler et al. have explored another approach in an attempt to produce p-mannoside [31] or a-glucoside [32] linkages. A succinyl linker between the glycosyl donor and the glycosyl acceptor was used to tether the glycosyl donor and acceptor together. It is noteworthy that the glycosyl acceptor bears a free hydroxyl group.
460
18 Intramolecular Glycosidation Reactions
-
,OBn
-
OBn B O nFo* BnO BnO
44
LDA,THF, PhCHO, -80°C &O -nB BnO
R-OTf
Wh(1:I)
NaH, lkmwn-5 45
O
.B BnO n o
OBn
& c q
Ph
R
-
49
R-OTf
BnO
BnO
BnO
,OBn
-0H
H. 'Ph
46
47
Bno*ORBnO Bnb
48a-c
R-OTf
/OTf
B-rrlucoovranosides
Yield
48a
90%
P
48b
7Ph
P:a8:1
a
C,,H,OTf
b (OTf
Scheme 13. Leaving group-tethered glycosidation.
In contrast to the intramolecular aglycone-delivery strategy the glycosidic bond is here formed directly between the free hydroxyl group and the glycosyl donor producing a bridged disaccharide. Finally the tether can be removed. Treatment of thioglycoside 50 with 10 equivalents of succinic anhydride 51 gave mannopyranoside derivative 52 in almost quantitative yield (Scheme 14). Reaction of 52 with glucose derivative 53 then regioselective opening of the benzylidene acetal with NaCNBH3 gave the tethered saccharide 54 in good yield (Scheme 15). Activation of 54 with NIS and a catalytic amount of TMSOTf gave the bridged a-mannopyranoside 55 in 540/0yield (Scheme 16).
18.3 Reactions in which the Tether does not Participate in the Reaction
461
Scheme 14. Succinoylation of a glycosyl donor.
BnO,
I ) DCC, CH2C12,60%
52
+
HO
2) NaCNBH3 THF HCI in ether, 87%
53
SEt
54
Scheme 15. Tethering with a succinic acid tether.
0
BnO
54
0
55
Scheme 16. Succinic acid-tethered glycosidation.
The effect of connecting the succinyl linker to positions other than 0 - 2 and 0-3’ was also investigated and prearranged saccharides 56 and 57 were prepared by use of the same strategy as described above. Activation with NIS and TMSOTf gave mixtures of bridged a- and b-mannopyranosides 57 and 59 (Scheme 17). There has also been much interest in the formation of P-L-rhamnosides (6-deoxy13-L-mannopyranosides) in which the glycosyl donors and acceptors are tethered with succinyl, malonyl, or phthaloyl bridges [33-351. It has been pointed out that not only the configuration of the glycosyl donors but also the configuration of the glycosyl acceptors plays an important role in the anomeric selectivity obtained. Comparison of reaction 57 + 59 with reaction 57a + 59a, in which the glycosyl acceptor has been changed from a D-glucose derivative to an L-glucose derivative, reveals that the anomeric selectivity has changed significantly (Scheme 18) [36].
462
18 Intramolecular Glycosidation Reactions
Bnr+hr&$, 0
~~0OBn NIS, MeCN,-30°C cat. TMSOTft
BnO
BnO SEt
56 0 NIS, cat.TMSOTf MeCN, -30°C BnO BnO
BnO BnO SEt
45%~ 25%p
0 59
51
Scheme 17. Succinic acid-tethered glycosidation
22Ya
NIS,cat. TMSOTf McCN, -30°C
OBz
BnO SPh 57a
0 59a
Scheme 18. Stereodifferentiation in succinic acid-tethered glycosidation.
It has recently been found that anomeric selectivity is sometimes strongly dependent on the nature of the activating agent. Thus treatment of 60 with MeOTf or NIS resulted in the corresponding a- or p-glycopyranosides, respectively (Scheme 19). It was, furthermore, found that use of galactopyranoside derivatives with a free hydroxyl group in the 4 position as glycosyl acceptors gave P-mannopyranosides exclusively [ 371. One of the main drawbacks of all these strategies is the time-consuming task of synthesizing monosaccharides with the desired protection group patterns. To avoid too much protection-group chemistry Valverde et al. investigated the possibility of having more than one unprotected hydroxyl group on the glycosyl acceptor [38]. Tethered saccharides 61 and 62 were prepared by using phthalic anhydride to tether the glycosyl donor and glycosyl acceptor [39]. Activation with NIS-TfOH resulted in the bridged a-mannopyranoside derivatives 63 and 64,with stereo- and regiocontrol, in good yields. The tether was finally removed and the free hydroxyl groups acetylated to give the corresponding a-mannopyranosides 65 and 66 (Scheme 20).
463
18.3 Reactions in which the Tether does not Purticipate in the Reaction
Brio>
-I 00 -
60
I
OBn
Brio\ 5 eqv. NIS, TMSOTflcat.) CH&N, -3OoC, 10 min.,
OBn
/OBn
BnO
z%
Scheme 19. Dependence of anomeric selectivity on activating agent. (OTBDPS
/ 1)NaOMelMeOH
2) A%OPyridine
OAc OMe
Me0 AcO-
OMe
65
MeO. M
nu
62
64.
n,n/
YLV
Ad)
1OMe
66
,
Scheme 20. Phthalic acid-tethered glycosidation.
Interestingly, it was found that different anchoring positions on the glycosyl acceptor resulted in differences between the selectivity of the free hydroxyl groups. Thus anchoring to the 0-2' position gave the 3' a-glycoside 65 whereas anchoring to the 0-6' position gave the 4' a-glycoside 66. Treatment of 67 with CpzHfClz and AgOTf has been found to give the regioselective intramolecular glycosidation product 68 in 37% yield (Scheme 21) [40]. This regioselectivity was in agreement with Monte Carlo calculations.
I OMe
464
18 Intramolecular Glycosidution Reactions
C&HfClz, AgOTf
CH,CI,, 4A-MS, 31%
68
67
Scheme 21. Phthalic acid-tethered synthesis of a trisaccharide.
a, a’-dibromo-rn-xylene has recently been used to produce the tethered compounds 69 and 70 which, upon activation with NIS/TMSOTf, resulted in the macrocyclic derivatives 71 and 72 containing 14- or 15-membered rings respectively (Scheme 22). In both instances p( 1-4)-linkages were formed exclusively [41].
18.4 Conclusion Since the first report of an intramolecular glycosidation reaction in 1991 chemists have shown much creativity within this area. The new and interesting chemistry
1.3 eq. NIS
BnO BnO OBn
x
x = Pentenyioxy
BnO BnO
BnO BnO
OBn OMe
71
69
X = SEt
1.3 eq. NIS
BnO BnO
1 eq. TMSOTf 84%
SEt
OBn
OBn
70
Scheme 22. rn-Xylene-tetheredglycosidation.
BnO BnO
OBn
OBn
72
References
465
which has been developed reveals much about glycoside reactions and also demonstrates the difficulty in stereoselective formation of glycosidic bonds. For everyday use the PMB method seems very attractive; it is the only method extended to solid-phase chemistry. So far, however, interest has been shown only in the formation of P-mannopyranosides and fLfructofuranosides, whereas the use of silyl tethers been very well documented and successfully used in stereoselective formation of a variety of glycosidic linkages. Methods in which the glycosyl acceptor contains more then one unprotected hydroxyl group have occasionally been shown to be stereoselective but the selectivity is relatively unpredictable. Occasionally, however, theoretical calculations have been consistent with the results obtained.
References 1. P. Preuss, K-H. Jung, R. R. Schmidt, Liebigs Ann., 1992, 377-382. 2. M. Nakata, T. Tamai, T. Kamio, M. Kinoshita, K. Tatsuta, Tetrahedron, 1994, 35, 19, 30093102. 3. M. Nakata, T. Tamai, T. Kamio, M. Kinoshita, K. Tatsuta, Bull. Chem. Soc. Jpn., 1994, 67, 3057- 3066. 4. F. Barresi, 0. Hindsgaul, J. Am. Chem. Soc., 1991, 113, 9376-9378. 5. F. Barresi, 0. Hindsgaul, Synlett, 1992, 759-761. 6. F. Barresi, 0. Hindsgaul, Can. J. Chem., 1994, 72, 1447-1465. 7. Y. lto, T. Ogawa, Angew. Chem. Int. Ed. Engl., 1994,33, 17, 1765-1767. 8. Y. Oikawa, T. Yonemitsu, Tetrahedron Lett., 1982, 23, 885-888. 9. R. Johansson, B. Samuelsson, J. Chem. Soc. Chem. Commun., 1984,201-202. 10. A. Dan, Y. Ito, T. Ogawa, J. Org. Chem., 1995,60,4680-4681. 11. A. Dan, Y. Ito, T. Ogawa, Tetrahedron Lett., 1995, 36, 41, 7487-7490. 12. A. Dan, M. Lergenmiiller, M. Amano, Y. Nakahara, T. Ogawa, Y. Ito, Chem. Eur. J., 1998,4, 11, 2182-2190. 13. Y. Ito, Y. Ohnishi, T. Ogawa, Y. Nakahara, Synlett, 1998, 1102-1 104. 14. Y. Ito, T. Ogawa, J. Am. Chem. Soc., 1997, 119, 5562-5566. 15. C. Krog-Jensen, S. Oscarson, J. Org. Chrm., 1996, 61, 4512-4513. 16. C. Krog-Jensen, S. Oscarson, J. Org. Chem., 1998, 63, 1780-1784. 17. M. Bols, T. Skrydstrup, Chem. Rev., 1995, 95, 1253-1277. 18. G. Stork, G. Kim, J. Am. Chem. SOC.,1992,114, 1087-1088. 19. G. Stork, J. J. la Clair, J. Am. Chem. SOL..,1996, 118, 247-248. 20. M. Bols, J. Chem. SOC. Chem. Commun., 1992,913-914. 21. M. Bols, J. Chem. Soc. Chem. Commun., 1993, 791-792. 22. M. Bols, Tetrahedron, 1993, 49. 44, 10049-10060. 23. M. Bols, Acta Chern. Scand., 1993, 47, 829-834. 24. J. Madsen, M. Bols, Angew. Chem., 1998, 110, 22, 3348-3350. 25. M. Bols, H. C. Hansen, Chem. Lett., 1994, 1049-1052. 26. M. Bols, Acta Chem. Scand., 1996, SO, 931-937. 27. M. E. Behrendt, R. R. Schmidt, Tetrahedron Lett., 1993, 34, 42, 6733-6736. 28. C. Mukai, T. Itoh, M. Hanaoka, Tetrahedron Lett., 1997, 38, 26, 4595-4598. 29. T. Iimori, T. Shibazaki, S. Ikegami, Tetrahedron Lett., 1996, 37, 13, 2267-2270. 30. G. Scheffler, R. R. Schmidt, Tetrahedron Lett., 38, 17, 2943-2946. 31. T. Ziegler, G. Lemanski, A. Rakoczy, Tetrahedron Lett., 1995, 36, 49, 8973-8976. 32. T. Ziegler, A. Ritter, J. Huttlen, Tetrahedron Lett., 1997, 38, 21, 3715-3718. 33. T. Ziegler, R. Lau, Tetrahedron Lett., 1995, 36, 9, 1417-1420. 34. R. Lau, G. Schiile, U. Schwaneberg, T. Ziegler, Liebigs Ann., 1995, 1745-1754. 35. G. Schule, T. Ziegler, Liebigs Ann., 1996, 1599-1607.
466
18 Intramolecular Glycosidution Reactions
36. T. Ziegler, G. Lemanski, Eur. J. Chem., 1998, 163-170. 37. T. Ziegler, G. Lemanski, Angew. Chem., 1998,110, 22, 3367-3369. 38. S. Valverde, A. M. Gomez, A. Hernandez, B. Herradon, J. C. Lopez, J. Chem. SOC. Chem. Commun., 1995, 2005-2006. 39. S. Valverde, A. M. Gomez, J. C. Lopez, B. Herradon, Tetrahedron Lett., 1996, 37, 7, 11051108. 40. H. Yamada, K. Imamura, T. Takahashi, Tetrahedron Lett., 1997,38, 3, 391-394. 41. U. Huchel, R. R. Schmidt, Tetrahedron Lett., 1998,39, 7693-7694.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
19 Classics In Total Synthesis of Oligosaccharides and G1ycoconjugates Jean-Maurice Mullet and Pierre Sinuy
19.1 Introduction Several chapters in this volume illustrate various selected methods which have been developed over the years to achieve efficient glycosylation reactions. It is quite amazing to witness the progress which has been made in the art of oligosaccharide construction, probably the most central challenge in carbohydrate chemistry. Our aim in this brief chapter is to highlight these achievements and illustrate various strategical aspects such as the judicious choice of protecting groups and of the activation of the anomeric centre, while describing a few ‘classic’ syntheses of complex oligosaccharides of potential biological importance. One inherent difficulty associated with such a chapter is the selection of what should be considered as ‘classic’ in the art of synthetic oligosaccharide construction. We are sure that the carbohydrate community will understand that many alternative brilliant accomplishments may have equally been selected.
19.2 Syntheses of Nod factors 19.2.1 Introduction
The two Nod factors 1 and 2 (Scheme 1) are secreted by bacteria fixing atmospheric nitrogen [ l , 21. The synthetic challenge associated with 1 or 2 can be summarized as follows: how to selectively differentiate, as shown in Scheme 2, hydroxyl and amino groups present in a chitin type fragment. More precisely, the strategical problems inherent to the total synthesis of Nod factors are as follows:
468
19 Classics In Total Synthesis of Oligosaccharides and Glycoconjugates OS03Na
H HO O&o&,O& HO O~oH HN
AcHN
HO
HO AcHN
-
O
1 2
AcHN
A
R=H R=Ac
Scheme 1. The structure of two classical Nod factors.
0-acetylation (Nod 2 only)
sulfation
HO HO NH2
NH2
NH2
Scheme 2. What has to be achieved to build up the Nod factors.
1. The four primary hydroxyl groups have to be carefully differentiated; the presence of a sulfate group and, in compound 2, of an additional acetate, forces the introduction of suitable temporary protecting groups on the primary positions. 2. Similarly, one amino group has also to be differentiated from the others. 3. Last, but not least, the presence of double bonds in the final product precludes the classical use of hydrogenation conditions, subsequent to the introduction of the unsaturated chain. This means for example that the very classical benzyl ether cannot be used as a protecting group in the last steps. This kind of problem is similar to the one met in the synthesis of sphingoglycolipids where a double bound is also present in the sphingosine moiety. 19.2.2 The K. C. Nicolaou Synthesis (1992) [3] The key protected tetrasaccharide to be synthesized in this approach is compound 3, the retrosynthetic analysis being presented in Scheme 3. This strategy illustrates the classical elongation process from three monosaccharidic blocks 4, 5 and 6, starting from the protected reducing end, that is compound 4. The three required blocks 4, 5 and 6 are easily prepared from a common glucosamine precursor 7 (Scheme 4). There are two noticeable features in the assembly of the monosaccharidic blocks which have to be commented for the benefit of the practitioner. First the use of the
469
19.2 Syntheses of Nod factors ,OTBDMS
OTBDPS ' MB &$LOMP NPhth
M B & M & M & M & o M p
MB
NPhth
AcHN
AcHN
AcHN
3
,OMP F
,
$ &
- O c A MB NPhth 5
Scheme 3. Retrosynthetic analysis of the Nicolaou's strategy. Abbreviations: MP (p-methoxyphenyl), MB (p-methoxybenzyl), NPhth (N-phtalimido), TBDMS (tertbutyldimethylsilyl), TBDPS (tertbutyldiphenylsilyl).
phtalimido group (see specific comments in Box 1) and second the use of glycosyl fluorides for the construction of the glycosidic linkage. The combination Cp2MC12AgC104 (M = Zr, Hf) is highly efficient for the activation of glycosyl fluorides [4], Zr-system exhibiting the most powerful reactivity. In this piece of work, the silver perchlorate has been replaced by silver triflate (Scheme 5). The blockwise synthesis selected here allows the transformation in due time of three phtalimido groups into three N-acetyl groups (see compound 3) and of the last one into the required N-acyl group: the problem of differentiation of one amino group among four is thus easily solved, as illustated in scheme 6. The rational for the choice of the various protecting groups present in blocks 4, 5 and 6 - that is
A Ac c O A S P h
Aco&oAc Ac
NPhth
a
1
NPhth 7
steps
5 and 6
+1:n
LAcoAc
9
OMe
steps
4
Scheme 4. Reagents and conditions: a) PhSH, SnC14, CH2C12, 25"C, 74%; b) MeOC6€&0H, SnC14, CHzC12, 25 "C, 72%.
470
19 Classics In Total Synthesis of Oligosaccharides and Glycoconjugates
"'AF
OTBDPS
MB
+
OTBDPS
5
H*MB oMp
NPhth
4
5
AcO&O&& MB
NPhth
MB NPhth
NPhthOMP
OTBDPS
MB
NPhth 5
MB
+ MB
MB NPhth
6
A NPhth M
d
O NPhth M
P
11
OMP NPhth
MB NPhth
Id
12
H&ooMP&oMp
MB NPhth
M
Ic
Aco&&e
FS. . .
H
+
MB
MB
AcHN
AcHN
AcHN
13
Ie
M M & BM &M &G MB
OMP NPhth
AcHN
AcHN
AcHN
3
Scheme 5. Reagents and conditions: a) 5, AgOTf, Cp2ZrCl2, 2,6-ditertbutyl-4-methyl pyridine, MS 4A, CHzC12, 0-25"C, 56%; b) K2C03, THF, 25"C, MeOH; c) 5, AgOTf, Cp2HfCl2, 2,6-ditertbutyl-4-methyl pyridine, MS 4A, CH2C12, 0-25"C, 60%; d) N2H4, EtOH, PhH, 100°C then AczO, MeOH, CH2C12, 72%; e) 6, AgOTf, CpzHfCl2, 2,6-ditertbutyl-4-methyl pyridine, MS 4A, CH2C12,0-25 "C, 50%.
in the resulting protected tetrasaccharide 3 - is fully apparent when contemplating the chemical conversion of 3 into the two final targets 1 and 2, as illustrated in Scheme 6 . Final comments: An interesting feature of the Nicolaou's strategy is the judicious choice of three different protecting groups for the primary alcoholic positions, which on one hand withstand the glycosylation conditions, and on the other hand can be sequentially removed. Such a nicely tuned strategy indeed allows the only synthetic access described so far to the acetylated Nod factor 2. Weaker points are the moderate yields resulting from the use of the fluoride glycosylation method and
19.2 Syntheses of Nod factors M B o g M & M & M & o M p
a
3
47 1
MB NHZ
AcHN ,4
AcHN
AcHN
M B o g M & MB&M&M&Mm?DFg -
RCOOH
MB
b
HN 0
-
AcHN
AcHN
-
AcHN
15
C
OMP
MB
H 0
-
AcHN
AcHN
-
AcHN
16
d
M B o & M & M & M & OMP
MB HN - - - - - -O
e --t
2 (Nod-factor 2)
AcHN
f
AcHN
-
AcHN
17 1 (Nod-factor 1)
Scheme 6. Reagents and conditions: a) N2H4, EtOH, lOO"C, 87%; b) RCOOH (3eq), 2-chloro-lmethyl pyridinium iodide, Et3N, CH3CN, 25 "C, 73%; c ) PPTS, EtOH, 25 "C then Ac20, Et3N, DMAP (cat), CH2C12, 2 5 T , 72%; d) TBAF, THF then Me3NSO3, Pyr. 25"C, 85%; e) CAN, CH3CN, H20, 25"C, 30%, f ) MeONa, MeOH, 2 5 T , 75%.
the rather low yield of the simultaneous removal of MB and MP protecting groups on a complex molecule. 19.2.3 The J.-M. Beau Synthesis (1994) 112)
This is another example of synthesis of an oligosaccharide through stepwise addition of monosaccharide blocks. But in sharp contrast to the previously described achievement, it is representative of a nonconventional construction of a linear oligosaccharide starting from the nonreducing end of the oligomer. The protected tetrasaccharide is built up from two precursors as shown in Scheme 7. The detailed synthetic route is shown in Scheme 8.
472
19 Classics In Total Synthesis of Oligosaccharides and Glycoconjugates
19.2 Syntheses of Nod factors ,OPiv
l
,OPiv
,OPiv
473
,OAc
18
,OAc
Scheme 7. Retrosynthetic analysis of the Beau’s strategy. Abbreviations: Z (benzyloxycarbonyl), Bn (benzyl), Piv (Pivaloyl).
The 1,2-trans selectivity of the first glycosylation reaction is insured by a carbamate participating group, which is not so frequently used (see Box 2) compared to the now very classical phthalimido group. It is worth noting that the acetolysis of l16-anhydrosugars is a rather smooth process, compatible with glycosidic linkages present in the molecule. Such a behaviour qualifies the 1,6-anhydro ring for this rather atypical type of left to right elongation. A feature of this strategy is the daring choice of a nonparticipating azido group at C-2 to successfully achieve a 1,2-trans glycosylation through a clean SN2 process. There are now more and more examples of this, so that such an option should not be disregarded in a synthetic strategy (see Box 3). Looking at the final steps of this strategy (Scheme 9), it is apparent that the fatty acid chain is introduced at the very last step, permitting the use of benzyl ethers as temporary blocking groups. Final comments: It has been stated in the previous section (see Box 1) that N-phthalimido group insures a perfect (3-selectivity, but is sometimes difficult to remove. Especially, such a removal is not compatible with acetates. The J.-M. Beau synthesis highlights an attractive option: the use of the nonparticipating azido group, the selectivity being intrinsically insured by a clean S N process ~ under appropriate experimental conditions. 19.2.4 The T. Ogawa Synthesis (1994) 1161
The strategy designed by T. Ogawa et al. uses three blocks, one being a disaccharide donor (Scheme 10). This strategy (Scheme 10) is rather close to that described by K. C. Nicolaou. The central block 37 has been prepared from a chitobiose derivative (171. The option of the N-phthalimido participating group has also been selected (see Box 1). The in-
19 Classics In Total Synthesis of Oligosaccharides and Glycoconjugates
414
B KBn r & & , ZHN
&g a
+
OCNHCC13
OH
19
C
N3
N3
21R=Ac 22R=Piv
NHZ
-
B"&m+
Bn&o+
d
Bn
NHZ
NHZ
N3 OAc
23
N3 OCNHCCI,
24
R
20
0
__t
NHZ
e
bl
Bn
20
Bn
*n&O
25
B Bn n & B * BNHZ 4
N3
f
N,
20 __t
'
26
"&&Bn ~&o
'"OCNHCCI,
9
N3 NHZ
N3
h
1
27R=Ac 28R=Piv
N3
Scheme 8. Reagents and conditions: a) BF3 EtzO, Toluene, -78°C > -2O"C, 91%; b) MeONa, MeOH then PivC1, DMAP, Pyr. 2 0 T , 100%; c) Ac20, TFA, rt; d) BnNH2, Et20, rt, then CCl3CN, DBU, CH2C12, rt, 73% 3 steps; e) 20, BF3 EtzO, CHzC12 -78°C > -2O"C, 85%; f ) Ac20, TFA, rt; 73%; BnNH2, Et20 rt, then CC13CN, DBU, CH2C12, rt, 73% 3 steps, g) 20, BF3 Et20, CH2C12 -78°C > -2O"C, 91%; h) MeONa, MeOH then PivC1, DMAP, Pyr. 20"C, 100%.
troduction of the unsaturated fatty acid is performed at the very last step, a feature which allows the initial protection with benzyl ethers (see also J.-M. Beau). The protected tetrasaccharide is obtained as described in Scheme 11. The deprotected tetrasaccharide 34 is easily obtained as depicted in Scheme 12 and is identical to the one previously obtained by J.-M. Beau. Final comments: As persistently stated in this chapter, the stability of protecting groups during the removal of N-phthalimido has to be carefully considered while selecting a strategy. This work points out the stability of a primary sulfate function during hydrazinolysis of the N-phthalimido group (Scheme 12).
19.2 Syntheses of Nod factors
A
A
C
O
S
S
~+
HJ
COW
NH-CO-Ct+CCI,
EE rt
-r
415
AK Al-Jg&Jcorm
NH-CC-CH&CI~ 85%
19.2.5 The Y. Z. Hui Synthesis (1992) [lS] The strategy used by Hui et al. illustrates the so-called block synthesis, more precisely the synthesis of a protected tetrasaccharide from two appropriate disaccharidic moieties, as shown in Scheme 13. The disaccharidic glycosyl donor 49 was first prepared as described in Scheme 14. As previously pointed out in Box 3 and exemplified in J.-M. Beau et al. ~ approach, it is possible to achieve a 1,2-trans glycosylation through an S N process,
19 Classics In Total Synthesis of Oligosaccharides and Glycoconjugates
476
a
28
OR NHZ
C
N3
bJ
N3
N3
N3
N3
NHAc
OBn NHAc
NHAc
NHAc
NHAc
NHAc
29 R= AC 30 R = OCNHCC13
B n & B & & B &
OBn NHZ
N3 18
d
Bn&B&&Boi& NHZ
-
NHAc
Bn&B&&Boscg
31
.
8
OBn NHZ
NHAc
32
-----t f
Bn&Bn &B&&OBn NHZ
NHAc
33
-
H & & & &
9
OH NH2
AcHN
AcHN
h
AcHN
34
Scheme 9. Reagents and conditions: a) TFA, Ac20, 81%; b) BnNH2, Et20 then CC13CN, DBU, CH2C12;c) BnOH, BF3.Et20,toluene, 79%; d) NaBH4, NiC12, DME-EtOH, then Ac20, Fyr.; e) MeONa, MeOH, 52%; Me3NS03, DMF, 50"C, 98%; f ) MeONa, MeOH, 88% g) H2, Pd/C AcOEt, MeOH, H20; h) N-acylation 60%.
using a nonparticipating azido group at C-2. Another feature of this synthesis is the very classical choice of the ally1 group as a temporary protecting group of an anomeric centre. The disaccharidic acceptor 50 is obtained as described in Scheme 15. A noticeable aspect of this synthetic sequence is the use of a monochloroacetate protecting group, which is easily removed under mild conditions fully compatible with N-phtalimido and pivaloyl groups.
19.2 Syntheses of Nod factors
471
AcO AcO NPht
NHAc
NHAc
fl AcO AcO &F
NHAc
35
A BnO c o & O ~ CBnO N H C C I 3 NPht
NPht 37
36
HBnO o g
NPht
o
B
n
38 NPht
Scheme 10. Retrosynthetic analysis of the Ogawa's strategy.
AcO BnO
CNHCC13
+
-
HO BnO
MP
NPht
BnO NPht
NPht
39
40
41
NPht
PMP
b
I t "
AcO AcO*O*CNHCC13 BnO Rnn BnO &O&CNHCCl3 BnO NPht 37
AcO BnO NPht
NPht
d
OBnAcOAcO +F
BnO NHAc
*
-
A0&.& BnO NHAc
n
C
OBn NPht
NPht
HO
BnO
-
Ho*OBn4 O B Brio Brio 38 NPht
U
NPht
*
NHAc
e
43
35
Scheme 11. Reagents and conditions: a) BF3.Et20, CH2C12, -78 "C?82%; b) CAN, CH3CN, H20, then CC13CN, DBU; c) BF3.Et20, CH2C12, -78"C, 69%; d) N2H4, EtOH, then Ac20, MeOH, 99%; e) Cp2HfCl2, AgOTf, (CH2Cl)2,79%.
I9 Classics In Total Synthesis of Oligosaccharides and Glycoconjugates
478
35
a
AcO AcO NPht
NHAc
NHAc
OBn NHAc
NH2
NHAc 46
NHAc
OBn NHAc
1
45 R= S03Na
C
HO HO
d
HOHO &o
- & o &HOo g o H NH2
HO
AcHN
HO AcHN
AcHN
34
R4-h 47 0
Scheme 12. Reagents and conditions: a) CAN, CH3CN, H20; b) Et3NSO3, DMF, 50"C, 70% two steps, c) N2H4, EtOH; d) Pd-black, Hz, MeOH, H20, 88% two steps; e) MeOH, Hz0, 97%.
BnO BnO
N3
NPhth
BnO
Bn NPhth
NPhth
48
BnO BnO
-C(NH)CC13 N3
HBnO O & A OBnO B n
NPhth 49
Scheme 13. Retrosynthetic analysis of the Hui's strategy.
NPhth
50
NPhth
19.2 Syntheses of Nod factors
BnO% BnO
+
51
b
bC(NH)CC13
BnO BnO - &
a
HBnO O G , , ,
Bno&o&Al~ BnO
BnO
NPhth
N3
NPhth
52
& N3 O BnO 54 a
479
53
H NPhth L
BnO BnO &O&
N3 BnO
NPhth I
55
BnO-*BnO
BnO
NPhthO-C(NH)CCI,
N3 49
Scheme 14. Reagents and conditions: a) BF,.Et20, MS 4p\, CH2C12, -20"C, 61%; b) PdCI2, NaOAc, aq AcOH, 5O"C, 84%; c) (COC1)2,DMF (cat), 88%; d) CC13CN, DBU, CH2C12, 84%.
57
58
BnO
BnO
NPhth
59
O-C(NH)CC13 NPhth
60
DPiv Ho BnO *OBn
'\
61
HO BnO
BnO NPhth 50
NPhth
OBn NPhth
NPhth
62
Scheme 15. Reagents and conditions: a) ClCH2COC1, Pyr. O T , 88%; b) PdC12, NaOAc, aq AcOH, 50"C, 89%; c) (COC1)2,DMF (cat) ; 89%; d) CC13CN, DBU, CHzC12,85%;e) AgOTf, MS 4A, CHzC12, -1O"C, 58%; f ) BF3.Etz0, CHZC12, -15"C, 76%; g) Thiourea, Pyr, EtOH, 7 0 T , 86Yo.
480
19 Classics In Total Synthesis of Oligosaccharides and Glycoconjugates
The final steps of the synthesis are shown in Scheme 16. The imidate 49 appeared as a better glycosylationg agent than the chloride 55. It has to be pointed out that the final amine 34 is the one previously obtained by J.-M. Beau and T. Ogawa. Final comments: A recurrent theme in the synthesis of Nod factors is the differentiation of the terminal amino group. It is achieved here in a very simple way, using a combination of azido and N-phthalimido groups.
19.2.6 Conclusion There are always two questions when launching an oligosaccharide synthesis: 1. which protecting groups have to be used. 2. which glycosylation methods have to be selected. There are many glycosylating agents developed so far. Nevertheless, only two methods have been selected by the four groups: the fluoride method and the trichloroacetimidate method. As far as the protecting groups are concerned, a larger diversity has been exploited. A careful comparison of these four achievements illustrates many facets of the art of oligosaccharide synthesis.
19.3 Synthesis of the Antithrombin-Binding Pentasaccharide Sequence in Heparin (1984) [19, 201 19.3.1 Introduction Heparin is an extraordinary complex sulfated glycosaminoglycan (GAG) with well known anticoagulant and antithrombotic activities. The coagulation of blood is the result of a cascade of events implying a series of proteases called activated bloodcoagulation factors. Antithrombin (AT) is the main physiological inhibitor of these factors and heparin is known to bind to AT and to amplify its action. A breakthrough in heparinology came in the early 1980’s with the recognition [21, 221 of a specific pentasaccharide sequence within the polysaccharide, capable of binding to AT. The total synthesis of this pentasaccharide 67 (Scheme 17) has first been reported in 1984 [ 191, confirming the specific nature of the interaction heparin/AT. We can consider this selected piece of work as a typical classic because:
1. This is the first synthesis of an oligosaccharidic fragment of heparin with a prominent biological activity [23]. 2. It opened the door to a series of syntheses of other biologically active compounds containing variants of this pentasaccharide.
19.3 Synthesis of the Antithrombin-Binding Pentusacchuride Sequence 55
+
48 1
50
49 +50
b
BnO BnO N3
NPhth
NPhth
OBn NPhth
63
C
BnO BnO
N3
BnO
NHAc
NHAc
NHAc
NHAc
OBn NHAc
64 d __t
BnO BnO
N3
e
NHAc
65 HOHO +o
& o &HO o~:H NH2
HO
AcHN
L1
HO AcHN
AcHN
34
Scheme 16. Reagents and conditions: a) AgOTf, MS 4A, CH2C12, -2O"C, 50%; b) BF,.Et20, CH2C12, -15"C, 72%; c) N2H4, H20, EtOH, 9 5 T , then AczO, Pyr., 2 0 T , then KOH, MeOH, THF 20"C, 66% in 3 steps; d) Pyr.SO3, DMF, 50°C; e) Hz, 10% Pd/C, THF, EtOH, H20, 83%; f ) 66, Et3N (cat), EtOH, 45 "C, 45%.
3. This synthetic pentasaccharide and analogues are potential antithrombotic drugs devoid of certain detrimental side effects when compared with natural standard heparin [24].
19.3.2 An Overview of the Synthesis of the Protected Pentasaccharide 73 The synthesis, which is summarized in Scheme 18, is based on the use of three blocks: two disaccharides and one monosaccharide. The hydroxyl groups which are going to be sulfated are protected by acetyl groups, the hydroxyl groups being free in the final pentasaccharide are protected as benzyl ethers.
482
19 Classics In Total Synthesis of Oligosucchurides and Glycoconjugutes
OH
67
oso3-
Scheme 17. The structure of the specific heparin pentasaccharide sequence binding to AT.
19.3.3 Synthesis of the Disaccharidic Bromide Donor 68 The preparation of this bromide is described in Scheme 19. The bromide 75 is easily prepared from the corresponding hemiacetal using the Vilsmeier reagent. This represents a mild and convenient way to achieve this kind of transformation (see Box 4). The selective formation of a P-glucoside from 75 goes through a so called push-pull mechanism, that is an SN?process at the surface of an insoluble silver salt such as silver carbonate. This method complements the combination a-trichloroacetimidate/ BF3.Et20, which has been put into practice in the previous classic of this chapter.
19.3.4 Synthesis of the Disaccharidic Acceptor 69 The glycosylating species which has been used is the orthoester 79 (Scheme 20). This derivative of methyl L-iduronate is an interesting compound. The tertbutyl orthoester fulfills three functions: it insures the protection of positions 1 and 2, a necessary step for the selective synthesis of 81; it reacts with an alcohol under acidic conditions to give a 1,2-trans glycoside, with the concomitant introduction of an acetate function at position 2 (a progenitor of the sulfate group). This is an example of the orthoester procedure nicely developed by the Kotchetkov group in the 1960s [28]. Such a method is rarely used nowadays. The synthesis of 69 is presented in Scheme 20: the orthoester glycosylation is followed by the selective removal of the monochloracetate on position 4.
19.3.5 Synthesis of the Protected Pentasaccharide 73 The two glycosylation reactions 68 & 69 then 71 & 72 (see Scheme 18) illustrate the use of a bromide, in the presence of silver triflate, to synthesize an a-glycoside. It should be noted that the presence of a participating acetyl group at C-6 probably insures an excellent a-selectivity (Scheme 2 1). Without this participation, the re-
19.3 Synthesis of the Antithrombin-Binding Pentasaccharide Sequence
M Bn c A & A 4 BnO
q i
+ 68
N3Br
483
HO
1
69 ZHN OBn
OAc
a
O , Ac
70
I OAc
I b
t
.OAc
,OAc
72
lc 73
I
OAc
I OAc
Scheme 18. Reagents and conditions: a) AgOTf, collidine, CHZC12, -2O"C, 55%; b) thiourea, pyr. EtOH, 100 "C, 82%; c) AgOTf, collidine, CH2C12, -20 "C, 70%.
sulting selectivity is more difficult to predict and relies on subtle factors, such the nature of the solvent and the temperature. 19.3.6 Synthesis of the Active Site of Heparin
The final steps are shown in Scheme 22. This is now a classical sequence of reactions in the field of glycosaminoglycans. Final comments: With eight sulfonato groups on specific oxygen and nitrogen atoms of the molecule, with the presence of the very unusual L-iduronic acid residue, this molecule represents a formidable synthetic challenge. It is amazing to ob-
484
19 Classics In Total Synthesis of Oligosaccharides and Glycoconjugates
Bn
OH
BnO
-
Bn
Bn
---+
M~A&~& Bn
C
BnO
75
74
-
b 77
68
d
BnO 78
N3 O
A ~
Scheme 19. Reagents and conditions: BrCH=N+Me*, Br-, collidine, CH2C12, 0 "C, 90%; b) Ag2C03, MS 4 A , CH2C12, rt,70%; c) TFA, Ac20, rt, 86%; d) TiBr4, CH,Cl,/AcOEt, rt.
Box 4 Preparation of anomeric bromides
,OAc
79
\
80
Scheme 20. Reagents and conditions: a) 2,6-dimethylpyridinium perchlorate, PhCl, 130 "C, 40%; b) thiourea, Pyr. EtOH, lOO"C, 86%.
19.4 Totul Synthesis of VIM-2 Ganglioside
485
Scheme 21. A possible remote acetyl group participation in a glycosylation reaction.
,OAc
Scheme 22. Reagents and conditions: a) NaOH, CHCI3, MeOH, H20, rt then CH2N2 73%; b) Me3N:S03, DMF, 5 0 T , 90%; c) NaOH, MeOH, H20, 90%; d) H2, 10% Pd/C, MeOH,
H20, 93%; e) Me3N:SO3, H20 (pH = 9.5), 36%
serve that closely related analogues of this compound have now been prepared in bulk quantities as drug substances. Following excellent pharmacokinetics and tolerance studies in man [29], clinical investigations are currently underway to confirm the therapeutic potential of such complex carbohydrate derivatives. This is an excellent example of the power of organic synthesis in the field of glycoscience [30].
486
19 Classics In Total Synthesis of Oligosaccharides and Glycoconjugates
H
84
Scheme 23. Structure of a natural human sphingoglycolipid called VIM-2.
OTBDPS
f'
p- glycosylation
HO-p-13H27 N3
c
85
Reduction and fatty acid amidification
Scheme 24. Overall strategy used by Hasegawa et al. to synthesize VIM-2.
19.4 Total Synthesis of VIM-2 Ganglioside [31] 19.4.1 Introduction The so called VIM-2 ganglioside 84 (Scheme 23) is a natural sphingoglycolipid which is regarded as a tumor associated carbohydrate antigen. It has been isolated from human chronic myelogenous leukemia cells. Many compounds of this type have been synthesized. We would like to present in this section the achievement of the late Hasegawa. It is a typical example of the block synthesis approach of a sphingoglycolipid. 19.4.2 The Total Synthesis of VIM-2-a
General Strategy
A key step of the strategy is the glycosylation of compound 85 [32] (Scheme 24) with a complex glycosyl donor which is fully acylated. The presence of any benzyl ether protecting group is precluded at this point of the synthesis, due to the presence of an easily reducible double bond in compound 85; catalytic hydrogenolysis should be avoided. In the case of VIM-2, the fully acylated glycosyl donor is the trichloroacetimidate 87, which has been obtained from the 2- (trimethylsilyl)ethyl glycoside 86 (Scheme25). The 2-(trimethylsilyl) ethyl glycoside, popularized by G. Magnusson 1331 is often used as a temporary protecting group of the anomeric position, and is easily trans-
19.4 Total Synthesis of VIM-2 Ganglioside B
z
W
~
Bzo' OBz
OAcAc
NHAc 86
a, b
~
Ac OAc
487
&
~
dOAc 1 NHAc
OAcAc
0 OAc
OAc OAc
Scheme 25. Reagents and conditions: a) CF3COOH/CH2C12; b) CC13CN, DBU.
formed in a two step sequence here exemplified in an efficient glycosyl donor. It nicely complements in this respect the ally1 group (see the Hui's synthesis of the Nod factor).The main part of the synthesis is thus the preparation of the 2-(trimethylsilyl) ethyl glycoside 86. Let's now analyse the main characteristics of this achievement. 19.4.3 Preparation of the Key Protected Octasaccharide 86
A protected trisaccharidic Lewis X thiophenyl glycoside 92 is prepared as shown in Scheme 26. Several features of this synthesis have to be pointed out. In the diol89, the position 4 is more reactive than position 3, so that there is no need for a temporary protection at position 3, an observation which simplifies the synthetic scheme. This is due to the presence of the bulky neighbouring phthalimido group. This diol is thus an interesting pivotal acceptor for a double glycosylation sequence. The thiophenyl glycoside 89 can be glycosylated with the bromide 88 (so called orthogonality, which has not always been perfectly observed in this case). The bromide 88 is benzylated at the primary position, a feature which will be commented later on. The thiophenyl Lewis X trisaccharide 92 is now condensed without any problem with the easily available alcohol 93 to give the pentasaccharide 94, as shown in Scheme 27. De-0-benzoylation of 94 gave the triol 95. This triol is next glycosylated with the previously reported thiophenyl glycoside 90, as shown in Scheme 28. This critical step of the synthesis demonstrates that the
./
32
488
19 Classics In Total Synthesis of Oligosaccharides and Glycoconjugates
+
H&SPh
Bz
NPhth
Bzo Br
aa
a9
a
BZ 90
Bzs G
b
Bz
h
OBz
?
O
B
M
T
h
n
OBn OBn 92
Scheme 26. Reagents and conditions: a) AgOTf, toluene, -4O"C, 75%; b) NIS, TfOH, toluene, - 15 "C, 93%.
93
W
O
B
n
94
OBn OBn
Scheme 27. Reagents and conditions: a) NIS, TfOH, CH&, 98%.
-6O"C, 86%; b) MeONa, MeOH,
19.4 Total Synthesis of VIM-2 Ganglioside
90+95
-
489
a
BZ OBn
96 b
NHAc OBn
97
Scheme 28. Reagents and conditions: a) NIS, TfOH, CH2C12, -6O"C, 83% b) N2H4, EtOH, H 2 0 , reflux, then AczO, MeOH, 40 "C, 93%.
trio1 95 can be selectively glycosylated at position 3 , a feature which again considerably simplifies the strategy. We now understand why the primary position of the terminal galactosyl residue should be benzylated, and not benzoylated (see compound 88). The more reactive primary hydroxyl group has to be protected by a persistent blocking group. At this stage, the N-phthalimido groups are converted into N-acetyl groups, an operation which is more problematic when the sialyl residue is present. The sialylation of the polyol 97 is finally most easily achieved using the sialyl donor 98, as shown in Scheme 29. It is remarkable that the regioselective sialylation of the required hydroxyl group is achieved in 72% yield, in the presence of several non protected hydroxyl groups. Final steps are now uneventful and are shown in the last Scheme 30. Final comments: Such an overall strategy can be utilized to synthesize a variety of oligosaccharides of the polylactosamine type, with additional possible L-fucose residues. It is thus of general interest and can be considered as a classic. Many considerations in this synthesis have also been delineated by other groups, and this achievement reflects a nice strategical manipulation of many interesting observations done over the years.
19.5 Epilogue The aim of this chapter was to convince the reader that the chemical organic synthesis in solution of oligosaccharides of any complexity is now feasible. More than
490
19 Classics In Total Synthesis of Oligosaccharides and Glycoconjugates
97 +
BzM C02Me
a_
OBz
98
AcH
99
b
86
Scheme 29. Reagents and conditions: NIS, TfOH, CH3CN, -43"C, 72%).b) H2, Pd/C, MeOH/ EtOH/AcOH, then Ac20, Pyr., 63%.
85
+
87
AcH
OAc OAc
100
Scheme 30. Reagents and conditions: a) TMSOTf, CH2C12, 0"C, 47%; b) Ph3P, PhH, H20; c) octadecanoic acid, 2-chloro-1,3-dimethylimidazolium chloride, PhH. 80%; d) NBQF, CH3CN; e) MeONa, MeOH then water, 87%.
enough glycosylation methods are now available, and a great deal is known about the control of the selectivity. Nowadays, organic chemists are more than ever in a position to collaborate with glycobiologists, to contribute to the development of gly coscience. References 1. P. Lerouge, P. Roche, C. Faucher, F. Maillet, G. Truchet, J.-C. Promt, J. Denarie, Nature, 1990,344, 781.
References
49 1
2. P. Roche, P. Lerouge, C. Ponthus, J.-C. Prome, J. Biol. Chem., 1991,266, 10933; G. Truche, P. Roche, P. Lerouge, J. Vasse, S. Camut, F. De Billy, J-C. Prome, J. Denarie, Nature, 1991, 351, 670; M. Schultze, B. Quiclet-Sire, E. Kondorosi, H. Virelizier, J. N. Glushka, G. Endre, S. Gero, A. Kondorosi, Proc. Nat. Acud. Sci. USA, 1992, 89, 192. 3. K. C. Nicolaou, N. J. Bockovich, D. R. Carcanague, C. W. Hummel, L. F. Even. J. Am. Chem. Soc. 1992,114, 8701. 4. T. Matsumoto, H. Maeta, K. Suzuki, G.4. Tsuchihashi, Tetrahedron Lett., 1988, 29, 3567. 5. S. Akira, T. Osawa, Chem. Pharm. Bull., 1960, 8, 583. 6. R. U. Lemieux, T. Takeda, B. Y. Chung, In Synthetic Methods For Carbohydrates, Ed. Hs. El. Khadem, ACS Symposium Series. 39, 1976, 99. 7. P. L. Durette, E. P. Meitzner, T. Y. Shen, Tetruhedron Lett., 1979, 4013. 8. 0. Kanie, S. C. Crawley, M. M. Palcic, 0. Hindsgaul, Curbohydr. Res. 1993, 243, 139. 9. D. R. Bundle, S. Josephson. Can. J. Chem., 1979, 57, 662. 10. J. S. Debeham, R. Madsen, C. Roberts, B. Fraser-Reid, J. Am. Chem. Soc., 1995, 117, 3302. 11. a) J. C. Castro-Palomio, R. R. Schmidt, Tetrahedron Lett., 1995, 36, 5343. b) Zbid, Liebigs Ann., 1996, 1623. 12. a) D. Tailler, J.-C. Jacquinet, J.-M. Beau, XVltlz Intern. Carbohydr. Symp., Paris 1992, A298; b) D. Tailler, J.-C. Jacquinet, J.-M. Beau. J. Chem. Soc, Chem. Commun. 1994, 1827. 13. a) H. Paulsen, B. Helpap, Carbohydr. Res., 1991,216, 289. b) D. Tailler. J.-C. Jacquinet. A-M. Noirot, J. M. Beau, J. Chem. Soc. Perkin. I , 1992, 3163. c) M. Schultz, H. Kunz, Tetrahedron Lett. 1992,33, 5319. d) E. Kaji, F. Lichtenthaler, Y. Osa, S. Zen, Bull. Soc. Jp., 1995,68, 1172. e) A. Vargas-Berengel, M. Meldal, H. Paulsen, K. Bock. J. Chem. Soc. Perkin 1, 1994, 2615. 14. J. Banoub, P. Boullanger, D. Lafont, Chem. Rev., 1992, 92, 1167. 15. W. Kinzy, R. R. Schmidt, Carbohydr. Rex, 1989, 193, 33. 16. S. Ikeshita, A. Sakamoto, Y. Nakahara, Y. Nakahara, T. Ogawa. Tetrahedron Lett., 1994, 3123. 17. H. Kuyama, Y. Nakahara, T. Nukada, Y. Ito, Y. Nakahara, T. Ogawa, Carbohydr. Res. 1993, 243, C1. 18. a) L. X. Wang, Y. Z. Hui, XVIth Intern. Carbohydr. Symp., Paris A203. 1992; b) L. X. Wang, C. Li, Q. W. Wang, Y. Z. Hui, Tetrahedron Lett., 1993, 34, 7763; c) Ihid., J. Chem. Soc. Perkin I, 1994, 621. 19. P. Sinay, J.-C. Jacquinet, M. Petitou, P. Duchaussoy, I. Lederman, J. Choay, Carbohydr. Rex, 1984, 132, C5. 20. M . Petitou, P. Duchaussoy, I. Lederman, J. Choay, P. Sinay, J.-C. Jacquinet, G. Torii, Curbohydr. Res. 1986, 147, 221. 21. J. Choay, J.-C. Lormeau, M. Petitou, J. Fareed, Ann. N. Y. Acad. Sci. 1981, 370, 644. 22. L. Thurnberg, G. Backstrom, U Lindahl, Carbohydr. Res. 1982, 100, 393. 23. see also: C. A. A van Boeckel, T. Beetz, J. N. Vos, A. J. M. de Jong, S. F. van Aelst, R. H. van den Bosch, J. M. R. Mertens, F. A. van der Vlugt, J. Carbohydr. Chem. 1985, 4, 293. 24. M. Petitou, J.-P. Herault, A. Bernat, P.-A. Driguez, P. Duchaussoy, J.-C. Lormeau, J.-M. Herbert Nature, 1999, 398, 417. 25. D. R. Hepburn, H. R. Hudson, J. Chem. Soc. Perkin 1, 1976, 754. 26. H. Paulsen, C. Kolar, W. Stenzel, Chem. Ber. 1978, 111, 2358. 27. F. W. Lichtenhaler, E. Kaji, S. Weprek, J. Org. Chem. 1985, 50, 3505. 28. N. K. Kochetkov, A. J. Khorlin, A. F. Bochkov, Tetrahedron, 1967, 26, 693. 29. B. Boneu, J. Necciari, R. Cariou, A.M. Gabaig, G. Kieffer, J. Dickinson, G. Lamond, H. Moelker, T. Mant, H. Magnani, Thromb. Huemost., 1995, 74, 1468. 30. P. Sinay, Nature, 1999, 398, 377. 31. T. Ehara, A. Kameyama, Y. Yamada, H. Ishida, M. Kiso, A. Hasegawa, Curbohydr. Res., 1996,281; 237. 32. A. Hasegawa, T. Nagahama, H. Ohki, M. Kiso, J. Carbohydr. Chem., 1992, 11, 699. 33. K. Jansson, S. Ahlfors, T. Frejd, J. Kihlberg, G. Magnusson, J. Org. Chem. 1988, 53, 5629.
Part I Volume 1
I1 Synthesis of Oligosaccharide Mimics
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
20 Synthesis of C-Oligosaccharides Troels Skrydstrup, Boris Vnuzeilles, and Jean-Marie Beau
20.1 Introduction Cell-surface carbohydrates are well known for participating in key molecular recognition events with protein receptors [l]. Such interactions can be beneficial, as in cell-to-cell recognition and binding, or detrimental, as in bacterial [2] and viral infections 131, and inflammation [4]and metastasis 151, the initial steps of which can involve carbohydrate-protein interactions. To combat such diseases it would be ideal to prepare small, soluble carbohydrate-based pharmaceuticals which inhibit these recognition events by binding preferentially to the protein receptors. The potential instability of such small, soluble oligosaccharides to extra- and intracellular glycosidases has, however, prompted many chemists to develop hydrolytically stable carbohydrate mimics [ 6 ] . C-Glycosides are an important class of such glycomimetics, and are defined as carbohydrates in which the interglycosidic linkage has been substituted by a methylene group. This minor but important modification renders these analogs completely resistant to hydrolysis [7]. Because of their close analogy with the parent 0glycoside, C-disaccharides and C-oligosaccharides have become important compounds for study of carbohydrate-protein interactions in lectins and glycosidases. The incorporation of a methylene group in the glycosidic linkage not only enables binding of the C-oligomer to the receptor without sensitivity to hydrolysis but might also give information about the conformation of the bound sugar around the glycosidic linkage by 'H NMR analysis [8, 91. Realizing that C-glycosides seem to be promising drug candidates against the above detrimental cellular interactions, or are important biological tools for the study of carbohydrate-protein interactions in lectins and glycosidases 19, lo], several groups have developed interesting methodologies for the construction of such compounds. The synthesis of these compounds is not always easy, considering that a C-C bond generation is required not only at the terminal sugar but also at the reducing end.
496
20 Synthesis of C-Oligosuccharides
In this review the different methods for the synthesis of C-disaccharides and higher C-oligomers will be highlighted, covering the literature to the end of 1998. The synthetic approaches to these glycomimetics will be divided into four chapters depending on whether the methodology applied by individual groups was: 1) by direct coupling of intact carbohydrate units via (i) anionic or (ii) radical pathways; 2) by the de novo synthesis of one .or all of the sugar units in the C-disaccharide or C-oligomer; or 3) by applying a cycloaddition reaction to connect the two sugar units. We include only work which represents a true mimic of the sugar, i.e., mimics with the correct number of linking carbons and endocyclic oxygen atoms. A great many series of disaccharide analogs have been synthesized either having no carbon linkage or one carbon more than the correct mimic, or where the ring oxygen has been replaced by nitrogen [ 1 I , 121. For these studies the reader is referred to several other reviews [7, 131.
20.2 The Anionic Approach 20.2.1 C5-Alkynyl Anions An approach to the first synthesis of a true C-disaccharide was reported by Rouzaud and Sinay in 1983 [14]. Having previously worked on the synthesis of C-(alkyny1)-Pglucosides [ 151, the Sinay group took advantage of this chemistry for the synthesis of a Glc(P1-6)Glc C-disaccharide employing a C5-alkynyl anion as shown in Scheme 1. This method is highly effective for the construction of 1,6-linked C-disaccharides owing to the ease of preparation of the starting sugars and the high-yielding coupling steps exemplified below. The glucose derivative 1 was converted in two steps
4
BnO BnO
1. DMSO, (COC1)2,
Br
2. CBq, PPh3 0
BnO OMe 2
1
2+3
-
BuLi BnO 92% B
n BnoOH
BnO O 4
Scheme 1.
OMe
OBn 3
2. 1. EgSiH, H,, PdKBF3.Et20 HHO O L h BnO 52% HO OMe
(
o
i 0
5
l
20.2 The Anionic Approach
BuLi 0
BnO BnO 6
75%
BnO
497
BnO
7
8 1. Et,SiH, BF,.OEt, 2. Hz, Pd/C
74%
OH
I 6, BuLi 67% 1 Me0
9
APBn
n..
Bnow BnO[uB:
OBn
BnO BnOW
\ \O
O
OBn B
*
n
HO
74%
OMe HO
4 steps 29%
BnO B n
0
&
~
&
OBn
BnO BnO BnO BnO
'?
OBn
BnoOH
H
OBn 71% 12
Scheme 2.
to the vinyl dibromide 2 which was transformed to the acetylenic anion with 2 equiv. BuLi at -50 "C. After treatment with the glucopyranolactone 3, the hemiacetal4 was obtained in high (92%) yield. Stereospecific reduction of the hemiacetal with Et3SiH and BF3.Et20 involving hydride attack on the u face of a cyclic oxonium ion intermediate then afforded the b-C-disaccharide 5 after catalytic hydrogenolysis. Applying this methodology, the same group was able to prepare the Ctetrasaccharide 12 in an iterative sense, in addition to the dimer 9 and trimer 11 obtained from intermediates along the synthetic route (Scheme 2) [ 161. Starting this time with the vinyl dibromide 6 and galactopyranolactone 7, a similar reaction sequence as described above was adopted for the preparation of the protected Cdisaccharide 8. After removal of the anomeric hydroxyl group and selective reduc-
OMe HO
498
20 Synthesis of C-Oligosaccharides 1. AllylMgBr
3. oso4, 2. MeZC(OMe)Z, NMO H+
mu-n OBnOG0 B BnO BnO 0 3
BnO% BnO
~
-0BnO
BnO OH 13
84% 84%
6 8 I
0
'
A
+ diastereomer (2:3)
PivCI, Pyr BnO,
B n O S
2, BuLi NaOMe, MeOH HCI C1C02CCI,, Py
86%
OPiv
B n O p un
15
36%
-)-0
14
0
1. C02(CO)* 2. BF,:OEt, 3. CAN
55%
I. TEA, Py, H20 2. PdC, H?
RO- ' "
Ro
17 R=H
OMe
(a$=1:l)
8R
Aczo'Py
R=Ac(9l%)
16
Scheme 3.
tion of the alkyne functionality, a two-step hydrolysis and oxidation sequence gave the corresponding lactone which could be coupled again with 6 to give a Ctrisaccharide intermediate 10. In the same manner, these steps were repeated and followed by a deprotection step to produce the C-tetrasaccharide 12, the parent 0glycoside of which is known as a plant elicitor. Schmidt and coworkers have recently extended this approach for the synthesis of C-disaccharides of ketoses as depicted in Scheme 3 [ 171. In their attempt to prepare a glycosyl transferase inhibitor the Schmidt group reacted the Sinay intermediate 2 with the hemiacetall3, readily prepared in three steps from the glucopyranolactone 3. Although, no coupling was observed in this case, the reaction proceeded when 13 was first transformed to the open-chain compound 14 with pivaloyl chloride and then treated with the alkynyl anion. This led to the formation of two propargylic alcohols, as a 3 :2 diastereomeric mixture in a 61% yield, which were subjected to protecting group manipulations to give 15. Ring closure was best achieved by per-
20.2 The Anionic Approach
499
forming a Nicholas reaction whereby the alkynes were first transformed to their dicobalthexacarbonyl complexes. Thus treatment of either diastereomer with BF3.Et20 readily led to the formation of a stabilized carbocation with immediate cyclization to give a 1 : 1 mixture of a : p anomers 16 (only a anomer shown) after decomplexation with cerium ammonium nitrate. A two-step deprotection protocol of either anomer then gave the corresponding unprotected C-disaccharides 17 (only a anomer shown). Interestingly, with the diastereomer of ketone 14 only one stereoisomer was obtained upon reaction with the vinyl dibromide 2. In addition, ring closure of the coupling product is highly selective, affording an a : p mixture in a 10: 1 ratio. Employing the monosubstituted alkyne 19, easily prepared from vinyl dibromide 2, van Boom recently developed a stereoselective ZnC12-assisted ring-opening of epoxide 18, enabling rapid access to the C-isomaltoside 21 via the alkyne 20 (Scheme 4) [18]. This surprising cis selectivity observed in the epoxide opening is rationalized by the initial formation of a zinc acetylide species (see Scheme 5), followed by an intramolecular delivery of the alkyne moiety in the ion-pair 23 or the p chloride 24. A Nicholas reaction [ 191 involving the triflic acid-induced isomerization [20] of the dicobalthexacarbonyl complex of alkyne 20 can be performed to access the p anomer 22.
4
BnO BnO
+
BuLi, ZnC1, BnO
0
BnoOMe
18
59%
19 O *:B i BnoOMe
H,, Pd/C
80%*
R
E
O
20
X
RoOMe
21 R=H Aczo’Py
R=Ac(92%)
BdOLMe
Scheme 4.
22
500
20 Synthesis of C-Oligosaccharides Sugar-Li
i
B
n
O
G
Sugar
OBn
*ZnCl
BnO 0
18
23
(OBn
20
24
Scheme 5.
20.2.2 C1-Glycal Carbanions An attractive method employing stabilized C1-glycal anions for the synthesis of pC-disaccharides was developed by Schmidt and coworkers (Scheme 6). The Konstanz group had observed earlier that the deprotonation of protected glycals is greatly facilitated by the presence of a phenylsulfinyl group at the C2 position [21]. To obtain a glycal derivative, such as 25, an easy two-step synthesis from tribenzylD-glucal was employed with a 63% overall yield. C1-substituted glycals are then formed by the coupling of the corresponding anion 26 with aldehydes. Reductive removal of the phenyl sulfinyl group and a stereospecific hydroboration/oxidation step affords P-C-glycosides 27 with an equatorial C2-hydroxyl group. This strategy was first reported in 1989 for the preparation of a C-cellobiose derivative as illustrated in Scheme 7 [22]. The electrophilic species was prepared
OBn 1. PhSCI; DBU BnO BnO&
0 2.mCPBA
63%
B g ( 3 0 q
LDA-100°C
Bpo)
Li
PhSO 25
PhSO 26
RCHO R PhSO 21
Scheme 6.
501
20.2 The Anionic Approach
HBnO O
1. DMSO, DCC 2. Ph3P=CH2 3. H3B.Me2S;H202
q
* 26%
BnOOBn 28
30
29
OBn
1. Raney Ni
25,LDA
BnO
___)
3. PdC, H2
74%
OH
OBn 31
32
Scheme 7.
starting from the appropriately protected glucoside 28. DMSO-assisted oxidation of the C4 hydroxyl group then Wittig reaction led to formation of an exocyclic alkene, which upon borane addition, employing the borane dimethylsulfide complex, and subsequent oxidation, afforded in modest yield the C-branched sugar 29 with the gulucto configuration in addition to the reversed borane addition product. Steric control from the C3 and C5 positions is undoubtedly the cause of the high stereoselectivity observed in the hydroboration step. Oxidation of 29 then gave the corresponding aldehyde which could be isomerized by mild basic treatment to the isomer 30 with the gluco configuration. Addition of the C1-anion to aldehyde 30 gave compound 31 in good yield with high diastereoselective control at the new stereogenic center (10 : 1). Transformation of the Cl-substituted glycal to the C-cellobiose derivative 32 then required a threestep sequence involving reductive removal of the phenylsulfoxide with Raney nickel, a stereoselective hydroboration-oxidation step, and finally catalytic hydrogenation. Since this preliminary example, the Schmidt group has adapted this approach for the construction of other p-C-glycosides employing either the 2-(phenylsulfinyl)-~glucal 25 or the galactal counterpart 33 (Scheme 8) [23-261. Many improvements BnO B
n
OBn O
e HO
PkO
HO
HO
35
OH 36
Scheme 8.
OMe
34
OH
HO
Ho I
HO
33
502
20 Synthesis of C-Oligosaccharides 1. BnBr, NaH 2. CS,, NaH; Me1
33 + 30
steps
4.H2. Pd/C
BnO
OH
OBn
Scheme 9.
were made to increase the yields of the synthesis of the electrophilic coupling partner, notably in the alkenylation and hydroboration/oxidation steps. It was found that Tebbe’s reagent gave good yields of the desired alkene where the Wittig reagent failed, whereas 9-BBN proved much more effective than borane, securing consistently higher regioselectivities in the hydroboration step. In one example a cyano group was used for the introduction of a C4-branched formyl glycoside [25], an approach which was also reported by Vasella [27] and Armstrong [28] for onecarbon homologations of uloses. Several hydroxy C-disaccharides, 34-36, have thus been prepared by this approach, as illustrated in Scheme 8. The presence of the hydroxyl group in the carbon linkage might even be an advantage in that it enables the introduction of other functional groups at this position, and provides a conformational preference around the two exocyclic C-C bonds. In one case, the hydroxy C-disaccharide was deoxygenated using the Barton protocol (NaH, CS2, MeI, then Bu3SnH, AIBN) providing an efficient overall entry to C-lactose 37 (Scheme 9) [24]. 20.2.3 Anomeric Samarium Species Application of a Cl-lithiated sugar with a protected C2-hydroxyl group, and its direct coupling with a carbonyl substrate at low temperature, does not lead to the formation of the C-glycoside owing to a rapid, competing p-elimination which leads instead to the corresponding glycal [29]. In contrast, anomeric samarium species have proven to be much more resistant to this process, even at room temperature, and hence have been used for the rapid assembly of C-disaccharides. In 1995, Skrydstrup and Beau reported the synthesis of the C-disaccharide derivative 40 of a-C-mannopyranosyl( 1-2)-~-glucopyranose, via the reductive samariation of the mannosyl pyridyl sulfone 38 in the presence of aldehyde 39 (Scheme 10) [30]. Because this aryl sulfone group has a sufficiently low-lying LUMO energy level, electron transfer from the mild reducing agent, samarium diiodide, is facilitated, resulting in a fragmentation step with concomitant formation of the thermodynamically more stable a-anomeric radical (the anomeric effect), as illustrated in Scheme 11 [31]. A second electron transfer to 49 from an additional equivalent of samarium diiodide then leads, under kinetic control, to the organosamarium species 50 which is configurationally stable because of a fast conformational change, placing the C1 and C2 substituents in a more stable equatorial position, as in 51. Its
20.2 The Anionic Approach
oms
BF%
+
~,g-&+
2.Bu4NF 1. Sm12
503
~
BnO
38
OB n
75%
o'
S02PY
OBn
HO
OBn 40
39
BnO
~$3 ~{3 BnO
S02PY 41
OMe
73%
BzO BnO
42
BnO
1. Im2CS 2. FjPhOH, Ph3SnH
70%
2. I.B MeONa u~NF
OTBS
.,,H BzO BzO BnO
3. H2, Pd/C
+H
*
79% HO HO
.O
-0 I
OMe
OMe
44
Bz% + BnO
B BnO
45
q P
*
oJ S02PY 46
OMe 47
48
OMe
Scheme 10.
B;;oEh BnO
Scheme 11.
syn-elimination to glycal
I
504
20 Synthesis of C-Oligosuccharides
53
Ph
Ph
1. O3 2. Bu$nH, AIBN
,
39
BzO
63% P h d P h
OMe
OMe 42
Scheme 12.
coupling with aldehydes under Barbier conditions therefore produces the a-Cdisaccharides, 52, only [30]. The synthesis of the electrophilic partner 39, reported in 1993 by Jung and Choe, is interesting because of its originality in respect of the introduction of a formyl group into the sugar ring (Scheme 12) [32]. In this work, the authors demonstrated that substrate 53, obtained by iodoglycosylation of tribenzylglucal with 1-phenylprop2-en01 and then ozonolysis, readily underwent a 5-ex0 radical cyclizationfragmentation process resulting in the intramolecular transfer of a formyl group to C2 and generation of a more stable benzyl radical. Not only was the formyl group transferred stereospecifically to give the 1,2-cis relationship in 39, owing to the 5-membered cyclic intermediate in the transfer mechanism, but the original functionality at the anomeric center was then transformed to the well-known benzyl protecting group. An adaptation of this approach has recently been exploited by Skrydstrup and Beau for the preparation of aldehyde 42 required in the synthesis of methyl a-Cmannobioside 45, a C-glycoside analog of the Mycohacterium tuberculosis capping disaccharide (Scheme 10) [33].Rather than a formyl group transfer from C1, the C3 position was selected affording the derivatized sugar 42 with a CZaxial substituent (Scheme 12). Subsequent coupling with the mannosyl pyridyl sulfone 41 under the influence of SmI2 afforded only one C-glycoside, 43, which was transformed to the unprotected C-disaccharide 45 via a deoxygenation step to 44 and subsequent deprotection. Recently, the C-dimer of u-D-Man(1-3)a-~-Man48 has been prepared by a similar approach employing the mannosyl pyridyl sulfone 46 and aldehyde 47 P41. Other extensions of this C-glycosylation methodology have been reported for the preparation of two C-linked 1,6-disaccharides. Reaction of the pyridyl sulfone of N acetylglucosamine 54 with aldehyde 55 produced mainly the a-C-disaccharide 56 (Scheme 13) [35].The stereoselectivity at the anomeric center is explained in this case by internal coordination of the N-acetyl group with the metal ion of the intermediate glycosyl samarium species 57, retarding anomerization to the ther-
20.2 The Anionic Approach
505
Bnr% AcHN SO2Py
BnO
bMe Bnb
54
55
bMe
56 (a$= 4: I )
57
Scheme 13.
modynamically more stable p isomer as observed for the corresponding glucose derivative. Reductive samariation of tetrabenzylmannosyl chloride 58 (X = C1) or phosphate 58 (X = OPO(OPh)2) in the presence of 55 also served as a useful reaction for the synthesis of C-disaccharides as exemplified in the synthesis of C-dimer 59 (Scheme 14) [36, 371. Recently the glycosyl pyridyl sulfones have been applied by the Linhardt group for the synthesis of a C-disaccharide containing N-acetylneuraminic acid (Scheme 15) [38]. The coupling of glycosyl pyridyl sulfone 60 with the previously reported aldehyde 63 gave a high yield of the (1,3)-C-disaccharide64 with complete stereoselectivity both at the anomeric center and at the new exocyclic stereocenter. It is important to point out that these reactions proceed through a samarium enolate intermediate 65 in contrast to 2-deoxy sugars lacking a C1 carboxylate group which involve true anomeric samarium species and which are non-selective at C1. The coupling step can even occur with the glycosyl phenyl sulfone 61 or chloride 62 because the initial homolytic cleavage step after the first electron transfer is favored by the stability of the radical formed (captodative effect). An extension to a KDNcontaining disaccharide derivative 66 has recently been published by the same team with the same aldehyde 63 having identical stereoselectivity [39].
55
+
goq
F o x
Sm12
b
x 58
Scheme 14.
x=c1
X= OPO(OPh),
54% 73%
BnO
BnO 59
BnO
OMe
506
20 Synthesis of C-Oligosaccharides
OAc
OMe
60 X= ~lS02pV 61 X= aS02Ph 62 X= PCl
63
64
63, SmI2 Bn
OAc
X= aS02Ph
66
OAc
a-face
65
x=SCl Scheme 15.
20.2.4 C-Branched Carbanions p-Glycosyl nitromethanes are effectively prepared from the parent sugar in two steps [40]. Because the corresponding nitronate anions do not undergo ring opening, chain elongation at the a carbon is possible and has been exploited by Martin and Lai for the synthesis of both p(1-6)- and p,p(l-l’)-linked disaccharides [41, 421. Treatment of the C-glucoside 67 with KF in the presence of the galactose-derived aldehyde 68 led to the rapid formation of the derivatized C-dimer which was transformed to the unsaturated nitro compound 69 (Scheme 16). Further reduction and denitration involved two steps with eventual deprotection providing the meth1-6)-~-Gal. ylene bridged analog 70 of p-~-Glc(
A AcO c
O
G NO2
~
To%
OAc
67
68 ,OAc
1. N a B Q 2. BqSnH, AlBN 40%
Scheme 16.
1. KF, 18-crown-6 2. Ac20, Py * 47%
20.2 The Anionic Approach
507
1.80% AcOH 2. AclO, BF,.Et,O
1.67, KF, 18-crown-6 2. A c ~ OPy ,
c
36%
44% OPiv 71
OPiv
u I 70
OH 74
73
Scheme 17.
Elaboration of this approach for the synthesis of C-P,P-trehalose was successfully achieved as illustrated in Scheme 17. The nitroaldol condensation of 67 with the aldehyde-glucose derivative 71 provided an epimeric mixture of unstable nitroalditols which were immediately dehydrated to give mainly the E-nitroalkene 72. The sensitive deacetalization and cyclization step is best brought about by heating 72 under reflux in 80% AcOH. In this way, both deprotection and ring formation take place to produce the second p-C-glycoside of the required C-disaccharide via attack of the internal nucleophile on the si face of the double bond. A substantial amount of the C-pyranosyl-C-furanosyl isomer was likewise isolated. Radical denitration and deprotection then afforded the C-p, b-trehalose 74. Reduction of the nitro group in 73 also gave rise to a C-disaccharide containing a bridging methylenamine group; similar compounds were demonstrated by Schmidt to be good glycosidase inhibitors. A similar approach was also published by Witczak and collaborators for the preparation of 3-deoxy-C-cellobiose (Scheme 18) [43]. The starting glycosyl nitro-
-? B
n
O
w +
BnO
Qo
BnO NO,
0
BnoOH 75
76
1. BqSnH, ABCN 2. BF3:OEt2, Et&H 3. L-Selectride 5 . Pd(OH)& 4. EgSiOTf, AczO C,H,,, EtOH
-
-& AAcO
28% OAc 78
Scheme 18.
508
BBnO n
20 Synthesis of C-Oligosaccharides
O
G
+
k0
BnO BnO
BnoOCH3
OBn
KF, 18-crown-6 52%
BnO-- ..BnO
BnO
BnO
80
79 1. BuLi, PhOC(S)Cl
2. Bu$nH, AIBN 3. TsNHNH2, AcONa 4.Na, NH3 10%
81
HO
B"bbCH3
OH
HO
Scheme 19.
methane 75, easily prepared in two steps, underwent stereospecific conjugate addition to the sterically less hindered exo face of the bicyclic enone 76 under mild basic conditions (KF or triethylamine). Tributyltin hydride reduction of the nitro group in 77 followed by removal of the anomeric hydroxyl group with Et$iH/BF3.Etz0 and stereoselective reduction of the ketone subsequently afforded the C-disaccharide 78 after a deprotection-acetolysis sequence. Reversing the roles of the two coupling partners in the nitroaldol condensation is also an option for construction of C-linked (1-6)-disaccharides [44]. In this instance aldehyde 80 was coupled to the C6-nitro derivative 79 to afford adduct 81 (Scheme 19). Tin hydride-induced reduction of the corresponding thionocarbonate then led to the C-dimer 82 after two reduction steps. Schmidt and coworkers, on the other hand, have developed an approach to Clinked (3-disaccharides employing CCbranched carbanions which can be prepared by lithium-halogen exchange as exemplified with the 1,6-anhydro sugar 85 in Scheme 20 [45]. This precursor was prepared via a stereoselective hydroborationoxidation of the exocyclic alkene in 84 obtained in two steps from alcohol 83. Addition of the anion generated from 85 to gluconolactone 3 gave a hemiketal which was stereoselectively reduced to the C-disaccharide 86. Further standard manipulations provided the octaacetate derivative of (3-~-Glc( 1-4)-~-Gal87. The peracetylated derivative of p-D-Gal(1-4)-~-Gal88was prepared in a similar manner. 20.2.5 CdPhosphoranes Wittig olefination is another attractive and rapid route to (1,6)-C-disaccharides. The Dondoni team demonstrated that the readily accessible phosphonium ylides at C6 of sugars coupled well with C-formyl glycosides available through thiazole-based formylation of glyconolactones [46]. In Scheme 21, a representative of this approach is shown for the synthesis of the C-dimer of p-~-Gal(l-6)-~-Gal [47]. Hence, treat-
20.2 The Anionic Approach
1. DMSO,DCC 2. Ph3P=CH2
HO
83
pq
39%
OBn
;:;wy;
H202
+ 67%
OBn
I
1. Ac20, TFA 2. HZ, WIC
3.Ac20,Py
B 0
%no
40%
OBn
OBn
86
+ . &+& o ? i A
OBn 85
84
1. BuLi 2. Et3SiH, BF3.Et20 85 + 3 * 34%
509
OAc
87
OAc
OAc
88
AcO
OAc
OAc
Scheme 20.
BnO
6-step formylation
53%
BnO BnO 80
80 + 89
Scheme 21.
ment of the phosphonium salt 89 with base and then aldehyde 80 led to the formation of the cis olefin 90, which upon Pd-catalyzed hydrogenation subsequently afforded the C-disaccharide 91. A set of ten (1-6)-linked p-C-disaccharides could therefore be prepared by means of this approach starting from C-glycosyl formaldehydes 80 and 93-98 with the phosphonium salts 89 and 92 (Scheme 22) [48]. The basic conditions of the Wittig reaction nevertheless prohibit the formation of the corresponding u-C dimer; e. y. starting with the u-C-glucosyl precursor, the corresponding P-C-disaccharide is obtained by initial isomerization at the anomeric center.
510
20 Synthesis of C-Oligosaccharides 'PPh3 I-
BnO% BnO
BBnO n BnoOMe 92
y
%:nBBnO
' a0
93
96
BnOR R
94 R = H, R = CHO 95R= CHO,R'=H
98
91
Scheme 22.
steps
BnO
c
P
BnO
70%
68
99
BE+ 101
-P
102
Ho
Scheme 23.
The reverse strategy has recently been reported by the same group for the preparation of a similar C-mimic of the p(1-6) tetragalactoside as shown in Scheme 2 [49]. Condensation of the D-galactopyranosyl-derived phosphonium salt 99 and aldehyde 68 led to the unsaturated dimer 100 (Scheme 23). Repetition of the sequence twice with phosphonium salt 99 furnished tetramer 101. These steps are, however, characterized by low coupling efficiencies (36% for the trimer and 11% for the tetramer) mostly because of the base-induced a,p-unsaturation of the aldehyde substrates. The desired tetrasaccharide 102 was then obtained after standard deprotection and double-bond hydrogenation.
70
20.3 The Radical Approach
51 1
20.3 The Radical Approach 20.3.1 Intermolecular Anomeric Radical Addition In principle, intermolecular radical-mediated addition of anomeric radicals to an exocyclic double bond of the C-glycosyl acceptor should provide an efficient route to C-oligosaccharides leading directly to the desired mimic. In practice, however, these reactions with unactivated alkenes are not favorable as they lead solely to products of direct H-abstraction. Reducing the LUMO of the alkene with electronwithdrawing groups, typically a,P-unsaturated carbonyl compounds or similar derivatives, greatly improves the overall efficiency of these coupling reactions. For example, Giese demonstrated that the anomeric radical obtained by reaction of acetobromoglucose with Bu3SnH adds to the a-methylene y-lactone 103 in good yields and with high a-selectivity to give the C-dimer 104 (Scheme 24) [50].In addition, hydrogen abstraction at the new radical center at C2 of the reducing sugar is stereoselective leading to the predominant formation of the product with an equatorial oriented CZsubstituent. Further modification involving reduction of the lactone to lactol and acetylation afforded the protected C-mimic of kojibiose 105. Two other examples 106 and 107 have also been successfully prepared, as shown in Scheme 24. In a similar approach, Vogel described the use of ‘naked sugars’ such as 108 as radical acceptors, as shown in Scheme 25 [51, 521. The coupling product 109 was further elaborated before a true C-disaccharide was reached, with the key step being Baeyer-Villiger oxidation to introduce the fully oxygenated ring system. Several
Ace% OAc
AcO
nBo- \+
Bu$nH, AIBN 70%
AcoB,
OBn 104
103 1. Na[A1(OC2H,0Me)2(OEt)H1 2. AQO
64%
OBn
105
BnO BnO 106
Scheme 24.
AcO
Me0B-n
I OAc AcO
OAc
107
5 12
20 Synthesis of C-Oligosucchurides
1. Bu3SnH, AIBN 2. mCPBA, NaHC03 OAc AcO OAc 1 0 9 ( a : p =5.5:l) OAc
66%
108
C02Me
1. NaHC03, MeOH 2. A c ~ O DMAP, , Py
1.NaBH4 2. HCI OAC 3. A c ~ ODMAP, , Py
*
* 36%
0 0
n
Acd
OAc
Scheme 25.
variations of this approach have now been reported for the synthesis of several Cdimers with different substitution patterns of the radical acceptor in addition to the use of acetobromogalactose (Scheme 26) [ 531. In a different approach, Martin reported the use of an exocyclic radical derivative The as a means of preparing the a(1-6) C-linked disaccharide 113 (Scheme 27) [54]. corresponding radical from the cobaltoxime derivative 111, prepared from iodide
1. Bu3SnH, AIBN + pl,sed\
Atgo% AcO AcoBr
c;
0
2.NaBH4
PhSe, 48%
1. mCPBA 2. Ac7O 3. osb, 4. Ac,O
I\
r5
c
OH
86%
R mCPBA
steps
R
AcO 0
OAc
Scheme 26.
20.3 The Radical Approach
2. 1. A hv,c NaOH, ~O 95% E
t *O
111 + 112 30%
"
"
"
~
Bk'
Bu3SnH
AcO AcO
*
AcO
AcO
AcO
OMe
513
113
AcO
OMe
Scheme 27.
Bu3SnH
Bno%I BnO
+
BnoOH
114
Q 0 76
ABCN 26% 0
BnO BnO
Scheme 28.
110, was generated photochemically and coupled to the nitronate derivative of the C6 functionalized glucoside 112. Tin hydride-promoted denitration then afforded the desired C-dimer. An exocyclic radical approach to C-disaccharides was also employed by Witczak starting with the readily available iodide 114 (Scheme 28) [43]. An exo-facial selective radical addition to levoglucosenone 76 afforded, in low yield, the C-dimer 115, with direct reduction being the major competing pathway. This compound was then converted to the 3-deoxy derivative of C-cellobiose as depicted in Scheme 18. 20.3.3 Intramolecular Anomeric Radical Addition
If the radical addition reaction is performed in an intramolecular fashion, olefin activation with an electron-withdrawing group will not be a prerequisite for Cglycosylation, because the cyclization event competes well with hydrogen abstraction. In such examples, ring size and the stereochemistry of the ring substituents greatly influence the stereoselectivity and efficiency of the cyclization reaction. With the above points in mind, Sinay's group developed a direct approach to Clinked disaccharides employing a temporary silicon tether approach to graft the
5 14
20 Synthesis of C-Oligosaccharides
o& &no
SePh
BuLi, Me$iC12; Imidazole
BnO
OH
116
117
2. 1. HF, BqSnH, THF AIBN *
BnbAMe
.
95%
BnO& BnO
SePh
9,Sic
B&(30% B
40%
n
BnO
118
4 BnoOMe
BnoOMe
Scheme 29.
radical donor and acceptor together [55]. These reactions are surprisingly efficient considering the medium-sized ring intermediates formed, which upon liberation from the silicon linker afford the C-dimer. Illustrative of this approach is the synthesis of a C-linked analog of methyl amaltoside (Scheme 29) [56]. Linkage of the two glucose derivatives 116 and 117 with dimethyldichlorosilane followed by tin hydride-promoted 9-endo-cyclization afforded the C-disaccharide 118 in 40% yield after liberation from the linker. The anomeric effect seems to dominate in this reaction with exclusive formation of the a anomer, whereas H-abstraction by the resulting intermediate radical after cyclization affords the equatorially substituted product. The nature of the attacking sugar radical has a substantial impact on the cyclization yields and stereoselectivites of these reactions-when the method was extended to the galacto series 60% yield of three isomeric Clinked disaccharides was obtained in the ring-closure step [57]. The ring size and tethering positions are equally important, as is observed in the 8-endo cyclizations of the gluco and galacto derivatives 119 and 121, respectively (Scheme 30). In the first case an inseparable mixture of all four isomers 120 was obtained [56],whereas the C4-epimer 121 led to the C-linked disaccharide 122 in 45% yield [ 5 8 ] . Quite remarkably, the anomer was furnished in the latter case,
6 1
OBn Bno& -BnO $S -,ePh
OBn
/
BnO
\
BnO !&&SePh
121
Scheme 30.
O
w
o
B OBn
120
OBn
\
B BnOn
50%
119
\&
OBn
O\Si//
OBn
2. 1. Bu+nH, HF, THF AIBN
OMe
1. BusSnH, AIBN 2. BQNF
45% (tethering incl.)
OBn 122
n
515
20.3 The Radical Approach
~ ~ 0 % SO2R B n g o M C
~1. .SmI2 HF
BnO
\si’o
/
50%, R = Ph 70%, R = Py
\
BnO
118
OMe
Scheme 31.
Br:o&
0
CSA, 4..4 MS
Tebbe’s reagen:
81%
87%
BnO
SePh
iePh
-
117
123 1. BqSnH, AIBN
BnO
Brio\
OH
BnO
BnO
35% 124
125
+
pproduct (3%)
BnOOMe
Scheme 32.
where there was undoubtedly a strong conformational bias induced by the tether because attack of the anomeric radical species occurred on the p face. The standard tin hydride conditions for generating an anomeric radical can be replaced by the divalent samarium-induced reductions of glycosyl aryl sulfones [ 59, 601. In one instance the Sinay group employed the 2-pyridyl sulfone group, introduced by Skrydstrup and Beau in 1994 [30, 311, which was rapidly reduced by SmI2 at room temperature (Scheme 31). In this way, a significant increase in the cyclization yield of 118 was achieved [60]. The ketal connectors which were exploited earlier in 0-glycosylations proved equally efficient in the synthesis of C-linked disaccharides as shown in Scheme 32 [61]. With acetate 123 a two-step sequence via vinyl ether formation with Tebbe’s reagent and acetalization with alcohol 117 produced the linked sugars 124. Upon tin hydride-promoted cyclization and removal of the acetal tether the a-D-Man(1 4 ) - ~ - G l cC-disaccharide 125 was obtained in 35% yield. Similarly, a benzylidene acetal linkage as in 128 could be produced via a DDQmediated oxidation of the p-methoxybenzyl group in 126 in the presence of the fucose derivative 127 (Scheme 33) [62], a strategy introduced in 0-glycosylations by Ito and Ogawa [63]. Subsequent radical cyclization and acetal hydrolysis afforded the a-~-Fuc(lL2)-~-Glc analog 129 in good (60%) yield. A phosporamidic linker also proved viable, as reported in the synthesis of the Cdisaccharide analog of a-~-GalNAc( 1-4)-~-Glc(Scheme 34) [64]. Intermediate 131 prepared in two steps from the galactosamine derivative 130 and the unsaturated -
5 16
20 Synthesis of C-Oligosucchurides
1. Bu3SnH, AIBN 2. DDQ, HZO
60%
129
Scheme 33.
1. PhPCl,, Et3N, THF 2. tBuOOH
BnO
BnO 80%
OMe
SePh
130
117
1. Bu+nH, AIBN 2. HCI, MeOH 3. MeONa, MeOH 4.AQO, Py
BnO OBn
+ p product (4%)
BnOQ+++
16% 132
OMe
Scheme 34.
alcohol 117 underwent 9-endo cyclization to give the C-dimer 132 after a deprotection and acetylation sequence. An approach to P-C-disaccharides was reported by the same group whereby the roles of the two tethered sugars are interconverted as exemplified with silaketal 133 in Scheme 35, in which the radical acceptor has an anomeric exo methylene [65]. Radical addition to the double bond led to formation of an a-oriented anomeric radical which subsequently abstracts a hydrogen leaving the C1 carbon branch in the j3 position as in the C-disaccharide 134. Because of the facile access to these C-dimers by use of this intramolecular approach, multigram quantities of the analogs are available and have been exploited for the synthesis of mixed C/O-glycosides of biologically relevant oligosaccharides. In this way, the analogs of sialyl LewisXand the pentasaccharide re-
517
20.3 The Radical Approach (OBn 1. Bu$nH, AIBN 2. B u ~ N FTHF ,
OMe
37% (tethering incl.) '
OH
-
\
OH 134
133
Scheme 35.
135
136
Scheme 36.
sponsible for the antithrombin I11 binding region of heparin, 135 [66] and 136 [67], respectively, have been prepared (Scheme 36). Predictable stereochemistry at C 1 would be expected when applying h x o cyclizations in silicon-tethered radical reactions for the construction of Cdisaccharides. A sole example has been provided by Skrydstrup and Beau using glycosyl pyridyl sulfones for the generation of the anomeric radical [31]. This approach has been successfully exploited in the synthesis of peracetylated methyl Cisomaltoside 137 (Scheme 37).
1. SmL 2. B u 4 h 3. H2, PdC 4. A c ~ OPy ,
,OAc
a
*
48%
AcO AcO 137
Scheme 37.
Ac'O 1 OMe
5 18
20 Synthesis of C-Oligosaccharides
20.4 The Partial de Novo Approach In the partial de nuuu synthesis of C-oligosaccharides one of the carbohydrate units is constructed from smaller building blocks. The advantage of this approach is that in the designed synthesis of a C-mimic little variation can easily lead to other stereoisomers, which has resulted in the preparation of a large collection of C-di- and trisaccharides. Much of the work in this area has been dominated by Kishi and coworkers, primarily because of their interest in studying the solution conformation of the parent oligosaccharides and their bound conformation on carbohydrate binding proteins [9], an interest which was sparked from their phenomenal synthetic work on the marine toxin, palytoxin [68]. Kishi and coworkers found that the solution conformations of C-disaccharides strongly resemble those of the corresponding O-glycosides and can therefore be exploited to predict the conformational preferences of the natural sugars. Because the C-analogs have two protons on the connecting carbon atom their solution conformations are readily extracted from simple 'H NMR experiments, which is not the case for the parent sugars. As a result of other NMR experiments Jimenez-Barber0 suggested that C-disaccharides are more flexible around the methylene bridge, and it was observed that in certain instances such compounds are bound to lectins in conformations different from that of the normal disaccharide [ 10, 691. In general, the de nuuu approach applied by the Kishi group for the synthesis of C-oligosaccharides relies on acyclic stereocontrol for the construction of the contiguous chiral centers of at least one of the carbohydrate moieties. The first examples of this approach were reported by this group in 1987 and involved the synthesis of four C-disaccharides, including the C-analogs of methyl maltoside and cellobioside, from a common synthetic intermediate [70]. Initial Wittig reaction of the readily available arabinose derivative 138 and the phosphonium salt 139 led to the cis alkene 140 (Scheme 38). Stereoselective dihydroxylation, exploiting the 1,3-allylic strain effect, then monoprotection of the diol, oxidation and cleavage led to the cyclic hemiacetal 142 after protection of the primary alcohol. Reduction of the hemiacetal was best performed with n-Pr$iH, affording exclusively the p anomer 143. Deprotection and acidic methanolysis then led to the C-disaccharide analog 144 of P-~-Man(l-4)-~-Glc. On the other hand, simple oxidation of the C2-hydroxyl group in 143 followed by stereoselective reduction converted the mannu series to the gluco series with the eventual formation of C-cellobioside 145. Preparation of the corresponding a isomers of these C-dimers requires only slight modification of the syntheses, as illustrated in Scheme 39 by simple reversion of the order of the synthetic steps in the preparation of the acyclic precursor. Hence, the isopropylidene in intermediate 141 was hydrolyzed, followed by diol cleavage and preparation of the anomeric p-nitrobenzoate 146. Introduction of the C6' carbon could be achieved by stereoselective allenylation resulting in formation of the manno derivative 147 after chair inversion. Oxidative cleavage then reduction of the allene moiety and subsequent debenzylation and methyl glycoside formation led to the a - ~ - M a n1-4)-~-Glc ( carbon analog 148. Access to methyl C-maltoside 149 was
519
20.4 The Partial de Novo Approach
138
139
140
1. DMSO, (COC1)2, Et3N 2. HC1 3. BzCl, Py
1. Os04 2. PMBBr, NaH
B z * BnO 75% BnO
O
G
Y
n
OH 142
2. 1. DMSO, BH3.Et3N (COCI),, Et3N
nPr$iH, BF3.Et20
B
z
O
G
BnO BnO OH 143
90%
1
1. H2, Pd(OH)2/C 2. MeOH, HC1 90%
1
145
Scheme 38.
1. AcOH, HzO 2. Pb(OAc)4 3. pN02PhCOCl
//SiMe,,
*
141
PNBO
*
I OBn
1. 03; NaBH4 2. Hz, Pd(OH)2/C 3. MeOH, HC1
-
HO
146
H 148
Scheme 39.
BnO BnO
BF,.Et,O
,OH
W
HoOMe
:
,
,
520
20 Synthesis of C-Oligosaccharides 1. 0 3 ; MezS 2. CBr4, PPh? 3. BuLi 4. 12, morpholine 5 . KOzCN=NC02K. AcOH
I
BnO
150 1. Hz, WA1203
DHP, PPTS 3. B u ~ N F
OBn
151
OBn
/OBn
L.
- '\?oI?;1,
1. NaH, Me1 2. Hz, Pd(OH t>/I
5 . DMSO, 4. TsOH (COC1)2, Et3N
BnO% BnO BnO
'
'"'OH
OBn BnO
OTBS
152
- OH
nn
153
" HO :
o
UDll
x
HO 21
154
HoOMe
Scheme 40.
achieved by a sequence of reactions similar to that used for the synthesis of the Ccellobioside 145. The Kishi-Nozaki reaction [71] is the key step in the preparation of methyl Cisomaltoside (Scheme 40) [72]. Coupling of vinyl iodide 150 with the xylose derivative 151, readily available in six steps from L-xylose, afforded the corresponding allylic alcohol with a diastereomeric ratio of 15 : 1 in favor of 152. Finally, selective hydrogenation of the double bond and oxidation of the primary alcohol led to the C-disaccharide 153, which could be converted to its methyl glycoside 21. A useful dideuterated derivative of C-isomaltoside 154 for solution conformation studies was also accessible from this synthetic approach via the cis-deuteration of the alkene intermediate 152 with Pt on A1203 and D2. A similar strategy was applied for efficient synthesis of the C-glycoside analog of sucrose, starting from the vinyl iodide 155 and the aldehyde 156 (Scheme 41) [73]. The wrong diastereomer at the C3' position is obtained in the anti-selective Cr(I1)mediated reaction; this can nevertheless be corrected via a two-step Mitsunobu reaction and hydrolysis sequence. An anti-favored epoxidation and a regioselective acid-catalyzed ring formation by attack of the C5' hydroxyl group at the quaternary center of the epoxide finally completes the C-disaccharide 157 after a debenzylation step. Nicotra and coworkers have reported another synthesis of a C-glycoside analog of sucrose, as shown in Scheme 42, although missing the C2/-hydroxymethyl group [74].Wittig reaction of the stabilized phosphorane 159, prepared in four steps from C-ally1 glucose 158, with the isopropylene derivative of D-glyceraldehyde 160 rep-
521
20.4 The Partial de Novo Approach
1. DEAD, PhCOOH
2. KzCO~,MeOH 3. mCPBA
-OH 1. CSA
+
30% overall from 155
OH OH 157
Scheme 41.
Bi:oT
kc
Hg(OAc)2, NaCl97%t B g 3 0 Y o H i : 3. PPh,, Et,N
Brio
t
36%
HgCl
158
BnO0v
?%
-
160
88%
BnO
\
1. oso4, NMO 2. AczO, Py
-
159
PPh,
,OBn
52%
161 0 162
FeCI,.SiOz
-Bi:o%
1. Florisil 2. K2C03, EtOH 3. H2, Pd/C
HoOAc
78%
OAc
89%
OH
163
OH
164 OH
Scheme 42.
resents the key coupling step in this synthesis. Subsequent stereoselective dihydroxylation of alkene 161 afforded adduct 162 as the major product (4: 1 ratio) after acetylation; this was subjected to acid hydrolysis furnishing the pyranose derivative 163. Florisil treatment and two-step deprotection then gave the C-sucrose analog 164 in which the furanose and pyranose forms are in equilibrium. The C-dimer of trehalose (cL-D-G~c(~-~’)-cY,-D-G~c) can also be prepared by use of
522
20 Synthesis of C-Oligosaccharides 1. 03;Me$ 2. (Me0)2P(O)CHN2, tBuOK , i B 3. 12,morpholine t
QBn Y
BnO
88%
158
O
G%
O
165
166
1. H2, Lindlar cat. 2. BnBr, NaH
165 + 166
Bn
PBn
57%
(OBn 1. oso4 2. BuzSnO;
CsF, PMBCI, TBAI 3. A c ~ ODMAP, , Py
* 85%
*
63%
PMB~ 169
168 1. DDQ 2. TsOH
1. DMSO, (COC1)z. Et3N 2. BH,.THF 3. AcOH, H20 4. Hz. Pd(OH)I/C *
-
4. 3. MMTr, KzCO3, Py MeOHBi:o\
76%
OMMTr
74%
Hgo%
'OH
HO HOoH
BnOoBn
170
171
Scheme 43.
a Cr(II)/Ni(II)-mediated coupling reaction as reported by Kishi and Wei in 1994 (Scheme 43) [75].Condensation of the alkynyl iodide 165, prepared in three steps from C-ally1 glucose 158, with the four-carbon fragment 166 favored the formation of the syn-propargylic alcohol 167 with a diastereomeric excess of 60%. Further cis reduction of the trip bond and a hydroxyl group-protecting step, set the stage for a stereoselective dihydroxylation exploiting the 1,3-allylic strain effect. Regioselective protection of the diol then gave the protected polyol 168, which could be transformed in three steps to the primary epoxide 169. Exclusive formation of the tetrahydropyran 170 was then achieved by removal of the p-methoxybenzyl group and subsequent acid-catalyzed epoxide opening. Finally, by inverting the C2'stereocenter via an oxidation-reduction sequence and deprotection, the C-analog of a,a-trehalose 171 was made available.
20.4 The Partial de Novo Approach
523
woMe
HO
OH
X M e P O H HOoH
172 X = OH, Y = CH20H 173 X = H, Y = CH20H 174 X = OH, Y = H 175 X = Y = H
1
y
-
Me@OH
HO 176 X = OH, Y = CH2OH 177 X = H, Y = CH2OH 178 X = OH, Y = H 179 X = Y = H
Scheme 44.
A most impressive illustration in the application of the de nouo approach to the synthesis of C-oligosaccharides was provided by the same group in their synthesis of the C-trimer of the type II(H) cellular antigen trisaccharide (a-~-Fuc( 1-2)p-DGal(1-4)(3-~-GlcNAc(Scheme 44). In contrast to other stratagems, a sufficiently flexible aldol route was adopted to introduce specific structural modifications, so that a series of trimer analogs 172-179 could be prepared [8b, 761. As shown in Scheme 44, the bond disconnections require a glucosamine (or glucose) unit with a three-carbon appendage on the C4 carbon, a Cl-formyl fucose unit, and a fourcarbon acyclic fragment representing the future central galactose sugar. To prepare the glucosamine unit, the epoxide 180 was transformed in six steps to the C4branched sugar 182, including opening of the epoxide via a Cu(1)-catalyzed Grignard reaction and subsequent treatment with base to give the C2,C3 epoxide 181 (Scheme 45), which was then subjected to azide opening, benzylation, azide reduction, and phthalimide formation. Opening of the I ,6-anhydro derivative 182 and allylation at the C1 position led stereoselectively to the (3-C-glycoside 183; the stereochemical outcome can be explained by the anchimeric assistance of the C2phthalimido group. A five-step procedure then converted 183 to the ketone 184 which coupled smoothly with aldehyde 185 affording the aldol product 186. A sequence involving desilylation, thioglycoside formation, reduction and oxidation then set the stage for the next aldol coupling between the C-dimer 187 and aldehyde 188. In this reaction, optimum equilibrating conditions are required to produce the equatorial alcohols at the C2-position of the galactose unit in 189. Two stereoselective reductions on the enone product 190, obtained by base-promoted elimination of the corresponding mesylate of 189, led to the C-trimer 191 which was fully deprotected and converted to the polyol 176 in the form of its lithium carboxylate. Armstrong and Sutherland have reported a combinatorial approach to Cdisaccharides in which one sugar unit was kept constant while the other was varied to provide all possible stereoisomers [77]. To illustrate this concept, the C-allylated glucose derivative 158, was chain-extended to give the diene ester 192 in four conventional steps (Scheme 46). Dihydroxylation and DIBALH reduction afforded four stereoisomeric lactols with the DIL-galacto and the D/L-ido configurations
524
20 Synthesis of C-Oligosaccharides 1. NaN3, TBAI
I
1. A M g C I , CuI
3. Hz. Lindlar cat. 4.phthalic anhydride; Ac20 48%
12% 180 OTS
1. DBU, MeOH 2. BnBr, NaHMDS 3. 03;PPh3 4.PMBO(CH2)gPPh3Br,BuLi 5. Hz, (Ph,P),RhCl
1. Ac20, BF3.EtzO
~
-
sTMSOTf ,
w
14%
NPhth
183
NPhth
YMB
(CH2)s
OPMB
184
186
1. B u ~ N F 2. MeSH, BF3.Et20 3. Ph3SnH, AIBN 4.DMSO, (COC1)2, Et3N
t
187
OBn
Me OBn BnO
B
p
(
61%
p
H
1. MsCl, 2,6-lutidine 2. NH,
OBn
BnOOBn
189
HO OBn OBn
1 OBn BnO
191
)
8
,
OPM B
80% Me
OBn
2
OBn
t
1. Ph3SnH. AIBN 2. NaBH4
Scheme 45.
OPMB
OBn
-0PMB
OH
Po
OBn BnO LHMDS, TMEDA. MgBr2
85%
BnO
-
26%
OTBS
& Bn
Bn
OPMB
OBn 190
1. NH2NHz; A c ~ O 2. DDQ 3. (Ph3P)3RuCIZ 4.NaC1OZ,NaH2P04 5. Hz. Pd(OH),; LiOH
-
34%
176
20.4 The Partial de Novo Approach I. 9-BBN;HzOz 2. DMSO, (COC1)2, Et3N 3. (Et0)20P*COOEt, 4.12, PhH
tBuOK
525
I . 0~01. NMO
OEt 2. DIBALH
*
20%
69% 158
192
OH OH 0
QH OH 0 P H + \ * H
+
\
OH OH
OH OH
OH OH 5: 1
D/L-gaiacto
OH OH
D/L-ido
HO
HO HO
HO L-Gal
D-Gal 193
OH
HO
194
D-Id0 195
L-Id0 196
Scheme 46.
(represented in their acyclic form), which were subjected to hydrogenolysis to give the corresponding C-disaccharides 193-196. The same group has provided another example in their synthesis of C-glycoside analogs of the Lewis type I blood group determinant, of general formula a - ~ Fuc( 1-2)hexose( 1-3)p-~-GlcNAc,whereby combinations of the central hexose unit are constructed by non-selective transformations to give a mixture of diastereomers [ 781. On the other hand, predictable stereochemical transformations enable access to a sole C-trisaccharide. This strategy begins by the Nozaki-Kishi reaction between vinyl bromide 197, prepared in three steps from L-fucose, and the aldehyde 198, obtained in four steps from D-glucosamine, to give, after silylation, a 2: 1 mixture of allylic alcohols 199 and 200, respectively (Scheme 47). Diastereoselective hydroboration was performed on the major isomer 199, which after oxidation, then homologation with ally1 Grignard reagent and subsequent desilylation led to the diols 201 and 202 (2 : 1 ratio). Cyclization of 201 by initial epoxidation of the alkene and then deprotection affords a 1 : 1 mixture of C-trisaccharides 203 and 204. In this way, 11 C-trimers were synthesized. To access single components of reactions affording inseparable diastereomeric mixtures, these non-selective steps (marked by
OH
526
20 Synthesis of C-Oligosucchurides
Me OBn 197
Me
198
200(1) 1.9-BBN, H202 2. periodinane *3. A M g B r 4. BQNF 199
BnOoBn *l. mCPBA 2. cat. CSA 3. Hi,Pd(OH)$ 201
Me
OBn 201 (2)
-
OBn
I OBn BnO
202 ( I )
+
55% h Z U d O H
Me
I OH HO
203 (1)
HAOH
OH 204 (1)
Scheme 47.
an asterisk in Scheme 47) are substituted with selective steps, an approach described by the authors as a recursive stereochemical deconvolution strategy. Applying a conceptually different approach to the de novo design of Coligosaccharides, Vogel and coworkers described the use of stereoselective transformations on the rigid oxa-norbornane bicyclic system; some examples, shown in Schemes 25 and 26 in Section 20.3.1, rely on radical reactions for assembly of the two sugar units [51-531. Recently, Vogel and Gerber have also applied an ionic transformation, depicted in Scheme 48, starting from the optically pure ketone 205 [79]. A six-step procedure converted 205 to enone 206, a precursor of both ring units of the target C-dimer. Stereoselective conversion of this enone to the poly-oxygenated system 207 was followed by regioselective Bayer-Villiger oxidation, opening of the uronolactone, and subsequent dithioacetal hydrolysis to give the methyl uronic ester derivative 208. Aldol condensation of this aldehyde with the aluminum enolate 209, prepared from enone 206, gave rise to the C-disaccharide precursor 210 after carbonyl reduction. Application of a similar sequence of steps as for the preparation of 208 then afforded the protected form of the C-linked ~-~-mannuronic(l-3) dimer 211, which was ready for the introduction of a third sugar unit by the above-described aldol reaction.
20.5 The Cyclouddition and Rearrangement Approach
527
1. t-BuOOH, DBU 2. NaBH4 3. BzCI, Py 4. B u ~ N F
30%
60%
BnO
205 1. periodinane 2. CF$OOH, H,O 3. AcCI, Py
79%
1. mCPBA, NaHC03 2. EtSH, TfOH; MeOH 3. Hg(C104). H20; Ag2CO3
* 59%
O&iZ OAc BnO
OBz
MeOOC OAc Bz 0 208
207 1.208
Me2AISPh 206
2. NaBH4
6 steps
BnO
TBs%sph
OAIMe2
210
209
AcO BzO 211
I SEt COOMe
Scheme 48.
20.5 The Cycloaddition and Rearrangement Approach In a rather different strategy to access C-disaccharides, Paton and co-workers applied the 1,3-dipolar cycloaddition of nitrile oxides derived from pyranoses with unsaturated sugars as the key step for linking the two sugar units [SO, 811. As shown with nitrile oxide 213 (Scheme 49), generated by the dehydration of the nitromethyl sugar derivative 212, its cycloaddition to alkene 214 led to the regioselective formation of the isoxazoline cycloadducts 215 in a 4: 1 ratio. Deacetylation of the major isomer and two sequential reduction steps yielded a mixture of diastereomers, with the major one, 216, being hydrolyzed to the hydroxymethylene-bridged analog O f p-D-Xyl(1-6)p-D-Glc 217. In another example, nitrile oxide 213 was cyclized with an excess of the 2,3unsaturated sugar 218 generating, after a similar sequence of steps, a 1 : 1 mixture of p(1-2)- and P(1-3)-C-disaccharides 219 and 220 (Scheme 50). In this example, the facial selectivity is controlled by the substituents of the em-pyranoside.
528
20 Synthesis of C-Oligosucchurides
'
bo OBn
"20-X OAc
tolylene di-isocyanate Et,N Reflux *
93%
Ob
212 X = CH2NO2 213 X = CNO 1 . KCN, MeOH 2. H2, Raney Ni, B(OH), 1
.,.n,l
I I
0-
215 (dr = 4: 1)
(4 equiv)
OH
H
.I\
ot\
214
g
o
a
O
OH B
l rn \
92%
216
HO
Of\
217
Scheme 49.
OAc 0
steps
213 +
-1 218
OEt
OEt
HO.
OH
HO
OH 219
1
Hd
OH
OEt
220
Scheme 50.
References 1. (a) T. Feizi, Curr. Opin. Struct. Bio. 1993, 3, 107; (b) A. Varki, Proc. Nail. Acad. Sci. USA 1994. 91, 7390.
2. J. Beuth, H. L. KO, G. Pulverer, G. Uhlenbruck, H. Pichlmaier, GlycoconjugateJ. 1995, 12, 1, and references therein. 3. S. A. Wharton, W. Weis, J. J. Skehel, D. C. Wiley, The Influenza Virus; Plenum, New York, 1989. 4. (a) P. Sears, C.-H. Wong, Proc. Nutl. Acad. Sci. USA 1996, 93, 12086; (b) L. L. Kiessling, N. L. Pohl, Chem. Biol. 1996,3, 71; (c) L. Lasky, Annu. Rev. Biochem. 1995, 64, 113. 5. (a) S. Hakamori, Curr. Opin. Zmmun. 1991,3, 646; (b) T. Toyokuni, A. K. Singhal, Chem. SOC. Rev. 1995,24, 231. 6. For some examples, see: C.-H. Wong, F. Moris-Varas, S.-H. Hung, T. G. Marron, C.-C. Lin, K. W. Gong, G. Weitz-Schmidt, J. Am. Chem. Soc. 1998, 119, 8152, and references therein. 7. (a) M. H. D. Postema, C-Glycoside Synthesis; CRC Press, Boca Raton, 1995; (b) D. E. Levy, C. Tang, The Chemistry of C-Glycosides; Pergamon Press, Exeter, 1995; (c) G. Casiraghi, F. Zanardi, G. Rassu, P. Spanu, Chem. Rev. 1995, 95, 1677. 8. (a) T.-C. Wu, P. G. Goekjian, Y . Kishi, J. Org. Chem. 1987, 52,4819; (b) P. G. Goekjian, T.-C. Wu, H.-Y. Kang, Y. Kishi, J. Org. Chem. 1987, 52, 4823; (c) S. A. Babirad, Y. Wang, P. G. Goekjian, Y. Kishi, J. Org. Chem. 1987, 52, 4825; (d) A. Wei, A. Haudrechy, C. Audin, H.-S. Jun, N. Haudrechy-Bretel, Y. Kishi, J. Org. Chem. 1995, 60, 2160 and previous work. 9. (a) A. Wei, K. M. Boy, Y. Kishi, J. Am. Chem. Soc. 1995, 117, 9432; (b) R. Ravishankar, A. Surolia, M. Vijayan, S. Lim, Y. Kishi, J. Am. Chem. SOC.1998, 120, 11297. 10. (a) J.-F. Espinosa, F. J. Caiiada, J. L. Asensio, H. Dietrich, M. Martin-Lomas, R. R. Schmidt, J. Jimenez-Barbero, Angew. Chem. 1996, 108, 323; Angew. Chem., Int. Ed. Engl. 1996, 35, 303;
Rejerences
529
(b) J.-F. Espinosa, F. J. Caiiada, J. L. Asensio, M. Martin-Pastor, H. Dietrich, M. MartinLomas, R. R. Schmidt, J. Jimenez-Barbero, J. Am. Chem. Soc. 1996, 118, 10862; (c) J.-F. Espinosa, E. Montero, A. Vian, J. L. Garcia, H. Dietrich, M. Martin-Lomas, R. R. Schmidt, A. Imberty, J. Caiiada, J. Jimenez-Barbero, J. Am. Chem. Soc. 1998, 120, 1309. 11. 0. Martin, L. Liu, F. Yang, Tetrahedron Lett. 1996, 37, 1991. 12. (a) B. A. Jones, Y . T. Pan, A. D. Elbein, C. R. Johnson, J. Am. Chem. Soc. 1997, 119, 4856; (b) A. Baudat, P. Vogel, J. Org. Chem. 1997, 62, 6252. 13. (a) J.-M. Beau, T. Gallagher, Topics Curr. Chem. 1997, 187, 1; (b) P. Vogel, R. Ferritto, K. Kraehenbuehl, A. Baudat, in Curbohjdrate Mimics (Ed. Y . Chapleur), Wiley-VCH, Weinheim, 1995, p. 19. 14. D. Rouzaud, P. Sinay, J. Chem. Soc. Chem. Commun. 1983, 1353. 15. J.-M. Lancelin, P. H. Amvam Zollo, P. Sinay, Tetrahedron Lett. 1983, 24, 4833. 16. Y.-C. Xin, Y.-M. Zhang, J.-M. Mallet, C. P. J. Glaudemans, P. Sinay, Eur. J. Org. Chem. 1999, 471. 17. H. Streicher, A. Geyer, R. R. Schmidt, Chem. Eur. J. 1996,2, 502. 18. M. A. Leeuwenburg, C. M. Timmers, G. A. van der Marel, J. H. van Boom, J.-M. Mallet, P. Sinay, Tetrahedron Lett. 1998, 38, 6251. 19. K. M. Nicholas, Acc. Chem. Res. 1987, 20, 207. 20. S. Tanaka, M. Isobe, Tetrahedron 1994, 50, 5633. 21. R. Preuss, R. R. Schmidt, Liebigs Ann. Chem. 1989, 983. 22. R. R. Schmidt, R. Preuss, Tetrahedron Lett. 1989, 30, 3409. 23. R. R. Schmidt, A. Beyerbach, Liebigs Ann. Chem. 1992, 983. 24. H. Dietrich, R. R. Schmidt, Liebigs Ann. Chem. 1994, 975. 25. T. Eisele, H. Ishida, G. Hummel, R. R. Schmidt, Liebigs Ann. Chem. 1995, 2113. 26. B. Patro, R. R. Schmidt, Synthesis 1998, 1731. 27. J. Alzeer, C. Cai, A. Vasella, Helv. Chim. Acta 1995, 78, 242. 28. S. M. Daley, R. W. Armstrong, Tetrahedron Lett. 1989, 30, 5713. 29. (a) J.-M. Lancelin, L. Morin-Allory, P. Sinay, J. Chem. Soc. Chem. Commun. 1984, 355; (b) V. Pedretti, A. Veyrieres, P. Sinay, Tetrahedron 1990, 46, 77. 30. D. Mazeas, T. Skrydstrup, J.-M. Beau, Angew. Chem. 1995, 107, 990; Angew. Cheni. Int. Ed. Engl. 1995,34, 909; T. Skrydstrup, 0. Jarreton, D. Mazeas, D. Urban, J.-M. Beau, Chem. Eur. J., 1998, 4 , 655. 31. D. MazCas, T. Skrydstrup, 0. Doumeix, J.-M. Beau, Angew. Chem. 1994, 106, 1457; Angew. Chem. In?. Ed. Engl. 1994, 33, 1383; T. Skrydstrup, D. Mazeas, M. Elmouchir, G. Doisneau, J.-M. Beau? Chem. Eur. J. 1997,8, 1342. 32. M. E. Jung, S. W. T. Choe, Tetrahedron Lett. 1993, 34, 6247. 33. 0. Jarreton, T. Skrydstrup, J.-M. Beau, J. Chem. Soc., Chem. Commun. 1996, 1661; 0. Jarreton, T. Skrydstrup, J.-F. Espinosa, J. Jimenez-Barbero, J.-M. Beau, Chem. Eur. J., 1999, 5, 430. 34. S. L. Krintel, J. Jimenez-Barbero, T. Skrydstrup, Tetrahedron Lett., 1999, 40, 7565. 35. L. Andersen, L. M. Mikkelsen, J.-M. Beau, T. Skrydstrup, Synlett 1998, 1393. 36. S.-C. Hung, C.-H. Wong, Tetrahedron Lett. 1996, 37, 4903. 37. S.-C. Hung, C.-H. Wong, Angew. Chem. 1996, ION, 2700; Angew. Clzem. Int. Ed. Engl. 1996, 35, 2611. 38. I. R. Vlahov, P. I. Vlahova, R. J. Linhardt, J. Am. Chem. Soc. 1997, 119, 1480; Y. Du, R. J. Linhardt, Carbohydr. Res. 1998, 308, 161; T. Polat, Y . Du, R. J. Linhardt, Synlett 1998, 1195. 39. Y. Du, T. Polat, R. J. Linhardt, Tetrahedron Lett. 1998, 39, 5007. 40. (a) L. Petrus, S. Bystricky, V. Bilik, Chem. Zvesti 1982, 36, 103; (b) A. Fortsch, H. Kogelberg, P. Koll, Carbohydr. Res. 1987, 164, 391. 41. 0. R. Martin, W. Lai, J. Ory. Chem. 1990, 55, 5188. 42. 0. R. Martin, W. Lai, J. Org. Chem. 1993, 58, 176. 43. Z . J. Witczak, R. Chhabra, J. Chojnacki, Tetruhedron Lett. 1997, 38, 2215. 44. W. R. Kobertz, C. R. Bertozzi, M. D. Bednarski, J. Org. Chem. 1996, 61, 1894. 45. R. Preuss, R. R. Schmidt, J. Carbohydr. Chem. 1991, 10, 887; R. Preuss, K.-H. Jung, R. R. Schmidt, Liebigs Ann. Cliem. 1992, 377. 46. A. Dondoni, M.-C. Scherrmann, J. Org. Chem. 1994,59, 6404.
530 47. 48. 49. 50.
20 Synthesis of C-Oligosucchurides
A. Dondoni, A. Boscarato, H. Zuurmond, Tetrahedron Lett. 1996,37, 7587. A. Dondoni, H. Zuurmond, A. Boscarato, J. Org. Chem. 1997, 62, 8114. A. Dondoni, M. Kleban, H. Zuurmond, A. Marra, Tetrahedron Lett. 1998, 39, 7991. B. Giese, T. Witzel, Angew. Chem. 1986, 98, 459; Angew. Chem. Int. Ed. Engl. 1986, 25, 450; B. Giese, M. Hosh, C. Lamberth, R. R. Schmidt, Tetrahedron Lett. 1988,29, 1375. 51. R. M. Bimwala, P. Vogel, Tetrahedron Lett. 1991, 32, 1429. 52. R. M. Bimwala, P. Vogel, J. Org. Chem. 1992, 57, 2076. 53. R. Ferrito, P. Vogel, Tetrahedron Asym. 1994, 5, 2077. 54. 0. R. Martin, F. Xie, R. Kakarla, R. Benhamza, Synlett 1993, 165. 55. P. Sinay, Pure & Applied Chem. 1997, 69, 459; P. Sinay, Pure & Applied Chem. 1998, 70, 407. 56. Y.-C. Xin, J.-M. Mallet, P. Sinay, J. Chem. Soc., Chem. Commun. 1993, 864. 57. A. Mallet, J.-M. Mallet, P. Sinay, Tetrahedron Asym. 1994, 5, 2593. 58. G. Rubinstein, J.-M. Mallet, P. Sinay, Tetrahedron Lett. 1998, 39, 3697. 59. A. Chenede, E. Rekai, E. Perrin, P. Sinay, Synlett 1994, 420. 60. A. Chenede, Ph. D. Thesis, Universite Pierre et Marie Curie, 1996. 61. B. Vauzeilles, D. Cravo, J.-M. Mallet, P. Sinay, Synlett 1993, 522. 62. E. D. Rekai, G. Rubinstein, J.-M. Mallet, P. Sinay, Synlett 1998, 831. 63. Y. Ito, T. Ogawa, Angew. Chem. 1994, 106, 1843; Angew. Chem. Int. Ed. Engl. 1994,33, 1765; A. Dan, Y. Ito, T. Ogawa, J. Org. Chem. 1995, 60, 4680. 64. G. Rubinstein, J. Esnault, J.-M. Mallet, P. Sinay, Tetrahedron Asym. 1997, 8, 1327. 65. B. Vauzeilles, Ph. D. Thesis, Universite Pierre et Marie Curie, 1996. 66. G. Rubinstein, Ph. D. Thesis, Universite Pierre et Marie Curie, 1996. 67. A. Helmboldt, J.-M. Mallet, M. Petitou, P. Sinay, Bull. Soc. Chim. Fr. 1997, 134, 1057; A. Helmboldt, M. Petitou, J.-M. Mallet, J.-P. Htrault, J.-C. Lormeau, P. A. Driguez, J.-M. Herbert, P. Sinay, Bioorg. Med. Chern. Lett. 1997, 7, 1507; M. Petitou, J.-P. Herault, J.-C. Lormeau, A. Helmboldt, J.-M. Mallet, P. Sinay, J.-M. Herbert, Bioorg. Med. Chem. Lett. 1998, 8, 1509. 68. R. W. Armstrong, J.-M. Beau, S. H. Cheon, W. S. Christ, H. Fujioka, W.-H. Ham, L. D. Hawkins, H. Jin, S. H. Kang, Y. Kishi, M. J. Martinelli, W. W. McWhorter Jr., M. Mizumo, M. Nakata, A. E. Stutz, F. X. Talamas, M. Taniguchi, J. A. Tino, K. Ueda, J.4. Uenishi, J. B. White, M. Yonaga, J. Am. Chem. Soc. 1989,111, 7525 and 7530. 69. (a) J.-F. Espinosa, H. Dietrich, M. Martin-Lomas, R. R. Schmidt, J. Jimenez-Barbero, Tetruhedron. Lett. 1996, 37, 1467; (b) J. F. Espinosa, M. Bruix, 0. Jarreton, T. Skrydstrup, J.-M. Beau, J. Jimenez-Barbero, Chem., Eur. J. 1999, 5, 442. 70. S. A. Babirad, Y. Wang, Y. Kishi, J. Org. Chem. 1987, 52, 1372; Y. Wang, S. A. Babirad, Y. Kishi, J. Org. Chem. 1992, 57, 468. 71. (a) H. Jin, J. Uenishi, W. J. Christ, Y. Kishi, J. Am. Chem. Soc. 1986, 108, 5644; (b) K. Takai, M. Tagashira, T. Kuroda, K. Oshima, K. Utimoto, H. Nozaki, J. Am. Chem. Soc. 1986, 108, 6048. 72. P. G. Goekjian, T.-C. Wu, H.-Y. Kang, Y. Kishi, J. Org. Chem. 1987, 52,4823; P. G. Goekjian, T.-C. Wu, H.-Y. Kang, Y. Kishi, J. Org. Chem. 1991, 56, 6422. 73. U. C . Dyer, Y. Kishi, J. Org. Chem. 1988, 53, 3384; D. J. OLeary, Y. Kishi, J. Org. Chem. 1993, 58, 304. 74. M. Carcano, F. Nicotra, L. Panza, G. Russo, J. Chem. Soc. Chem. Commun. 1989,642. 75. A. Wei, Y. Kishi, J. Org. Chem. 1994, 59, 88. 76. T. Haneda, P. G. Goekjian, S. H. Kim, Y. Kishi, J. Org. Chem. 1992, 57, 490. 77. R. W. Armstrong, D. P. Sunderlin, Tetrahedron. Lett. 1994, 35, 7743. 78. D. P. Sunderlin, R. W. Armstrong, J. Am. Chem. Soc. 1996, 118, 9802; D. P. Sunderlin, R. W. Armstrong, J. Org. Chem. 1997, 62, 5267. 79. P. Gerber, P. Vogel, Tetrahedron. Lett. 1999, 40, 3165. 80. R. M. Paton, K. J. Penman, Tetrahedron. Lett. 1994, 35, 3163. 81. I. M. Dawson, T. Johnson, R. M. Paton, R. A. C. Rennie, J. Chem. Soc. Chem. Commun. 1988. 1339.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
21 Synthesis of Oligosaccharide Mimics: S-Analogs Jon K. Fairweather and Hugues Driguez
21.1 Introduction It has been known for some time that carbohydrate-protein recognition is crucial in the biotransformation of natural oligo- and polysaccharides and, in recent years, it has also been considered that such interactions are the essential process for biological transfer of information into living organisms. Investigations of such recognition processes promote deeper understanding which might ultimately be applied to new concepts of enzyme engineering and glycotherapy. There are, essentially, two approaches to such investigations; site-directed mutagenesis of proteins and use of natural substrates or the use of native proteins and modified substrates. Whereas the former of these approaches, using the techniques of protein engineering, is becoming more popular [ l , 21, modification of substrates is somewhat more intriguing to the synthetic chemist. Some would argue that the introduction of specific alterations in the natural substrate and their interactions with native proteins are more challenging to analyze, and that only such an approach addresses the fundamental aspects of enzyme-substrate interactions-the nature of recognition, binding, and the catalytic process. Surely, one of the most versatile approaches for the study of glycosylhydrolases is the use of non-hydrolyzable oligosaccharide mimics, as competitive inhibitors, to map the active site. This can be achieved, in most respects, by substituting the natural glycosidic linkage (0)with a recognizable mimic-the analogs of choice thus far have been methylene groups (CH2) [3], free or functionalized amines (NR) [4], or sulfur atoms (S) [ 5 ] . This review will focus on thiooligosaccharides which have been synthesized with a biological application in mind.
532
21 Synthesis of Oligosaccharide Mimics: S-Analogs
21.2 General Synthesis The synthesis of thiooligosaccharides has traditionally been conducted via one of two strategies-(a) Lewis acid catalysed condensation between a glycosyl acceptor containing an SH group and a suitable glycosyl donor, or (b) S~2-likedisplacement of a leaving group of a glycosyl acceptor with a 1-thiolate or of a glycosyl halide by a sugar thiolate. Michael addition of thiolates to a,p-unsaturated systems [6], epoxide and aziridine ring openings [7], and chemoenzymatic approaches [8] have also been applied as alternatives to the traditional approaches. 21.2.1 Preparation of Thioglycoses 1-Thioglycoses
The most widely used synthons for the synthesis of thiooligosaccharides are the 1thioglycoses and their synthesis has been widely studied. Approaches for the synthesis of 1,2-trans-thioglycoses include nucleophilic sulfur substitutions at the anomeric center of the glycosyl halides with either pseudo-thourea derivatives or other thionucleophiles [9, 101. More recently, phase-transfer catalysis [ 111 and mild Lewis acid catalysed glycosidation of peracetylated sugars with thioacetic acid [ 121 have been applied. The first reported synthesis of peracetylated 1-thio-a-D-glucopyranose started from the Brigl’s anhydride [9] but traditionally, 1,2-cis-thioglycoses have been generated from the 1,2-trans-glycosylhalides by reaction of alkyl or benzyl xanthates in acetone [ 131, potassium thioacetate in hexamethylphosphoramide (HMPA) [ 141, or the tetrabutylammonium salt of thioacetic acid in toluene [15] (Scheme 1). A recent alternative is the peroxide-induced addition of thioacetic acid to hydroxy-glucal derivatives [ 161. 2-, 3-, 4-, 5, or 6-Thioglycoses
The action of thioacetate, thiobenzoate or thiocyanate anions on sugars bearing leaving groups (halides or sulfonyl), epoxides, and cyclic sulfates has afforded the thiosugars in good yield (Scheme 2).
+
-sx
____c
Br
Scheme 1. S~2-typesubstitution of glycosyl halides by thiolates.
21.2 General Synthesis
533
1,6-[17]
1,3 -[201
F-
TfO
sx
Scheme 2. General procedure for the synthesis of thioglycoses.
21.2.2 Selective S-Deprotection of Thioglycoses
The most widely used synthons for the preparation of thiooligosaccharides have been peracetylated 1-thioglycoses, which can be selectively deprotected either in a two step procedure or in situ and used as an activated species (Scheme 3). There are several methods for the selective S-deacetylation of thioglycoses; one of the most successful of these is demercuration of phenylmercury thio-a- or -P-Dglycoses [22, 231. Deprotection has also been achieved by use of sodium methoxide
R=Ac
Scheme 3. Activated thioglycosyl donors.
R=AcorH
R=AcorH
534
21 Synthesis of Oliyosaccharide Mimics: S-Analogs
at low temperature (less than -40°C) [24, 251 although 0-deacetylation is sometimes observed [24]. The thiolate anion is, however, more nucleophilic than its oxygen counterpart, and it should be noted that the fully deprotected 1-thiolates (usually generated in situ) can also be used in such reactions. A more preferential chemoselective deprotection has been achieved by the action of diethylamine in DMF [26], cysteamine in either acetonitrile [27] or HMPA containing dithioerythritol (DTE) [28], or hydrazinium acetate in DMF [ 111. The same procedures can also be used to obtain compounds with the free thiol at the 2-, 3-, 4-, 5-, or 6- positions. Although these derivatives are very prone to forming disulfide linkages, this can be overcome by treatment of such compounds with DTE in aqueous solution thereby generating the free thiol [29]. 21.2.3 Glycosylation Methods Both traditional and new approaches (often used in the preparation of 0-glycosides) have been applied to the establishment of thio linkages. Here, the synthesis of thiodisaccharides and thiooligosaccharides is described with reference to linkage specificity and method of generation.
21.3 Establishment of 1,6-Thio Linkages 21.3.1 6-Thiodisaccharides The first reducing thiodisaccharide synthesized was methyl a-thiogentiobioside (l), albeit in a very poor yield (19%), from the condensation of the tosylate (2) and the 1-thiolate (3) [30]. The low yield is probably attributable to the poor leaving ability of the tosylate group in acetone-water. Although the displacement of a glycosyl halide with a sugar thiolate overlaps the preparation of the 1-thioglycose itself, the synthetic utility of this reaction is noteworthy and, as an alternative, this work also compares the displacement of the glycosyl halide (4) with a sugar thiolate generated from 5 (Scheme 4). Methyl a-thiogentiobioside (1) was synthesized in this manner, in much higher yield (55%) [30].
OMe
2R=OTs 5R=SAc
3 R’ = SNa5R2 = H 4 R ’ = H. R = B r
OMe 1
Scheme 4. (i) K2CO3 (acetone/HzO); (ii) NaOMe (MeOH) then (acetone); (iii) AczO (pyridine).
535
21.3 Establishment of l,6-Thio Linkages
SH
R1 R2 R3 H OAc 7 OAc 9 OAc OAc H 10 NHAc OAc H
R1 R2 R3 8 OAc H OAc 11 OAc OAc H 12 NHAc OAc H
6
Scheme 5. (i) CsCO, (DMF).
Santoyo-Gonzales and co-workers [ 3 11 have recently developed a highly stereoselective and simple method for introducing thio linkages. Using the 4,6-cyclic sulfate acceptor 6, the galactosyl thiol 7 was condensed, in a cesium carbonatepromoted reaction, to yield the S-linked disaccharide 8 in excellent yield (89%) (Scheme 5). The versatile nature of this reaction has been applied to other donors; the glucosyl and N-acetylglucosaminyl thiols 9 and 10 have yielded the thiodisaccharides 11 and 12 in 52% and 68% yield, respectively. This reaction has also been generalized and a convenient route to P-1,4-gluco and P-1,3-allo thiodisaccharides has been established (see Section 21.4.1). An improved preparation of tri-O-acetyl-l,6-epithioP-D-glucose (13) [32] and the selective opening at C6 with 9 to yield the disaccharide 14 [33] (Scheme 6) could be an interesting alternative for the synthesis of complex thiooligosaccharides; the resulting thiodisaccharide or an analog refunctionalized from 15 can itself act as a donor for the synthesis of 0-glycosides. An approach to the synthesis of 1,6-thio linkages to mannose residues has recently been reported whereby the manno- 1,2-epoxide (16) was opened with the thiolate of 17 to generate the thiodisaccharide (18) in moderate yield (63%) [34] (Scheme 7). Although there are many other synthetic possibilities for the preparation of 1,6thio linkages, arguably none is easier or more effective than the displacement of a 6-deoxy-6-iodo derivative with a 1-thiolate. Recalling the poor yield of the methyl
i
AcO OAc
OAC
RO
SH OAc
SEt
RO ‘OR
13
9
Scheme 6. (i) Et3OBF4 (CH2C12); (ii) NaOMe (MeOH).
536
21 Synthesis of Oligosuccharide Mimics: S-Analogs
BnO ,OBn
\
OBn
16
17
18
Scheme 7. (i) NaH (DMF).
AcO AcO
OAc
ACO
AcO
SH AcO
-
19 R’ = OAc R2 = H 21 R’ = H, R’ = OAC
9 R’ = OAc, R2 = H 7 R’ = H, R2 = OAC
20
OAc
AcO
Scheme 8. (i) NaH (THF), then DMF; (ii) K2CO3 (acetone/H20).
a-thiogentiobioside (1) from 6-0-tosyl substitution (19%), the 6-thiogentiobiose derivative (19) has subsequently been obtained in good yield (73%) through halogen substitution of the 6-deoxy-6-iodoglucose (20) with the sodium salt of the thiol (9) in DMF [35]; 6-thioallolactose (21) was similarly obtained in moderate yield (59%) from the potassium salt of the donor 7 in acetone [36] (Scheme 8). Condensation of the iodide 20 with the thiolate 22 generated from 23, by selective S-deacetylationwith cysteamine in HMPA with DTE, afforded the 6-thioisomaltose (24) in good yield (69%) [37] (Scheme 9).
A AcOc
O OAc a OAc
+
AGO% ACO
i
AcO
R
AcO
Acfc+
AcO*AcO OAc OAc
20
Scheme 9. (i) cysteamine/DTE (HMPA).
22 R = S-
23 R = SAC
24
21.3 Establishment ojl,6-Thio Linkages ACO,
OAc 1
537 yOpMe
-
Acd
R' I
R*
R' R2 R3 25 OAc OMe H 26 NHAc OMe H 32 NHAc H O(CH2)2N3
R' R2 R3 R4 27 Br OAc OMe H 28 Br NHAc OMe H 31 OTS NHAc H O(CH2)2N3
29R=Ac 30R=Na
Scheme 10. (i) EtzNH (DMF); (ii) (DMF)
Other easily generated leaving groups have been investigated and two recent papers suggest that D M F is a good solvent for the effective substitution of tosylate and bromine at C-6 of sugars when used in conjunction with the appropriately activated donor. The N-acetylneuraminic acid derivatives 25 and 26 have been synthesized quite efficiently (82 and 75%, respectively) when the methyl 6-bromo-6-deoxya-D-glycosides (27 and 28, respectively) were coupled with the 2-S-acetyl-Neu5Ac (29) [26] (Scheme 10). The S-deacetylated and activated donor was prepared in situ by treatment of thioacetate 29 with diethylamine in DMF (room temperature). The sodium salt (30) could be isolated and was condensed with the tosylate (31) by heating in DMF, thereby generating the a(2-6) thiodisaccharide 32 in appreciable yield (76%) [38]. The sodium salt of 1-thio-a-L-fucopyranose (33) was generated by treatment of thiol 34 with NaH (DMF); subsequent condensation with the ally1 glycoside 35 afforded the a( 1-6) thiofucosyl disaccharide 36 in remarkable yield (99%) through a sequence of o-acetylation in the presence of dimethylancinopyridine (DMAP) and de-o-acetylation [7] (Scheme 11).
HO
-
Me%OH
i-iii
A AcOc Me&slA~ AcO
O
+
S0 NHAc
HO*HO
0 NHAc
33R=Na 34R=H
35
36
Scheme 11. (i) NaH (THF), then (DMF); (ii) Ac20-DMAP (pyridine); (iii) NaOMe (MeOH).
21 Synthesis of Oligosaccharide Mimics: S-Analogs
538
A AcO c
O
S
i OAc
AcO
38 R' = H,R2 = SC(=S)OEt 19 R' = H,R2 = OAc 39 R' = Br, R2 = H
37
Scheme 12. (i) NaOMe (CHC13), then (DMPU).
21.3.2 6-Thiooligosaccharides The dithiogentiotriose 37 was prepared by methoxide treatment of the thiodisaccharide 38, generated from 19 via the bromide 39, and the iodide 20 in 1,3dimethyl-2-oxohexahydropyrimidine (DMPU) (50%) [35] (Scheme 12). A similar approach has resulted in the synthesis of thiomaltose derivatives; the peracetylated 1-thio-a-D-glucopyranose 23 was coupled with 6-deoxy-6-iodomaltose (40) affording the thiotrisaccharide 41 (87%) [ 391; 6"-deoxy-6"-iodomaltose (42) afforded the thio-a-glycosyl derivative 43 in excellent yield (80%) [40]. In addition, the 6W-deoxy-6W-iodomaltooligosaccharides44 and 45 afforded the expected 6O-Sa-D-glucopyranosyl 6w-thiomaltooligosaccharides 46 (86%) and 47 (80%), respectively [41] (Scheme 13). The thiodisaccharide 18 (obtained as described earlier) was condensed in the presence of triethylsilyltriflate (TESOTF) with the trichloroacetimidate 48 to generate the thiotrisaccharide 49 in very good yield (88%) [34] (Scheme 14). This compound has been used as a probe of N-acetylglucosyltransferase [34].
21.3.3 Branched Thiocyclodextrins The selective deacetylation and in situ activation of thioacetates with cysteamine and DTE has been widely used for the synthesis of numerous cyclodextrin derivatives. The branched CDs 50 (82%), 51 (63%) and 52 (85%) were synthesized from 6-deoxy-6-iodocyclomaltoheptaose (53) and the S-activated glycoses of 23, 54, and 55, respectively [39, 421. Interestingly, only activated. 1-thio-a-D-mannopyranose (56) gave the expected CD (57) in high yield (83%) [42] (Scheme 15). Alternative routes to branched CDs have also been investigated. The branched thiocyclomaltoheptaoses 50 and 51 have also been obtained in 64 and 60% yield, respectively, from 6-0-tosylcyclomaltoheptaose (58) by s N 2 displacement with the I-thiolates 59 and 60 [43]. The utility of such a reaction for tethering cyclodextrins
21.3 Establishment of 1,6-Thio Linkages
AcO
+
539
Acfc*
k2c%&
23
OAc OAc
AcO
OAc OAc
40
41
AcO
+
23
Aco+oe) AcO
AcO
0 Ac
AcO
OAc
n
42n=1 44n=2 45n=3
43n=l 46n=2 47n=3
Scheme 13. (i) cysteamine/DTE (HMPA).
BnO
+
cc13
18
i
BnO BnO
O(CH2)7CH3
OBn 48
49
Scheme 14. (i) TMSOTf (CH2C12).
is exemplified by the more recent synthesis of a bis-thiocyclomaltooligosaccharide with an a,a-trehalosyl linker (61) upon condensation of 62 with 58 in DMF in the presence of sodium iodide [44]. Tosylate and bromide groups have a similar efficiency as leaving groups and the heptakis(6-bromo-6-deoxy)cyclomaltoheptaose (63) yielded the heptakis-6-thiogalactosylcyclomaltoheptaoses64 (80%) and 65
540
21 Synthesis of Oligosuccharide Mimics: S-Analogs , R’
R’ R2 53 I OAc 58 OTs OH 63 Br Br
R3 OAc OH OH
R2
23 54 55 56 59 60 66 67 62
R’ R2 R3 R4 R5 H SAC OAc H OAc SAC H OAc H OAc SAC H OAc H OAc H SH H OAcOAc SNa H OH H OH H SNa OH H OH SNa H OH H OH H SNa OH H OH
R7 OAc OAc H OAc OH OH H H
50 R‘ 51 R’ 52 R’ 57 R’ 64 R’ 65 R’
= a-S-Glc, R2 = OH = P-S-Glc, R2 = OH = p-S-Gal, R2 = OH = a-S-Man, R2 = OH
= R2 = a-S-Gal = R2 = p-S-Gal
61 R’ = o @ *:H i
:;*O~~OH HO 0
R6 H H OAc H H H OH OH
SNa
HO 0
OH ,R S-6p-CD
Scheme 15. (i) Cysteamine/DTE (HMPA), then NaOMe (MeOH); (ii) (DMPU), then NaOMe (MeOH); (iii) NaI (DMF), then NaOMe (MeOH).
(50%) although only when treated with the sodium salts of the 1-thiogalactoses 66 and 67, respectively, at elevated temperature [45] (Scheme 15). A new and exciting application to the synthesis of branched cyclodextrins has recently been reported, whereby acarbose (68), the known potent inhibitor of glucoamylase, has been tethered to a cyclodextrin via flexible spacers of different length [46]. The functionalized spacers (69 and 70) were S-deacetylated and the corresponding thiolates coupled easily to 6-deoxy-6-iodocyclodextrin(53),affording the branched fully acetylated cyclodextrins 71 (76%) and 72 (64%), respectively.
~=OH
21.4 Establishment ofl,4-Thio Linkages
541
These were refunctionalized as their iodides (73 and 74) and elongation of 73 with the spacer 69 under similar conditions generated the trityl ether 75 and thence the iodide 76. The thioacetate derivative of acarbose (77), was coupled to the cyclodextrin 53 and branched cyclodextrins 73, 74, and 76, and then subsequently deprotected, readily affording the acarbose tethered branched cyclodextrins 78 (63%), 79 (88%), 80 (83%) and 81 (84O), respectively, in a good yield (Scheme 16).
21.4 Establishment of 1,4-Thio Linkages 21.4.1 1,CThiodisaccharides General Approaches The first report of SN2-like displacement of glycosyl halides -y sugar thiolates for generating 1,4-thio linkages emerged in 1978 [ 141. Treatment of the 1,2-trans glycosyl halide 82 with the sodium salt generated from methoxide treatment of the methyl 4-thio-a-~-glucoside83, afforded methyl 4thiomaltoside (84), albeit in poor yield (34%) [ 141. Similarly, 4-thiocellobioside (85) and 4-thiodigalactobioside (86) were also synthesized but in only slightly better yield (52% and 56%, respectively) from the halides 4 and 87 and the corresponding thiolates from 83 and 88 [14] (Scheme 17). Methyl 4-thiolactoside (89) was also synthesized from the bromide 87 by a slight modification of this procedure. Somewhat surprisingly, however, the reverse coupling failed to give the expected thiodisaccharide [47]. A synthesis of the 4-thiogalabioside (90) has been reported whereby the thiolate of 91, generated by treatment with cesium carbonate, has been condensed with the 1,2-cis chloride 92 in good yield (85%) [48] (Scheme 18). The benzylated 1,6-anhydro P-D-glucose (93) has been coupled to trimethylsilyl4thio a-D-glucoside (94) under acidic conditions to afford the thiodisaccharide 95 in good yield (66%; a/P, 15: 1) [49] (Scheme 19). A noteworthy comment from the authors reveals, however, the poor reactivity of the 1,6-anhydro disaccharide (not shown). As eluded to earlier for the preparation of 1,6-thiodisaccharides (Section 21.3.1) the cyclic sulfate group has been targeted as a means of establishing thio linkages [31]. An extension of this has seen the thiodisaccharides 96 (73%), 97 (61%), and 98 (64%) produced from the regioselective opening of the 3,4-cyclic sulfate 99 by the thiols 7, 9, and 10, respectively (Scheme 20). On the other hand, nucleophilic attack of the same thiols (7, 9, and 10) on the corresponding methyl p-glycoside gave p(1,3)-thiodisaccharides in the gulo series. S~ZDisplacementon Triflates Whereas nucleophilic attack of an anomeric alkoxide on primary triflates is extremely efficient (50-80%), the attack on secondary triflates is somewhat lower
542
21 Synthesis of Oligosaccharide Mimics: S-Analogs
69 rn = 1, n = 2 70 m = 2. n = 3
68
I 77
I
vii-k
SAC AcO
Y
i-iii
71 rn = 1, n = 2, R = OTr 72 rn = 2, n = 3, R = OTr 73 rn = 1, n = 2, R = I 74 rn = 2, n = 3, R = I 75 rn = 4, n = 3, R = OTr 76 rn = 4, n = 3, R = I
J iv-vi
x-xi
CH3
HO
78rn=O 79 rn = 1, n = 2 80rn=2,n=3 81 m = 4 , n = 3
Scheme 16. (i) NaOMe (MeOH); (ii) (DMF); (iii) Ac20-DMAP (pyridine); (iv) HBF4/H20 (CH3CN); (v) MsCl (pyridine); (vi) NaI (DMF); (vii) A q O (pyridine); (viii) HBr/HOAc (CH2C12); (ix) KSAc (DMF); (x) Et2NH (DMF); (xi) NaOMe (MeOH), then NH40H.
21.4 Establishment of 1,l-Thio Linkages
a
civ
AcS% AcO
CI
AcO AcO
___t
OAC
OMe
83
82
84
-
83 AcO
543
i-iV
H HO
O
a HO
Br
4
OH
85 HO
Ace%
HO
i-iv
AcO
Br
OMe
87
88
83
OMe
86
i,ii,v,iv
HO "o
~
)geo"e
+
87
HO
89
OH
Scheme 17. (i) NaOMe (MeOH); (ii) (HMPA); (iii) Ac2O (pyridine); (iv) NaOMe (MeOH); (v) BzCl (pyridine).
Acop Ac;s .& [ii
AcO
OAc
~
OAc OEtTMS
+
HO*s 92
91
Scheme 18. (i) Cs2CO3 (DMF); (ii) NaOMe (MeOH).
90
OEtTMS
OH
544
21 Synthesis of OligosaccharideMimics: S-Analogs
i,ii OMe
OBn
BnO
OMe
95 (+ p-linked isomer)
94
93
Scheme 19. (i) ZnIz (CH2Cl2); (ii) KzC03 (MeOH).
i
SH
+
+& fs>
R
R3 2 e
;
V
o
M
e
AcO AcO OMe
R' R2 R3 7 OAc H OAc 9 OAc OAc H 10 NHAc OAc H
~
99
R'
OAc
R' R2 R3 96 OAc H OAc 97 OAc OAc H 98 NHAc OAc H
Scheme 20. (i) CsCO3 (DMF).
yielding. The main hindrance is the basicity of the alkoxide anion; this restricts the acceptor molecules used in the coupling reactions to those bearing only ethers and ketal protecting groups. The lower basicity and higher nucleophilicity of the sulfur atom, however, enables a I-thiolate to be generated in the presence of base-sensitive protecting groups. The majority of 1,4-thio linkages have, hence, been achieved by this method. 4-Thiomaltose (100) was obtained in good yield from either 4-O-triflate displacement of the 1,6-anhydro galactose (not shown) or of the methyl a-D-galactoside (101) with donor 60 [15]. These reactions were effected by condensation in HMPA and generated the thiodisaccharide 102 which was isolated and then subjected to acetolysis and deprotection (Scheme 21). 4-Thiocellobiose (103) was similarly obtained in an overall yield of 70% from the same acceptor (101) and the donor 59 via the thiodisaccharide 104 [50, 511 (Scheme 22). Tetra-O-acetyl-1-thio-P-D-galactoside (7), generated in situ from the corresponding pseudothiourea precursor, was coupled to the 4-O-triflyl-galactosyl sphingosine (105) yielding the thiodisaccharide (106) in 68% yield [52] (Scheme 22). The efficiency of this procedure extends to other series and under similar conditions, 4-thioxylobiose (107) was easily obtained (60% yield) by condensation of the thiolate (108) generated from the peracetylated l-thioxylose (109) and the triflate (110) [53]. A very recent investigation by Stick and co-workers [54] has afforded the pseudo-
21.4 Establishment of I,l-Thio Linkages
i,ii
545
-
BzO
OMe
60
101
102 R' = H,R2 = OMe, R3 = Bz, R4 = Ac
iii,iv
K 100 R',R2 = H,OH, R3 = R4 =OH
Scheme 21. (i) (HMPA); (ii) Ac20-DMAP (pyridine); (iii) H?S04/HOAc (AczO); (iv) NaOMe (MeOH).
R' R2 R3 R4 59Na OH OH H 7 H OAc H OAc
101 R' = H, R2 = OMe 105 R' = cerarnide, R2 = H
R3 R4 R5 R6 OBz OAc OAc H OH OH OH H OBz OAc H OAc
R' iili~104 H Io3 H,OH 106 cerarnide
TfO
BZO
108 R' = Na R2= H 109 R' = R'= Ac
110
A,+
"
\
OH
107
Scheme 22. (i) (HMPA); (ii) Ac20--DMAP (pyridine); (iii) H ~ S O ~ / H O A(Ac20); C (iv) NaOMe (MeOH); (v) K2C03 (acetone-MeOH).
thiodisaccharide 111; this was achieved in moderate yield (40%) by coupling of the condurithiol derivative 112 and the triflate 113 (Scheme 23). This molecule might prove interesting in xylosidase-inhibition studies. Thiodisaccharides in the N-acetylglucosamine (GlcNHAc) series have also been reported. N,N"-diacetylthiochitobiose (114) was achieved (50% and 63% yield respectively) via triflate substitution of the 2-acetamido-2-deoxy-galactosides 115 or 116 with thiolates generated from 117 or 10 [ 5 5 ] (Scheme 24). The ally1 glycoside 118 has also been coupled to the thiol 34 thereby generating the thiodisaccharide 119 in good overall yield (40%) [7] (Scheme 25).
546
21 Synthesis of Oligosaccharide Mimics: S-Analogs TfO
BnO-Q/ BnO
OBn SH
MOM.
+ BzO
BzO
112
i-v
H
~
'
~
113
s
~ OH O
111
Scheme 23. (i) DBU (toluene); (ii) NaOMe (MeOH-THF); (iii) Na (NH3(hq));(iv) Ac20-DMAP (pyridine); (v) NaOMe (MeOH).
OH
i-v -
AcO
HO H
a
t
v
AcHN o
AcNH
NHAc
117R=Ac 10R=H
O
R' R2 R3 115 OMe H NHAc 116 H OMeNHAc
H
OH
R4
114
OBz OBz
Scheme 24. (i) NaH (DMF) or cysteamine (DMF); (ii) NaOMe (MeOH); (iii) Ac20-DMAP (pyridine); (iv) HzSOd/HOAc (AczO); (v) NaOMe (MeOH).
HO
i-iii,ii,iv,ii,v
Me
OAC TBDMSO LI
AcO
NHAc
34
118
119
-
Scheme 25. (i) NaH (DMF); (ii) Ac20-DMAP (pyridine); (iii) B q N F (THF); (iv) H2S (pyridine/ H20); (v) NaOMe (MeOH).
21.4.2 1,4-Thiooligosaccharides Conventional Approaches
The peracetylated 1,4-thiodisaccharides of cellobiose [5 11 (120) and N,N"diacetylchitobiose [ 5 5 ] (121), obtained relatively easily by conventional chemical manipulation of their thiodisaccharide precursors, are key compounds in the synthesis of the 4,4"-dithiocellotrioside 122 and N,N'l,N1l'-triacetyldithiochitotrioside (123) by coupling with their respective triflates (101 and 116). The corresponding deprotected analogs (124 and 125) were obtained in good to fair yields (Scheme 26). An extension of this sequential modification and addition has afforded the tri-
M
547
21.4 Establishment ofl,4-Thio Linkages
120 R' = OAc 121 R1 = NHAc
101 or 116
R1 OMe OMe
122 123 124 125 126
OMe
127
H
R2 H H H H SCtjH4NH2
OMe H
(3)
R3 OAc NHAc OH NHAc OH
R4 Ac Ac H H H
OH
H
OH
H
OH
H
ggOMe 128
H AcHNOMe
129
H
:
e OH
H
f
j
HoOMe
Scheme 26. (i) Cysteamine/DTE (HMPA or DMF).
saccharide 126, the tetrasaccharides 127 and 128, and even a pentasaccharide 129 in reasonable yield [ 51, 55, 561. The tetrasaccharide 130 and the hemithiocellotetraoside 131 have also been obtained from the laminaribiosyl (132) or cellobiosyl (133) donor by use of the common lactosyl triflate acceptor (134) [57, 581 (Scheme 27). In the thiomaltose series, the first synthesis of thiomaltotriosides [59] (not shown) was considerably improved by the use of a versatile acceptor molecule. The trityl 1S-a-D-glycosides have masked a-thiol functionality at C-1 which could be easily and selectively activated for future condensation reactions [60, 611. The trityl 1-S-C~-Dglucoside 135 and galactoside 136 were easily obtained by S N displacement ~ of the p-chlorides 82 and 92 with the tetrabutylammonium salt of triphenylmethanethiol. The use of this aglycone proved exceedingly advantageous because it was easily transformed into the corresponding thioacetates 23 and 134 (Scheme 28). A synthesis of methyl 4,4"-dithiomaltotrioside (138) has subsequently been achieved in good overall yield (41%) by selective in situ deacetylation and activation of the thioacetate 23 and condensation with the trityl 1-S-galactoside 139 to yield the dithiodisaccharide 140. This in turn could be transformed into the donor 141 and condensed with the triflate 101 to yield the trisaccharide 142 [62] (Scheme 29). An extension of this work, using the donor 143 has resulted in the production of a suite of thio- and hemithiooligosaccharides up to degree of polymerization of 8 [63].
548
21 Synthesis of Oligosaccharide Mimics: S-Analogs
130
i,ii
132
TfO BzOs
B
z
.
z
.
O
M
e
134
ACO OAc
OAc
133
Scheme 27. (i) Et2NH (DMF); (ii) NaOMe (MeOH).
CI
iijii
A
OAc
82 R‘ = O A C ~ R =H ~ 92 R’ = H, R = OAc
~
STr
135 R’ = OAc R2 = H 136 R’ = H, Rp = OAc
SAC
23 R1= OAc, R2 = H 137 R’= H, R2 = OAc
Scheme 28. (i) BU4NSTr (toluene); (ii) PhHgOAc (MeOH/CH2C12); (iii) H2S (CH2Cl~/pyridine/ Ac20).
Chemoenzymatic Approaches Hemithiocellodextrins can essentially be viewed as a repeating chain comprising 4-thiocellobiosyl units linked by a P-1,4-O-glycoside or, conversely, as cellobiosyl units linked by P-1,4-S-glycosides. The enzymic oligomerization of 4-thio-Pcellobiosyl fluoride (144) by use of a cellulase in a mixed solvent system has resulted in the isolation of hemithiocellodextrins of different length (145 n = 1 (4.5%), n = 2 (7.5%), n = 3 (5.7%), n = 4 (5.0%), n = 5,6 (20%)) [64] (Scheme 30). A similar chemoenzymatic approach has been used for the synthesis of a series of
549
21.4 Establishment of 1,4-Thio Linkages
23
+
i
BzO
w
AcO
STr
140 R’ = Tr, R2 = Bz = Ac, R2 = BZ 143R’=R2=Ac
139
ii I 1 4 1 R’
141
+
Tfo%
i *
BzO
R20
OMe
101
R10
142 R’ = Bz, R2 = Ac 138 R’ = R2 = H
~.
17
R1oAM,
iii
Scheme 29. (i) Cysteamine/DTE (HMPA); (ii) PhHgOAc (EtOH), then H2S (Ac*O/CH*C12/ pyridine); (iii) NaOMe (MeOH).
hemithiocyclodextrins, viewed as a cyclic chain of repeating thiomaltose units linked through a-1,4-O-glycosides. The ability of cyclodextrin glucosyltransferase (CGTase) to use maltosyl fluoride (146) for the synthesis a-, p-, and y-cyclodextrins has been known for more than a decade [65],and more recent applications for the chemoenzymatic synthesis of regioselectively substituted cyclodextrins have also been applied [66, 671. In this fashion, the self condensation of 4-thio-a-maltosyl fluoride (147) in the presence of pure CGTases has afforded the hemithiocellodextrins (148 n = 4 (14%), n = 5 (l6%), n = 6 (16%)) [8] (Scheme 30). Michael Addition to Unsaturated Acceptors A recent application of Michael addition of sugar thiols to levoglucosenone (149) has been reported [6, 68, 691. The addition of the thiols 9 and 34 to levoglucosenone (149) resulted in the synthesis of the anhydrides 150 and 151. These were stereoselectively reduced, subjected to acetolysis conditions, and deprotected to yield the 3-deoxydisaccharides 152 and 153 in moderate yield (approximately 60% overall) [69]. The 2-acetamido-2-deoxyglucose derivative 154 was achieved by oximation of 151 which gave 155 before reduction and ring opening (Scheme 31).
21 Synthesis of Oligosuccharide Mimics: S-Analogs
550
OH
OH
OH
145
HO
HO
146X=O 147X=S
148
Scheme 30. (i) Cellulases (CH3CN/acetate buffer 0.05 M, pH 5.0); (ii) CGTases (phosphate buffer 0.2 M, pH 6.5).
Solid-Support Synthesis
Despite the popularity of the triflate displacement method, it does have one drawback-when the acceptor molecule bears an aglycone of the p-D configuration, H5 and H3 abstractions are known to occur. The yield of the desired coupled product and that resulting from elimination vary depending on the reaction conditions and the OH-6 and OH-3 protecting groups of the acceptor [7, 55, 701. For acceptors bearing a P-aglycone, the recent application of solid-support synthesis could be an important improvement [71]. The key feature of such a synthetic scheme is the use of bound and deprotected nucleophilic sugar 1-thiolates. Subsequent condensation with triflates gives the expected compounds in good to high yields. In the case where the aglycone of the acceptor molecule is in the p configuration, the usual by-products from the condensation are negated by the flushing the support after glycosylation, and the thiooligosaccharide is the only product observed when cleavage and isolation have been effected. The suitability of this method for the construction of trisaccharides is demonstrated below (Scheme 32).
551
21.5 Establishment of 1,S-Thio Linkages
9-
i
+
0
149
+ 34
. i * Me@OA~
HR -
SI
O ,H
ii-iv
or vi,iii,iv
1 OH
HO
AcO
v(
151 R = 0 155R=NOAc
153 R = O H 154 R = NHAc
Scheme 31. (i) Catalytic Et3N (benzene); (ii) L-selectride@ and Ac2O (pyridine); (iii) BF,.OEt2 (AczO); (iv) Et3N (MeOH/H20); (v) NHzOH.HC1 (EtOH/pyridine); (vi) 9-BBN (H202, NaOH) then Ac2O (pyridine).
21.5 Establishment of 1,3-Thio Linkages 21.5.1 1,3-Thiodisaccharides Conventional Methods Sp~2displacement of the activated allofuranose 160 has afforded the a- and 8-1,3thiodisaccharides of glucose and xylose [35, 37, 721. The donors (9, 23 or 162, 161) have been condensed with the triflate 160 thereby generating the thiodisaccharides 163 (84%), 164 (94%), and 165 (54%), respectively (Scheme 33). Deprotection then afforded the thiolaminaribiose 166, thionigerose 167 and, after standard procedure, the ~-1,3-thioxylobiose168. Cyclic Sulfamidate and Aziridine The sequence a-~-fuc( 1j 3 ) - ~ - G l c N H A cis a common constituent of Lewisxbearing glycoconjugates and the establishment of non-hydrolyzable analogs could prove invaluable for several biological processes. To this end the cyclic sulfamidate activating group has also been targeted for nucleophilic attack and thence genera-
552
21 Synthesis of Oligosaccharide Mimics: S-Analogs
Tfos
OTr -@ HO HO*sNa
+
BzO
SSEt
OBz 157
OH 156
OTr -@ H HO O - t e S S E t
OH
0 Bz
158
I H HO O & g e OH ; &
ii,iii,ii then 157, i,iW
OH
OH
SSEt
159 Scheme 32. (i) Crown ether 15-5 (THF); (ii) NaOMe (MeOH/THF); (iii) DTT (MeOH/THF/ Et3N); (iv) TFA (CH2C12).
tion of a 1,3-thio linkage to 2-acetamido-2-deoxyglycosides [73, 741 (Scheme 34). The 1-thiofucose 34 has been rapidly condensed with the acceptor 169, generating the expected disaccharide 170 (50% yield). The deprotection steps gave 171 in good overall yield. The reactive N-tosyl aziridine 172 has also been condensed with the 1-thiol fucose 34 to yield a mixture of the (11 2 ) and (1 '3) thiodisaccharides (2 : 1) [7]from which the desired compound (173) was isolated in 45% overall yield (Scheme 34). 21.5.2 1,3-Thiooligosaccharides
31,3rr-Dithiolaminaritriose (174) has been conveniently prepared (albeit in only 15% overall yield) from the bromide 175 which is derived from 166 [35]. Nucleophilic displacement on 175 with the sodium salt of 176 gave the tri-
21.6 Establishment of1,2-Thio Linkages
553
OH
SR
HO
162R=H i,V
164
I6O
AcO
\ SH
vi
167
Me
~
/ 'Me
OAc
9 R = C H OAc 161 R = d
163 R = CH~OAC 165 R = H
vii,i or viii,ix, vii,iii,i
166 R = CHpOH 168R=H
Scheme 33. (i) NaOMe (MeOH); (ii) HMPA; (iii) AczO (pyridine);(iv) NaH (THF), then (DMF); (v) (80% AcOH); (vi) NaH, Kript0fix~21(THF); (vii) (60% AcOH); (viii) (50% AcOH); (ix)NaI04 then NaBH4 (EtOH/H20).
saccharide 177 in 30% yield. The usual manipulations gave the free sugar (174; Scheme 35).
21.6 Establishment of 1,ZThio Linkages 21.6.1 1,2-Thiodisaccharides 1-Thiosucrose, the true analog of sucrose, was first obtained in 1984 [23] by Lewis acid catalysed glycosylation using tetra-0-acetyl- 1-thio-a-D-glucose (162) (not
554
21 Synthesis of Oligosuccharide Mimics: S-Analogs
i ii OR' OAc AcO
34 170 R' = OBn, R2,R3= PhCH, R4 = Ac
HO
172
iii,k
Ts
E 171 R' = OBn, R2 = R3 = R4 = H 173 R' = Pr, R2 = R3 = R4 = H
Scheme 34. (i) NaH (DMF); (ii) HzS04 (HzO/THF); (iii) H+ (MeOH); (iv) NaOMe (MeOH); (v) AczO, DMAP (pyridine); (vi) Pd/C, Hz (MeOH); (vii) Na (NH3(llq)/THF).
kO*:c AcO
OAc
175
Br
) *HOa:H i,ii
HO
OH
SR
HO
Me 0
M e x o &-bM ;e o
177 R =E x z b ;,s-hle o
1 iii
176 174R= HO
OH
Scheme 35. (i) Na (MeOH); (ii) crown ether 15-5 (THF); (iii) (60% AcOH).
shown). The reaction proceeded in very good yield (70%) but the isolation of pure 1-thiosucrose was laborious. Interestingly, the reverse condensation did not afford the desired compound. Conventional Methods Most 1,2-thio linkages have been established via S N displacement ~ of a triflate of either glucosyl or mannosyl acceptor molecules with 1-thiolates.
21.6 Establishment of 1,2-Thio Linkages
R +
AcO
162
i-iii
3
R
-
-c
OAc
R
q R1
R’ R2 178 H.OH 181 OAC H 183 OAll H 185 OBn H
555
R3 OH OAc OAc OH
179
79
+
i-iii
=
R’ R2 R3 R4 R’ 180 H,OH OH CH20H R2 182 184 OAc OAll H OAc EH20Ac CH20Ac
RkR+ R3R&s
R3
186 H,OH
OH
Scheme 36. (i) NaOMe (MeOH); (ii) (DMF) or (HMPA); (iii) AczO (pyridine)
There has recently been interest in synthesizing thio analogs of kojibiose, a known a-glucosidase inhibitor. The synthesis of thiokojibiose (178) was first achieved in very good yield (87%) by condensation of the triflate 179 with 1-thio-a-D-glucose (162) [27] (Scheme 36). Thiosophorose (180) was similarly produced from the pthiol 9 but when it was observed that deprotection of the peracetylated products of condensation (181 and 182) under Zemplen conditions was somewhat problematic, the easily removed allyl group was introduced at the anomeric position thus affording the glycosides 183 and 184. Shortly thereafter the acetylated 181 was converted to the benzyl kojibioside (185) by conventional methods [37]. A similar method (not shown) was used during the synthesis of the 2-thioxylobiose (186) [72] (Scheme 36). Other Approaches Pinto and co-workers [75] have chosen a more traditional approach toward establishing 1,2-thio linkages and ultimately the production of thiokojibiosides and related derivatives. The thiol 187 was coupled to the p-trichloroacetimidate 188 thereby yielding the protected allyl kojibioside 189 [75] (Scheme 37). Interestingly, when the w-trichloroacetimidate 190 was used as donor the thiosophorose derivative 191 was obtained as the major product (191 : 192, 2.3: 1 ) in a reaction proceeding via the stable orthoester 193. In addition, the thiol 194 was condensed with the 5-thiotrichloroacetimidate donor 195 to yield a mixture of compounds from which the dithiodisaccharide 196 was isolated in good yield (approximately 70%) [76] (Scheme 37). A further paper by the same authors reports the synthesis of the dithiodimannoside 197 from the appropriate precursors; the thiol 198 was condensed with 199 to afford 200 [77] in modest yield (37%) (Scheme 38).
556
R
21 Synthesis of Oligosuccharide Mimics: S-Analogs
R 2O
:
:
OR’
k
+
-
187
+
190
OR’
0TCC’3 NH
OR
SH
anomer 188 p 190 a 195 a
187 R’ = All, R2 = Bn 194 R’ = Pr, R2 = Bz
R20
i
%O :R
X 0 0 S
X 189 0 192 0 196 S
R Bn Ac Ac
R’ R2 R3 All All Pr
Bn Bn
Bn Ac
H
H
-
-
ON1
i
OAc 193
191
Scheme 37. (i) TESOTf (CH2C12).
phTq +
OMe
AcO
AcO A
c
t
q
Rq; Rzr% K i
3I‘ NH
R’ 0
198
Scheme 38.
(1)
TESOTf (CH2C12); (ii) HOAc/H,O; (iii) NaOMe (MeOH).
2,3-Epoxides have also been targeted for nucleophilic attack. Ring opening of the talopyranose 201 with a-fucosyl thiol (34) afforded a mixture of fucosyianhydrogalactoside and anhydroidosides (not shown). Separation and further reaction of the former compound gave 202 in moderate overall yield (50%) [7] (Scheme 39).
OMe
557
21.7 Establishment oj1,l-Thio Linkages
cvi
+
&d
HO
Aces
OAll
AcO
*
OAc AcO
Ms$e
OAc
OAc
AcO
202
34
201
Scheme 39. (i) NaOMe (MeOH); (ii) Aq0-DMAP (pyridine); (iii) H*SOd/HOAc (AczO); (iv) N2H4.AcOH (DMF); (v) CC13CN (DBU/CH2C12); (vi) AIlOH/BF3.OEtz (CH2C12).
21.7 Establishment of 1,l-Thio Linkages The syntheses of a,a-thiotrehalose (203) and a-D-glucopyranosyl 1-thio-a-Dmannopyranose (204) as substrate analogs for trehalase were achieved more than twenty years ago [ 141. This study was the first to note that the condensation of a thiolate or a glycosyl halide proceeds in HMPA without any neighboring group participation (Scheme 40). Na2S
Z
i,iii CI
AGO% AcO
82 OAC
W
o
H OH
HO
\-//
e
OH ‘s
1, 111
203
Y+ 60
82
57
SNa
ii,iii
Hr-HO
SH
Scheme 40. (i) HMPA (ii) K2CO3 (HMPA/H2O); (iii) NaOMe (MeOH).
&OH OH
s 204
OH
558
21 Synthesis of Oligosaccharide Mimics: S-Analogs
Hydrogen sulfide reacted with D-glucose (not shown) in hydrogen fluoride solution to yield predominantly compound 203 [78].
21.8 Establishment of Mixed Thio Linkages Ganglioside and Lewis A epitopes in which some of glycosidic oxygens have been substituted by sulfur have recently been obtained [38, 791. The thio-linked sialyl Lewis' analog and its precursor, the thio-linked ganglioside GM3 epitope have also been described [20, SO]. The coupling of the thiodisaccharide precursors 205 and 206 using well established methods has afforded the expected tetrasaccharide 207 in good yield. The thiolate 208 plays a pivotal role in this synthesis (Scheme 41). The branched sulfur-linked tetrasaccharides 211 and 212 have been conveniently prepared by s N 2 displacement of the triflate [35, 811 group of 160 or the iodide 20 with the sodium salt of the trisaccharide 213 (Scheme 42).
21.9 Thiooligosaccharides and Proteins Thioglycosides, and more commonly thiooligosaccharides, have played an important role in probing natural oligo- and polysaccharide recognition by proteins. Most of these studies have been performed during the last five years. These non-natural substrates have been used to define protein function often by intercepting the very mechanism by which they act, because these compounds are resistant to hydrolysis by O-glycosylhydrolases. Notably, there is only one class of naturally occurring thioglycoside, the glucosinolates, which are degraded under the action of myrosinases [82]. This enzyme is the only S-glucosidase known in nature, and recent studies reveal the remarkable similarity between this protein and the cyanogenic P-glucosidase [83].
21.9.1 The Conformation of Thiooligosaccharides in Solution The first step in carbohydrate-protein interaction is the recognition; this is dependent on the overall conformation of the oligosaccharide. It is, therefore, important to determine how closely these non-natural substrate analogs represent the natural compounds in aqueous solution. This can be achieved by a combination of NMR spectroscopy, molecular mechanics calculations, and X-ray crystallography. Conformational assessments of 4-thiomaltose [84-861, 4-thiogalabiose [48], a - ~ - 3 thioFuc( 143)GlcNHAc [ 871, a,a-thiotrehalose [88] and thiogangliosides [ 891 have all resulted in a consistent finding-the thio linkage provides a high degree of flex-
559
21.9 Thiooligosaccharides and Proteins
A AcHN cAcO"' o w
C
0
2
M
x
+
e
+ Me@
AcO
HS
O
kBzOo
m
AcO
y "J
209
208
OMPM OAc
210
v-xi
S(CH2)6CH3 AcHN
AcO
AcO
OAC
ACO
OAc AcO
205
206
207
Scheme 41. (i) NaH, Kryptofixm21(THF); (ii) TFA (H,O); AczO (pyridine); (iii) TBAF (THF); AczO (pyridine); (iv) HBr (AcOH); (v) TMSOTf (CHzC12); (vi) CAN (CH3CN/H20) then Ac20 (pyridine); (vii) EtSH, p-TsOH, then BzCN, NEtl; (viii) Tf20 (pyridine), KSAc (THF); (ix) TBAF (THF), then CC13CN, DBU; (x) HSC7H15, BF3.0Et2; (xi) NzH4.HOAc (DMF); (xii) NaH (DMF); (xiii) NaOMe (MeOH) then 0.2 M KOH.
ibility between sugar units and the thiooligosaccharides present a conformational equilibrium in solution between at least two major populations, whereas in the 0series only one rotamer is heavily populated. It is, however, possible to lock these substrate analogs by attachment of alkyl groups near the thio linkage into the rigid conformation required [90].
21 Synthesis of Oligosaccharide Mimics: S-Analogs
560
i-iii
160
211
SH
AcO
OAc
OAc
213
\
i,iii
O .H% &: ;& H O H HO OH
212
ko%OA~ AcO
HO HOo&
OAC
20 Scheme 42. (i) NaH (THF), then (DMF); (ii) 90% TFA (H20), then Ac20 (pyridine); (iii) NaOMe (MeOH).
21.9.2 Enzyme-Substrate Interactions The elucidation of structure-function relationships to facilitate the design and production of enzyme variants with altered properties has mainly been achieved by solution and X-ray studies of protein-substrate interactions. This section deals only with compounds previously described. a-Glucan-Active Enzymes
a-Amylases catalyze the hydrolysis of a-(1,4) glycosidic linkages of starch components. These enzymes are members of the glycosyl hydrolase family 13 [91], which also contains cyclodextrin gluconotransferases (CGTases) and pullulanases. The structures of pig pancreatic a-amylase and inactive CGTase in complexes with substrate analogs 138, Scheme 29, and 50, Scheme 15, respectively, have been recently solved [2, 921. 4-Thiomaltosyl fluoride (147, Scheme 30) was recognized as a substrate by CGTases and led to hemithiocyclodextrins in good yield [8].
H
21.9 Thiooligosaccharides and Proteins
561
Most fungal glucoamylases have a raw starch-binding domain (SBD) which is distinct from the catalytic domain. To obtain a better understanding of the role of this domain during the enzymatic process the inhibitors 43, 46, and 47 (Scheme 13) were used [40, 411. The thermodynamics of binding of compounds 78-81 (Scheme 16) to the two domains of glucoamylase GI from A . niger shows that the catalytic and starchbinding sites are in close proximity in space 1931. The hydrodynamic dimensions of GI alone and in interactions with these heterobidentate ligands were determined by quasi elastic light-scattering experiments [46]. From these results it seems that the presence of the bound bifunctional ligands stabilizes a more compact conformation of the enzyme. The motion between the two domains might achieve the processivity of this exo-enzyme which releases P-D-glucose units from the non-reducing ends of starch. Several ganglioside analogs containing an a-thiosialic acid residue, derived from compound 32 (Scheme 10) have been found to be potent inhibitors of sialidase activities of different subtypes of the influenza virus [96]. Disaccharide structures containing the a-thio-L-fucosyl residue-36 (Scheme 1 l), 119 (Scheme 25), 173 (Scheme 34), and 202 (Schcmc 39) -arc all inhibitors of a - ~ fucosidases from bovine kidney and epididymis with K, values of 0.65-5 mM [7]. Several 1,2-cis-thiodisaccharideswere evaluated for their capacity to inhibit HIVinduced cell killing and virus production in LEM or MT-2 cells. All the compounds were inactive except for the thiokojibiose octaacetate 181 (Scheme 36), but none was evaluated as an inhibitor of glucosidases I or I1 [ 3 7 ] . P-Glucan-Active Enzymes
Cellulases (cellobiohydrolases and endoglucanases) are glycosyl hydrolases able to cleave the p-1,4-linkages of cellulose. The catalytic domains of cellulases and related xylanases have been identified in 14 of the 63 families of homologous folds of all glycosyl hydrolases [91]. The structure of the endocellulase EGI from Fusarium oxysporum (family 7), complexed to the non-hydrolyzable thiocellopentaoside 129 (Scheme 26) has been determined by X-ray crystallography at 2.7 A resolution [94]. This study shows for the first time the same oligosaccharide molecule spanning the point of enzymatic cleavage and the distortion of the sugar residue in the - 1 subsite. The crystal structure of a processive endo-cellulase CelF of Clostridium cellulolyticum in complex with two molecules of 4"-thiocellotetraose 131 (Scheme 27) at 2.0A resolution has very recently been solved [95]. One inhibitor molecule was enclosed in a tunnel and the other was bound in an open architecture. They constitute the two parts of the active site. The P-thiocellobiosyl fluoride 144 (Scheme 30) was recognized as substrate by cellulases and afforded hemithiocellodextrins in good yield [64]. The usefulness of 4-thiochitobiose 114 (Scheme 24), 4,4"-dithiochitotriose 128 (Scheme 26), and their glycosides has been demonstrated in the characterization of the transport processes, the screening, isolation and kinetic study of the chitinolytic system of Vibriofurnissii [ 971. The free compound derived from the trisaccharide 49 (Scheme 14) was kinetically
562
21 Synthesis of Oligosacchavide Mimics: S-Analogs
evaluated as a substrate for N-acetylglucosaminyltransferase-V and found to be an acceptor with threefold increase in V,,, but higher K, value (330 PM) than the natural acceptor [34].
21.9.3 Lectin-Ligand Interactions
The in vitro flocculation of the yeast Kluyveromyces bulgavicus was inhibited by the branched thiocyclodextrin 64 (Scheme 15) with the same efficiency as the best known ligand [45]. On the other hand, the 4-thiogalabioside 90 (Scheme 18) has been shown to be ten times less recognized than its oxygen analog by a bacterial pilus adhesin [48].
21.10 Conclusion We have presented an exhaustive and up to date review on the synthesis of thiooligosaccharides which can be used to probe interactions with proteins. The ease of synthesis of this class of molecule should furnish new tools for glycobiochemists and glycobiologists.
Acknowledgments
Our own work described in this review has been supported by CNRS and several EU biotechnology programs (BI02 CT94 3008, 3018, B104 CT97 2303 and B104 CT98 0022).
References 1. Q. Wang, R. W. Graham, D. Trimbur, R. A. J. Warren, S. G. Withers, J. Am. Chem. Soc. 1994, 116, 11594-11595. 2. A. K. Schmidt, S. Cottaz, H. Driguez, G. E. Schultz, Biochemistry, 1998,37, 5909-5915. 3. J. M. Beau, T. Gallagher in Top. Curr. Cliem. 1997, 187, 1-54. 4. A. de Raadt, C. W. Ekhart, M. Ebner, A. E. Stiitz in Top. Curr. Chem. 1997, 187, 157-186. 5. H. Driguez in Top. Curr. Chem. 1997, 187, 86-116.
6. Z. J. Witczak, R. Chhabra, H. Chen, X. Q. Xie, Curbohydr. Res. 1997, 301, 167-175. 7. H. Hashimoto, K. Shimada, S. Horito, Tetrahedron: Asymm. 1994, 5, 2351-2366. 8. L. Bornaghi, J. P. Utille, E. D. Rekai', J. M. Mallet, P. Sinay, H. Driguez, Curbohydr. Res. 1997, 305, 561- 568, and references cited therein. 9. D. Horton, D. H. Huston, Adu. Curbohydr. Chem. 1963, 18, 123-199, and references cited therein. 10. D. Horton, Methods in Carhohydr. Chem. 1963, 2, 433-437.
References
563
11. W. K. C . Park, S. J. Meunier, D. Zanini, R. Roy, Curbohydr. Lett. 1995, I , 179-184. 12. J. Defaye, H. Driguez, E. Ohleyer, C. Orgeret, C. Viet, Curbohydr. Res. 1984, 130, 317-321. 13. M. Sakata, M. Haga, S. Tejima, M. Akagi, Chern. Pharm. Bull. 1964, 12, 652-656. 14. M. Blanc-Muesser, J. Defaye, H. Driguez, Carbohydr. Res. 1978, 67, 305-328. 15. M. Blanc-Muesser, J. Defaye, H. Driguez, J. Chem. Soc. Perkin I , 1982, 15-18. 16. A. Gadelle, J. Defaye, C . Pedersen, Carbohydr. Res. 1990, 200, 497-498. 17. M. Akagi, S. Tejima, M. Haga, Chem. Phurrn. Bull. 1962,10, 562-566. 18. R. L. Whistler, W. C . Lake, Methods in Cavbohydr. Chem. 1972, 6, 286-291. 19. S. D. Gero, R. D. Guthrie, J. Chem. Soc. ( C ) 1967, 1761-1762. 20. T. Eisele, A. Toepfer, G. Kretzschmar, R. R. Schmidt, Tetrahedron Lett. 1996, 37, 1389-1392. 21. J. Kocourek, Curbohydr. Res. 1967, 3, 502-505. 22. R. J. Ferrier, R. H. Furneaux, Curbohydr. Res. 1977, 57, 73-83. 23. J. Defaye, H. Driguez, S. Poncet, R. Chambert, M. F. Petit-Glatron, Carbohydr. Res. 1984, 130, 299-3 15. 24. A. Hasegawa, J. Nakamura, M. Kiso, J . Curbohydr. Chem. 1986, 5, 11-19. 25. S. Cottaz, P. Rollin, H. Driguez, Curbohydr. Res. 1997,298, 127-130. 26. S. Bennett, M. von Itzstein, M. J. Kiefel, Curbohydr. Res. 1994, 259, 293-299. 27. J. Defaye, J. M. Guillot, Carbohydr. Res. 1994, 253, 185-194. 28. M. Blanc-Muesser, H. Driguez, J. Chem. SOC.,Perkin Trans. I , 1988, 334553351, 29. C. Simiand, E. Samain, 0. R. Martin, H. Driguez, Curbohydr. Res. 1995, 267, 1-15. 30. D. H. Hutson, J. Chem. Soc. ( C ) 1967,442-444.
31. J. A. Calvo-Asin, F. G. Calvo-Flores, J. M. Exposito-Lopez, F. Hernandez-Mateo, J. J. Garcia- Lopez, J. Isac-Garcia, F. Santoyo-Gonzalez, A. Vargas-Berenguel, J. Chem. Soc. Perkin Trans. I , 1997, 1079-1081. 32. H. Driguez, J. C. McAuliffe, R. V. Stick, D. M. G. Tilbrook, S. J. Williams, Aust. J. Chem. 1996,4Y, 343-348. 33. I. Lundt, B. Skelbaek-Pederson, Actu. Chem. Scand. Sect. B, 1981,35, 637-642. 34. P. P. Lu, 0. Hindsgaul, H. Li, M. Palcic, Can. J. Chem. 1997, 75, 790-800. 35. M. 0. Countour-Galcera, J. M. Guilliot, C . Ortiz-Mellet, F. Plieger-Carrara, J. Defaye, J. Gelas, Carbohydr. Res. 1996,281, 99-1 18. 36. W. Boos, P. Schaedel, K. Wallenfels, Eur. J. Biochem. 1967, I , 382-394. 37. R. N. Comber, J. D. Friedrich, D. A. Dunshee, S. L. Petty, J. A. Secrist 111, Curbohydr. Res. 1994,262, 245-255. 38. A. Hagesawa, T. Terada, H. Ogawa, M. Kiso, J. Curbohydr. Chem. 1992, 11, 319-331. 39. S. Cottaz, H. Driguez, Synthesis, 1989, 10, 755-758. 40. S. Cottaz, H. Driguez, B. Svensson, Carbohydr. Res. 1992, 228, 299-305. 41. C. Apparu, H. Driguez, G. Williamson, B. Svensson, Carbohydr. Res. 1995,227, 31 3-320. 42. C. Lancelon-Pin, H. Driguez, Tetrahedron Lett. 1992,33, 3125-3128. 43. J. Defaye, A. Gadelle, A. Guiller, R. Darcy, T. O’Sullivan, Curbohydr. Res. 1989, 192, 251258. 44. B. Brady, R. Darcy, J. Carhohydr. Res. 1998, 309, 237-241. 45. L. de Robertis, C. Lancelon-Pin, H. Driguez, F. Attioui, R. Bonaly, A. Marsura, Biorg. Med. Chem. Lett. 1994, 4 , 1127-1 130. 46. N. Payre, S. Cottaz, C . Boisset, R. Borsali, B. Svensson, B. Henrissat, H. Driguez, Angew. Chern. Int. Ed. 1999, 38, 974-977. 47. L. A. Reed, L. Goodman, Curbohydr. Rex 1981, 94, 91-99. 48. U. Nilsson, R. Johansson, G. Magnusson, Chem. Eur. J. 1996, 2, 295-302. 49. L. X. Wang, N. Sakairi, H. Kuzuhara, J. Chem. Soc. Perkins Trans. 1, 1990, 1677-1682. SO. D. Rho, M. Desrochers, L. Jurasek, H. Driguez, J. Defaye, J. Bacteriol. 1982, 149, 47-53. 51. C . Orgeret, E. Seillier, C. Gautier, J. Defaye, H. Driguez, Curbohydr. Res. 1992, 224, 29-40. 52. B. Albrecht, U. Piitz, G. Schwartzmann, Curbohydr. Res. 1995, 276, 289-308. 53. J. Defaye, H. Driguez, M. John, J. Schmidt, E. Ohleyer, Curbohydr. Res. 1985, 139, 123-132. 54. M. McDonough, R. V. Stick, D. M. G. Tilbrook, Aust. J. Chem. 1999,52, 143-147. 55. L. X. Wang, Y. C. Lee, J. Chem. Soc. Perkin Trans. 1, 1996, 581-591. 56. C. Schou, G . Rasmussen, M. Schulein B. Henrissat, H. Driguez, J. Curhohydr. Chem. 1993, 12, 743-752.
564
21 Synthesis of Oligosaccharide Mimics: S-Analogs
57. V. Moreau, J. L. Viladot, E. Samain, A. Planas, H. Driguez, Biorg. Med. Chem. 1996,4, 18491855. 58. C. Reverbel-Leroy, G. Parsiegla, V. Moreau, M. Juy, C. Tardif, H. Driguez, J.-P. Belaich, R. Haser, Actu Crystallogr. Sect. D,1998, 54, 114-118. 59. M. Blanc-Muesser, J. Defaye, H. Driguez, G. Marchis-Mouren, C. Seigner, J. Chem. Soc. Perkin Trans. 1, 1984, 1885-1889. 60. M. Blanc-Muesser, H. Driguez, B. Joseph, M. C. Viaud, P. Rollin, Tetrahedron Lett. 1990, 31, 3867-3868. 61. M. Blanc-Muesser, L. Vigne, H. Driguez, Tetrahedron Lett. 1990, 31, 3869-3870. 62. M. Blanc-Muesser, L. Vigne, H. Driguez, J. Lehmann, J. Steck, K. Urbahns, Carbohydr. Res. 1992,224, 59-71. 63. M. Blanc-Muesser, H. Driguez, unpublished results. 64. V. Moreau, H. Driguez, J. Chem. Soc. Perkin Trans. I , 1996, 525-527. 66. E. J. Hehre, K. Mizokami, S. Kitahata, Denpun Kugaku. 1983, 30, 76-82. 66. S. Cottaz, C. Apparu, H. Driguez, J. Chem. Soc. Perkin Trans. 1, 1991, 2235-2241. 67. C. Apparu, S. Cottaz, G. Bosso, H. Driguez, Carbohydr. Lett. 1995, 1, 349-352. 68. B. Becker, J. Thimm, J. Thiem, J. Carbohydr. Chem. 1996,15, 1179-1181. 69. Z. J. Witczak, J. Sun, R. Mielguj, Biorg. Med. Chem. Lett., 1995, 5, 2169-2174. 70. V. Moreau, J. Ch. Norrild, H. Driguez, Carbohydr. Res. 1997, 300, 271-277. 71. G. Hummel, 0. Hindsgaul, Angew. Chem. Int. Ed. 1999,38, 1782-1784. 72. J. Defaye, J. M. Guillot, P. Biely, M. Vrianka, Curbohydr. Res. 1992, 228, 47-64. 73. B. Aguilera, A. Fernadez-Mayoralas, Chem. Commun. 1996, 127-128. 74. B. Aguilera, A. Fernadez-Mayoralas, C. Jaramille, Tetrahedron, 1997, 53, 5863-5876. 75. J. S. Andrews, B. M. Pinto, Carbohydr. Res. 1995, 270, 51-62. 76. J. S. Andrews, B. D. Johnston, B. M. Pinto, Curbohydr. Res. 1998, 310, 27-33. 77. B. D. Johnston, B. M. Pinto, Carbohydr. Rex 1998, 310, 17-25. 78. J. Defaye, A. Gadelle, C. Pedersen, Curbohydr. Res. 1991,217, 51-58. 79. T. Eisele, R. Windmiiller, R. R. Schmidt, Curbohydr. Res. 1998,306, 81-91. 80. T. Eisele, R. R. Schmidt, Liebigs AnnlRecueil. 1997, 865-872. 81. M. 0. Contour-Galcera, Y. Ding, C. Ortiz-Mellet, J. Defaye, Carbohydr. Res. 1996, 281, 119128. 82. G. R. Fenwick, R. K. Heaney, W. J. Mullin. CRC Crit. Rev. Food. Sci. Nutr. 1983, 18, 123201. 83. W. P. Burmeister, S. Cottaz, H. Driguez, R. Iori, S. Palmieri, B. Henrissat, Structure, 1997, 5, 663-675. 84. S. Perez, C. Vegelati, Actu Crystullogr. Sect. B, 1984, 40, 294-299. 85. K. Mazeau, I. Tvaroskd, Curbohydr. Res. 1992,225, 27-41. 86. K. Bock, J. 0. Duus, J. Refn, Curbohydr. Res. 1994,253, 51-67. 87. B. Aguilera, J. Jimenez-Barbero, A. Fernandez-Mayoralas, Carbohydr. Res. 1998, 308, 19-27. 88. K. Bock, J. Defaye, H. Driguez, E. Bar-Guilloux, Eur. J. Biochem. 1983, 131, 595-600. 89. A. Geyer, G. Hummel, T. Eisele, S. Reinhardt, R. R. Schmidt, Chem. Eur. J. 1996,2, 981-988. 90. S. Sabesan (du Pont de Nemours, E. I. ), US 5489675A6, 1996 (Chem. Ahstr., 1997, 124, 343979). 91. B. Henrissat, Biochem. J. 1991,280, 309-316. 92. M. Quian, S. Spinelli, H. Driguez, F. Payan, Protein Sci. 1997, 6, 2285-2296. 93. B. W. Sigurskjold, T. Christensen, N. Payre, S. Cottaz, H. Driguez, B. Svensson, Biochemistry, 1998,37, 10446-10452. 94. G. Sulzenbacher, H. Driguez, B. Henrissat, M. Schiilein, G. J. Davies, Biochemistry, 1996, 35, 15280-15287. 95. G. Parsiegla, M. Juy, C. Reverbel-Leroy, C. Tardif, J. P. Belakh, H. Driguez, R. Haser, EMBO J., 1998, 17, 5551-5562. 96. Y. Suzuki, K. Sato, M. Kiso, A. Hasegawa, Glycoconj. J . 1990, 7, 349-356. 97. L-X. Wang, N. 0. Keyhani, S. Roseman, Y. C. Lee, Glycobiology, 1997, 7, 855-860, and references cited therein.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
22 Saccharide-Peptide Hybrids Hans Peter Wessel
22.1 Introduction The term ‘saccharide-peptide hybrids’ (SPHs) encompasses sugar molecules linked by an amide group, e.y. a disaccharide with the interglycosidic oxygen formally replaced by an amide group (Scheme 1).
Scheme 1.
For oligomers, the monomer units are thus carbohydrate amino acids (CAAs). SPHs are interesting carbohydrate mimetics-although the linking unit is modified, typical carbohydrate epitopes are maintained. For the oligomerization process the synthetic advantage is obvious-the amide linkage is readily formed and, in contrast with glycosylations, avoids stereochemical complications because no anomers are formed. Another advantage is that the amide coupling reactions can be conducted without protection of the hydroxyl groups because the amino group is more nucleophilic than the hydroxyl groups. While solid-phase glycosylations are still in their infancy, amide bond formation by solid-phase chemistry has been well established for a considerable time. SPHs are therefore amenable to the combinatorial approaches which have become an indispensable tool in medicinal chemistry. With advances in glycobiology and glycochemistry interest in SPHs is increasing. The amide-linkage might render these carbohydrate mimetics more stable than oligosaccharides under physiological conditions, particularly towards glycosidases. The nomenclature of SPHs has not been used consistently. We prefer the term ‘saccharide-peptide hybrids’ [ 11 for reasons of clarity. As an abbreviated form, the
566
22 Saccharide-Peptide Hybrids
term ‘peptidosaccharides’ [2] has also been coined. The term ‘carbopeptoid’ [3] for an SPH is questionable because a ‘peptoid’ [4] is clearly defined as a non-chiral peptide mimetic with an N-substituted glycin as amino acid replacement. In this connection the term ‘glycopeptoid’ [5] for a peptoid carrying a carbohydrate residue has also been introduced. In the expression ‘glycotides’ [6] the suffix is not clearly connected with peptides. Alternatively, and in keeping with the hybrid character, SPHs can be viewed as peptide mimetics. One feature of oligomeric SPHs is their conformational rigidity because of the amide bond. CAAs have therefore been employed as turn mimetics in oligopeptides [7]. This type of molecule would fall into the class of ‘carbohydrate amino acid conjugates’, for which the expression ‘saccharopeptides’ has also recently been used [8].
22.2 Carbohydrate Amino Acids 22.2.1 Natural Carbohydrate Amino Acids Some carbohydrate amino acids occur in Nature as important construction elements. One example is muramic acid, R-2-amino-3-0-( 1-carboxyethyl)-2-deoxy-~-glucose (l),occurring in the peptidoglycan portion of Gram-negative bacterial cell walls in the form of the muramyl dipeptide, N-acetylmuramyl-L-alanyl-D-isoglutamine (Scheme 2) [9]. Glycosaminuronic acids, such as 2-acetamido-2-deoxyglucuronic acid, have also been found in bacterial cell walls [lo]; its N-acetylalanine analog [ 111 2-acetamido-2-deoxygalacturonicacid was found to be the main constituent of the Vi-antigen of E. coli and other bacteria [12]. Derivatives of glucosaminuronic acid were also detected in the cancomycin family of antibiotics [13]. Sialic acids are important constituents of glycoconjugates, often located peripherally on glycoproteins. This family of natural CAAs consists of N- and 0-acyl derivatives of neuraminic acid 2; the main substituents on nitrogen are the N-acetyl and N-glycolyl groups [ 141. Interesting natural CAAs were found in nucleoside antibiotics (for a review see Ref. [ 151). A furanoid amino uronic acid, 5-amino-5-deoxy-~-~-allofuranuronic acid, occurs in the polyoxins [16], e.g. polyoxin J (3) and in the nikkomycins [ 171. The polyoxins also contain a C-5 open-chain 2-amino-2-deoxy sugar acid, a derivative of 2-amino-2-deoxy-~-xylonic acid. A branched chain 6-amino-6deoxyheptopyranuronic acid is the central moiety of amipurimycin (4) and in the related miharamycins [ 18-20]. Two different 3-amino-3-deoxy uronic acids, acid and 3-amino-3,4-dideoxyderivatives of 3-amino-3-deoxy-~-gulopyranuronic D-xylohexopyranuronic acid, were found in ezomycin A1 (5) [211. 4-Amino-4deoxy-glucopyranuronic acid is the carbohydrate moiety in the aspiculamycin/ gougerotin nucleosides [22, 231.
22.2 Carbohydrate Amino Acids
567
HO W H2NC
O
O
H
HO
Me
1
2
3 Polyoxin J
Holln-.
4 Amipurimycin COOH
H2N
5 Ezomycin A,
I NHZ
Scheme 2.
22.2.2 Synthetic Carbohydrate Amino Acids In this chapter some synthetic approaches to CAAs will be described. The most straightforward route to CAAs is oxidation of an amino sugar, with the obvious limitation that few amino sugars, e.9. glucosamine, are readily available. Thus, Heyns and Paulsen [24], back in 1955, described the synthesis of glucosaminuronic acid by catalytic oxidation of the primary hydroxyl group, the method was later improved by Weidmann and Zimmermann [25]. Alternatively, hypochlorite oxidation in the presence of TEMPO can be employed (Scheme 3) to obtain CAA 7 in three steps (40%) [26]. Nucleoside analogs based on glucosaminuronic acid were also prepared in this fashion [27]. Other approaches have been to attach acid residues as substituents. Examples are a CAA 10, derived from activated chitin in four steps (26%) [28], or the demethyl
568
22 Saccharide-Peptide Hybrids
z
- E%
?
HO
OH
11
HO
NH,.HCI
HyOBn
HyOBn
Z
GlcN
Chit1n-H...
HOOC Ho-i
2
i
7
6
0-CHz-COOH O & o $ A
H
0-CH2-COOH k H
0
2 ACO%
[HO
6-
8
NH,.HCI
n
HzN Br 10
9
.HBr
vii
Vi
BuO'OC
HOOC
Z
11 GlcN
Z
12
- L&&cN ...
ix, x, xi
Vlll
AcO
H~&&cooH HO
NPhth
13 GlcNAc-
0-CH,-COOH
AcO
HO
NHAc
Z
xii, xiii
yo.0*
NH2.HCI
-
14
xiv, xv
OH NHBoc
Me0
15
OMe OMe
l6
Scheme 3. (i) ZCI, NaOH; BnOH, Tf20, 0°C to room temp., 4 days, 60%; (ii) NaOC1, TEMPO, KBr, Bu4NC1, HzO, CH2C12, O T , 1.5 h, 67%; (iii) Activation to alkali chitin; ClCHzCOOH, (CH3)2CHOH,room temp.; (iv) lysozyme, 45 "C, 3 days; 2 M HCl, reflux, 5 h, 70%; (v) AcBr, 55 "C, 90 min, 37%; (vi) PhCHO, ZnC12, room temp., 85%; BrCH*COO'Bu, KOH, dioxane, 55 "C, 2 h, 91%; (vii) KOCMe3, THF, H20, O"C, 2 min, 95%; (viii) four steps from GlcN [29]; (ix) 30% HBrAcOH, 0 ° C to room temp., 3 h, 85%; (x) Dowex 50W-X8 [HC], MeOH, 80"C, 16 h, 97%; (xi) LiOH, MeOH/H20 3: 1, 60"C, 16 h; 3 M HCI, reflux., 3 h, 95%; (xii) KOH, MeI, 18-crown-6, DMF, room temp., 16 h, 65%; (xiii) HCl, then di-'Bu-dicarbonate, NEt3, CHC13, room temp., 24 h, 90%; (xiv) PCC, 3 L% molecular sieve, CH2C12, room temp., 5 h, 85%; (xv) KOH, MeI, 18-crown-6, THF, room temp., 3 h; then 4 M HC1, room temp., 16 h, 49%.
22.2 Carbohydrate Amino Acids
569
analog 12 of muramic acid (five steps from glucosamine; 44%) [ 11. By carboxylation of the anomeric center of glucosamine via formation of an anomeric nitrile [29], the heptonic acid derivative 14 was synthesized in seven steps (36%). A longer way to an analogous heptonic acid derivative was chlorination followed by tin activation, lithiation, and carboxylation with carbon dioxide at the anomeric center of a protected glucosamine derivative. Direct PCC oxidation of the anomeric center of a protected glucosamine furnished the open-chain carbohydrate-derived a-amino acid derivative 16 (five steps from N-acetylglucosamine, 24%). The second obvious preparation of CAAs is the amination of a carbohydrate acid. Glucuronic acid was used to introduce an amino function at the anomeric center via an azide delivering a CAA building block on the solid phase in only four steps and excellent yield, giving the &-aminoacid 18 (Scheme 4) [30]. Kessler and colleagues [7] described a similar sequence from glucuronolactone. Also starting with glucuronolactone, the P-glycoside of glucosaminuronic acid was synthesized via glucal formation followed by azidonitration (12 steps, 6% yield) [31]. This approach is less efficient than that starting with glucosamine as described above, in comparison, a simple p-glycosidic analog was prepared in four steps and 49% yield [7].A furanoid CAA was prepared from D-galactonolactone (Scheme 4)-an azide was introduced at the primary position and reaction with triflic anhydride in pyridine, then with methanol, unexpectedly gave two products epimeric at C-5. The diastereomeric mixture was only separable after isopropylidenation to 22 and 23. The free carboxylates, e.g. 24 (18%), or amines were prepared to yield building blocks ready for amide coupling [32]. Although the number of carbohydrate acid derivatives available in reasonable quantity and price to be used as starting materials is limited, it should be pointed out that occasionally conversions to other configurations are straightforward. Thus, the ~-idufuranurono-3,6-lactone 25 is readily available after isopropylidene protection and inversion at C-5 [33]. This lactone was employed to synthesize the piperidine-type CAA 27 with a secondary amine as outlined in Scheme 4 [34]. Often CAAs have been synthesized from sugars, and both amino and carboxyl functionalities have been introduced by using a combination of methodologies described above, or others. Usually the amino function was introduced via an azide. Nitriles are particularly versatile functional groups which can be reduced to amines or hydrolyzed to carboxylic acids [35, 361. An obvious site for the modification of sugars is at the reactive anomeric center. As an alternative to anomeric nitrile chemistry, an anomeric aminomethyl group was introduced via nitromethane condensation (Scheme 5 ) to yield CAA 30 (six steps from glucose, 12%) [37]. Another method used for the introduction of a carboxylic acid was C-allylation followed by oxidative cleavage of the double bond, e.y. CAA 33 was synthesized from peracetylated ribofuranose (six steps, 9Yn) [ 61. Gurjar et al. converted a C-ally1 glycoside to a CAA by hydroxylation of the double bond followed by Jones oxidation [38]. An acid function was also introduced by reacting the free anomeric center with methyl(tripheny1phosphoranylidene) acetate in a Wittig reaction and followed by conversion to a C-glycoside by intramolecular Michael addition, CJ Scheme 5 for the preparation of 36 from 2,3 : 5,6-di-O-isopropylidene-~-mannofuranose (34) [6]. A disadvantage of this
570
22 Saccharide-Peptide Hybrids
HOOC
i, ii
HO OH
OH
HOOC N *oA c AcO
iii, iv
3
OAc
GlcA
OAc
17
19
20
18
21
1+
vii
N3\ + r p r o p
O
25
26
x
23
o
27
Scheme 4. (i) AQO, 12, 0°C to room temp., 2 h; (ii) TMSN3, SnC14, CH2C12, room temp., 16 h, 73%; (iii) 2-chlorotrityl-linked resin, DIPEA, CH2C12, room temp., 1 h, then MeOH; (iv) HS(CHl)zSH, NEt3, room temp., 10 h; (v) 61%; (vi) TfiO, Py, AcOEt, then MeOH, 46%; (vii) MezCHOH, HCI, then Me2CO; (viii) H2, Pd/C, 22 (65%), 23 (23%); (ix) TfiO, Py, CH2C12, -2O"C, then NaN3, DMF, -10 to -2O"C, 1 h; (x) H2, 10% Pd/C, AcOEt, then ZC1, NaHC03, AcOEt/HzO; (xi) CF3COOH-H20, room temp., then HI, Pd black, H20-AcOH 9 : 1 , 4 days, 44% from 25.
approach is the formation of a mixture of both anomeric C-glycosides; these were separated only after azide formation. Similarly, a Wittig reaction was applied to a 3-ketofuranose to introduce a carboxylate into a branched sugar [6]. Dondoni et al. [39] used their anomeric thiazole approach to furnish a-amino acids with the anomeric center as the a-carbon atom, see Scheme 5 for the synthesis of CAA 39. The coupling of a protected serine aldehyde, in which the amino acid functionalities are preformed, to a substituted furan was the key step of the preparation of
571
22.2 Carbohydrate Amino Acids
DGlc
-
-
iii, iv, v
HO HO OH
NO2
MeOOC
HOOC
L*OH HO OH
NHZ
H O HO
29
28
31
33
-
35
'COOMe
36
COOMe XVII-xx
37
NH2
30
32
34
OH
38
xxi
39
Scheme 5. (i) CH3N02, NaOMe; (ii) H+, H20; (iii) H2, Pd/C, ZC1; (iv) 0 2 , Pt/C; (v) MeOH, DCC-DMAP; (vi) NaOH, H2, Pd/C, 120/0; (vii) TMSAlI, TMSOTf, MeCN, 0°C to room temp., 6 h, 67%; (viii) LiOMe, MeOH, room temp., 3 h, 98%; (ix) TsC1, Py, room temp., 2 days, 53%; (x) NaN3, Bu4NI, DMF, llO"C, 4 days; (xi) AczO, NEt3, CH2C12, room temp., 1 day, 290/0; (xii) NaI04, RuC13, MeCN, CCld, H20, room temp., 15 min, 93%. (xiii) Ph3P=CHCOOMe, MeCN, reflux, 14 h, 90%; (xiv) AcOH, HzO, room temp., 16 h, 85%; (xv) MsC1, Py, DMAP, 0°C to room temp., 22 h, 76%; (xvi) NaN3, DMF, 70"C, 8 days, 84%, no yield given for the separation of anomers; (xvii) TMSN3, TMSOTf, CH2C12,4 A molecular sieve, room temp. 20 min, 84%; (xviii) MeOTf, MeCN, 4 A molecular sieve, room temp. 15 min, then NaBH4, room temp., 10 min; (xix) HgC12, MeCN/H20 10: 1, room temp.. 15 min; (xx) AgN03, aq. NaOH, THF, room temp., 36 h, then CH2N2, MeOH/Et20, O"C, 20 min, 53%; (xxi) H2, Pd/C, 'BuOH/H20 9: 1, room temp., 1 h, quant.
572
40
22 Saccharide-Peptide Hybrids
41
42
Scheme 6. (i) Baker’s yeast, 78%; (ii) LiOH, THF, 92%.
the 6-amino-6-deoxyheptopyranuronic acid of amipurimycin (4) [ 191. An interesting approach to a CAA from a non-carbohydrate starting material is by asymmetric synthesis using baker’s yeast. Keto-piperidine carboxylate 40, which is available by cyclization of an a-diazo P-keto ester precursor [40], was reduced to 41 [41] and, after ester cleavage, employed as carbohydrate mimetic 42 (Scheme 6) [42]. A dihydroxylated piperidine carboxylic acid mimicking galactose was prepared recently by syn-hydrogenation of an unsaturated precursor [43].
22.3 Amide-Linked Carbohydrate Polymers The condensation of carbohydrate amino acids was first performed in a polymerization mode, furnishing mixtures of compounds with different degrees of polymerization. Various techniques have been employed. Condensation of the ester 43 in methanolic sodium methoxide furnished a water-soluble polymer 44 which was not further characterized (Scheme 7) [44]. Polymerization of CAA 10 after activation of the acid with diphenylphosphoryl azide (DPPA) resulted in a chloroformsoluble polymer 45. Deacetylation and bromide hydrolysis with sodium methoxide in methanol gave a water-soluble polymer 46 with a free anomeric center readily reducing Fehling solution [28]. The estimated molecular weight was 15 kD as judged by SDS polyacrylamide electrophoresis and also by gel filtration on Sephadex8 G50. Similarly, an amphiphilic alkyl glycoside of glucosaminuronic acid was polymerized with DPPA to result in a degree of polymerization of only 2-13 as judged by MALDI-TOF mass spectrometry [31]. In another approach, the CAA 16 was activated with trichloromethyl orthochloroformate to give the isolable 1,3oxazolidine 47. This intermediate, on treatment with triethylamine in DMF, furnished a polymer 48 with an average molecular weight of 10 kD as judged by gel permeation chromatography [45]. At the structural limit of a CAA, the bicyclic oxalactam 8-oxa-6-azabicyclo[3.2.l]octan-7-one was polymerized in the presence of basic catalysts to give amino-‘uronic acid’ polymers devoid of hydroxyl groups [46]. Whereas the above examples start from CAAs, the construction of amide-linked polymers was also possible in a Nylon type condensation using sugar diamines derived from anhydroalditols [47] or hexoses [48] and dicarboxylic acids as building blocks. For example, methyl 2,6-diamino-2,6-dideoxy-a-~-glucopyranoside (49) was
22.3 Amide-Linked Curhohydrufe Polyrnevs
-
573
i
H2N& HO
COOMe OH
Me n
OH
43
44
..
45
Me0
46
iv
p::
OMe
6Me OMe 0
OMe
16
hH;j 47
vi T
Ho% HO CH,
49
48
HO
n
Scheme 7. (i) NaOMe, MeOH, 10O"C, 4 h, autoclave; (ii) PhZP(O)N3, NEt3, DMSO, 20°C; 4 days, 44%; (iii) NaOMe, MeOH, room temp., 30 min; (iv) C13CO-COC1, THF, 5 5 " C , 4 h, 95%; (v) NEt,, DMF, room temp., 2 days, precipitate. with EtzO; (vi) ClOC-R-COCl, Na2C03, H2OCC14, room temp., 2h.
coupled to aliphatic or aromatic diacids in an interfacial polycondensation [48]. Molecular weights of polymers 50 were in the range of 20 kD for aromatic spacers. The products were structurally not uniform, because 2-6, 2-2, and 6-6 linked polymers could be formed. This field of carbohydrate-derived polyamides has been expertly reviewed [49].
574
22 Succhuride-Peptide Hybrids
22.4 Amide-Linked Carbohydrate Oligomers 22.4.1 Solution Synthesis
A first synthesis of a saccharide-peptide hybrid in a directed synthesis was performed by Yoshimura et al. in 1976 [50]. The CAA 51 was activated with cyclohexylcarbodiimide and linked to amino sugar 52 to result in dimer 53. This glucuronic acid amide was less soluble in water than the corresponding mannuronic acid derivative. Dimer 53 could be converted, after removal of the anomeric protecting group by hydrogenation, to the amide-linked alditol 54 (Scheme 8) by reduction with sodium borohydride. This publication was not followed up at the time until the interest in carbohydrate mimetics rose [51]. The first SPH higher oligosaccharide mimetic was built up in a [ 2 + 2 ] block synthesis by Wessel et al. in 1995 [ 11. The benzyloxycarbonyl (Z) group was employed as the temporary amine protective group, and carboxylic acids were protected as tert-butyl esters. The CAAs of type 12 were coupled in very good yields (84-87%) using mixed anhydrides, and the dimeric building block 56 was activated with 2-chloro-4,6-dimethoxy-l,3,5-triazine (CDMT). Notably, this methodology enabled the coupling of a building block with four unprotected hydroxyl groups to give tetramer 57. In recent years different peptide coupling techniques have been used to assemble oligomers. Thus, McDevitt and Lansbury [6] coupled their protected CAAs with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) in dichloromethane in a stepwise fashion to result in trimer 59 and a number of dimeric SPHs. The amino function was protected as an azide. For the construction of a small carbohydrate mimetic library the group also prepared ‘spaced sugars’ [ 521 such as 60 on an aromatic template. For a better overview, the various coupling techniques are summarized in Table 1. Sialic acid derivatives have received particular attention. Sabesan [2] prepared an isosteric SPH analog 63 of the a-~-NeuAc(2-6)-P-~-Galp disaccharide, the terminal unit of numerous glycoproteins and glycolipids and the receptor ligand for influenza virus hemagglutinin and substrate for neuraminidase. The neuraminidase activity of 63 and analogs have not yet been reported. Gervay et al. described amino acid conjugates of neuraminic acid [6I], but also an amide-linked dimeric neuraminic acid 64 [55](Scheme 9). In this case the hydroxyl groups of the acid component had to be protected to avoid lactonization. The spaced neuraminic acid oligosaccharide mimetics 65 and 66 were synthesized by use of a glycine spacer. The Ishikawa group [ 621 also reported amide-linked oligomers with alternating CAAs and amino acids. The oligomer 67 was constructed with a BOP coupling approach as described above (Table 1, entry 6). For the synthesis of SPH 68, EDCI in combination with HOBt turned out to be advantageous coupling conditions. It is worth noting that the coupling yields for the higher oligomers did not decrease (Table 1, entry 11). This is a clear advantage of the amide coupling and in contrast with many oligosaccharide syntheses where yields are often getting lower for higher oligomers. The same coupling technique was employed by Fleet and colleagues [57]in the synthesis of a furanoid tetramer 69
22.4 Amide-Linked Carbohydrate Oligomers
-
HOOC Ho-i
+
HO HO
515
HO
H/NoBn Ad
51
52 53 ii, iii
I
Ac
,OAc
55
0
+
0 0
56
57
O
58 R = N , Ms
59 R =
MeOOC COOMe
H
60
Scheme 8. (i) DCC, Py-NEt3, room temp., 5 days, 710/0;(ii) Hz, Pd/C, HlO, HOAc, 80%; (iii) NaBH4, Hz0, room temp., 2 h, 99%; (iv) CDMT, N-methylmorpholine, DMF, 50"C, 18 h, then Ac20, Py, 400/; (v) NaOMe, dioxane-MeOH, room temp., 2.5 h, 81Y0.
516
22 Saccharide-Peptide Hybrids
0
OH H
HO
O
b N NHBoc 0
H
NHBoc
61 H HO O
AcOyOAc
OH
W NH I
COOH
BocHN AcO
64
AcHN
AcO
AcO
,OTr
68
Scheme 9.
22.4 Amide-Linked Carbohydrate Oliyomers
577
N3qE*~e 69
AcO*#
OAc
AcO
OAc
2
AcO
OAc
BOC
BOC
71
72
I
Me0
O(CH2)&OOMe
HO
bH
73
Scheme 10.
(Scheme 10). Even an octamer 70 could be assembled in very good yield in a block synthesis (Table 1, entry 13) [58]. By use of pyrrolidine carbohydrate mimetics and a 6-aminoglucose or glucuronic acid building block it was demonstrated that isomeric disaccharide mimetics can be prepared by formation of amide- and inverse amide-linked analogs such as 71 and 72 1591. SPH 73 and several analogs with different uronic acid moieties were prepaied- to serve as starting materials for higher oligosaccharide mimetics [60, 631.
578
22 Saccharide-Peptide Hybrids
Table 1. Conditions for coupling reactions of CAAs. Entry 1 2 3 4
5
6
Coupling conditions Dicyclohexylcarbodiimide, Py-NEt3, room temp., 5 days ClCOO'Bu, NEt3, THF-MeCN, room temp., 12 h; then AczO, Py CDMT, N-methylmorpholine, DMF, 50"C,18 h; then AczO, Py 2.5 equiv. acid, EDCI, NEt3, room temp., 24 h Acid chloride, NEt3, CH2C12 1.2 equiv. acid, BOP, DIPEA, DMF, room temp., 16 h
7
1.2 equiv. acid, DEPC, NEt3, DMF, 0 ° C to room temp.
8 9
CBMIT, MeCN, room temp., 4 days BOP, DIPEA, DMF, room temp., 48 h; then Ac20, Py BOP, HOBt, DIPEA, 1.2 equiv. amine, CH2C12-DMF, room temp., 48 h, then Ac20, PY BOP, DIPEA, CH2C12, room temp., 1 h EDCI, HOBt, THF, room temp., 10 h
10
11
12 13 14 15
EDCI, HOBt, DIPEA, DMF, room temp.; then Ac20, Py EDCI, HOBt, DIPEA, CH2C12 DCC, HOBt, NEt3, THF, 0 ° C to room temp., overnight HBPyU, NEt3, DMF, room temp., overnight
Block size
Yield ("YO)
Structure (Scheme)
71
53 (8)
? 90 85 81 45 27
61 (9)
Ref.
62 (9) 63 (9) 64 (9)
73 81 84 78
68 (9)
55
69 (10)
75 62
70 (10) 71 (10)
71
73 (10)
Abbreviations: CDMT = 2-chloro-4,6-dimethoxy-l,3,5-triazine; EDCI = l-ethyl-3-(3-dimethylaminopropy1)carbodiimide; BOP = benzotriazol-l-yloxy-tris-(dimethylamino)phosphonium hexafluorophosphate; DIPEA = diisopropylethylamine; DEPC = diethylphosphoryl cyanide; CBMIT = 1,l-carbonylbis(3-methylimidazoliumtriflate; HOBt = 1-hydroxybenzotriazole hydrate; HBPyU = 0-(benzotriazole-1 -yl)-N,N,N,N-bis(tetramethy1ene)uronium hexafluorophosphate.
22.4.2 Solid-Phase Synthesis With a view to application in combinatorial chemistry and to reduce solubility problems, Miiller et al. [26] developed a solid-phase approach to SPHs in 1995. Uronic acid 7 was coupled to benzhydrylamine polystyrene functionalized with
22.4 Amide-Linked Carbohydrate Oligomers
579
an amide linker to result in 74. The N-protection of choice was the fluoren-9ylmethoxycarbonyl (=Fmoc) group. Uronic acids were activated in situ with TATU in the presence of Hunig’s base, these coupling conditions did not require the protection of hydroxyl groups. The couplings were quantitative (Scheme 11), and the crude product after detachment from the resin was pure by NMR. Three coupling cycles and cleavage from the resin furnished the homotetramer 75. A similar strategy was adopted to link glucosaminuronic acids carrying a nucleo-base at the anomeric center [64]. The authors used 0-silyl protected CAA monomers and the TATU-related hexafluorophosphate ‘HATU’ (Scheme 11) as activating agent to generate amide-linked nucleic acid analogs as antisense agents. Oligomers up to 14mer 76 were prepared. The solid-phase-attached and O-protected CAA 18 (Scheme 4) was extended by azide-protected uronic acid 17 to give the dimeric SPH 77 (Scheme 11) which was further elongated with amino acids (see Chapter 28) [30]. With 3.6 equivalents of acid using Knorr activation and 25 equivalents of reducing agent (propane-l,3thiol) for the azide, nearly quantitative yields were reached for the dimer. In contrast, Long et al. pointed out that neither coupling nor azide reduction on the solid phase proceeded to completion when homologating their solid-phase-bound monomer 78 when using a diimide coupling. Even for the trimer 79 low yields were obtained (Scheme 11) [65]. With the piperidine carboxylic acid 42 and two related unhydroxylated amino acids (Boc-4-piperidine carboxylic acid and Boc-R-pipecolic acid) a small library was constructed on a 4-methyl benzhydrylamine resin by use of HATU activation in DMF-dichloromethane 1 : 1. N-Fmoc-protected neuraminic acid coupled to rink resin via an E-aminocaproic acid linker was homologated up to an octamer 80 (Scheme 11), and the low yields experienced during solution synthesis [ 551 were overcome; oligomer yields ranged from 44&55%(1.4 equiv. acid-more acid was added when required, BOP, DIPEA, NMP) [66]. 22.4.3 Biological Activity The main reason for the recent interest in SPHs is the topicality of carbohydrate mimetics, the attempt to mimic the biological function of carbohydrates. The advantage of SPHs would be the access in a synthetically facilitated fashion, and stability towards glycosidases would be expected. The oligomers 57 (Scheme 8) and 75 (Scheme 11) were sulfated to be investigated as heparinoid mimetics (unpublished data). A sulfated derivative of 61 (Scheme 9) fully blocked syncytium formation caused by HIV infection to CD4 cells at 50 J ~ Mconcentration [53], and the persulfated derivative of tetramer 62 (Scheme 9) had micromolar activity in the protection of MT2 cells from HIV infection [54]. The oligomeric conjugate 67 (Scheme 9) was evaluated in highly metastatic cell lines; inhibition of cell adhesion (Chemotaxis) and invasion were measured. In a chemotactic assay in which CD44-expressing lung carcinoma cells adhere to vitronectin, this pentamer with L-aspartate spacer and the analogous trimer with a
580
22 Saccharide-Peptide Hybrids
H2$f+ H
-
HNOBn
i,ii
7 -
74
H i s s ) HN OBn
Ho* HO
Fmoc
HNOBn
75
HO% HO H2N
HO & :H 2 HO
HNwBasi& -
...
111
OBn
HN
18
HO
76 n = 1 2 Base = Thymine or cytosine
HO
iv
Base
OAc
H2N
OH
H
AcO 2
OAc N
AcOe
78
OAcw
;
OAc p NH2
79
Hd
"
*'"
' 6
80
Scheme 11. (i) one cycle: 2 equiv. acid, 0-(7-azabenzotriazol-l-yl)-l, 1,3,3-tetramethyluronium tetrafluoroborate (TATU), DIPEA, DMF, room temp., 45 min, quant., then 20% piperidine, DMF, 2 x 7 min; (ii) TFA, CHzC12, room temp., 2 x 2 h; (iii) one cycle: 2 x 5 equiv. acid, G ( 7 azabenzotriazol-l-yl)-l,1,3,3-tetramethyluronium hexafluorophosphate (after structure revision: N[(dimethylamino)-lH-1,2,3-triazolo-[4,5-b]pyridin-l-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide [67], HATU), DIPEA, DMF, room temp., 20 min, 65-1000/0; (iv) 3.6 equiv. 17, 0-(benzotriazol-l-yl)-l, 1,3,3-tetramethyluroniumhexafluorophosphate (HBTU), HOBt, PIPEA, DMF, room temp., 1 h, quant.: (v) one cycle: diisopropylcarbodiimide, HOBt, DMF; dithiothreitol, DIPEA, DMF, 50 "C, 30%, lower yield for trimer; (vi) AczO, Fy;50% TFA-CH2Clz.
22.4 Amide-Linked Carbohydrate Oligomers
58 1
HOOC HO NHCO-Leu-Phe-Gly-Gl y -Tyr-H OH
81
HO OH HO HO
HO HO
82
HO HO
OH OH
83
Me?
Scheme 12.
D-aspartate spacer inhibited adhesion by ca 50% at 10 p~ [62]. In the tumor cell invasion assay, 67 slightly inhibited the invasion of A549 cells through an artificial membrane, by 64% at 10 p ~ . On the basis of the notion that the 6-glucuronide of morphine is distinctly more active in analgesia than morphine itself, glucuronide analogs of enkephalins were targeted, and the peptide conjugated SPH 81 (Scheme 12) was synthesized from 77 [301. In a mouse deferens assay, the sugar dimer-modified leucin enkephalinamide 81 was twice as active as the Leu-enkephalinamide standard. As an exact analog of the phytoalexin elicitor branched heptasaccharide 82 the branched SPH 83 was devised with four amide bonds replacing the 1 + 6 linkages of the pentasaccharide backbone and thus producing the same length. No elicitor activity was detected for 83, possibly because of the reduced flexibility or different conformation of the mimetic structure [56]. Mimetics of the sialyl Lewis' and sialyl Lewisa oligosaccharides have recently found increased attention. Baisch and
582
22 Saccharide-Peptide Hybrids
Ohrlein have prepared mimetics in which the acetamido group of the central glucosamine was replaced by methyl glycopyranosiduronamides [60, 631. Disaccharide mimetics of type 73 were accepted as substrates by the enzymes 2,3-sialyltransferase, p(1 + 4)galactosyltransferase and fucosyltransferases I11 and IV, so enzymatic synthesis of sialyl LewisXand sialyl Lewisa mimetics such 84 (Scheme 12) was feasible. It will be interesting to see the biological activity of these mimetics. NucleoSPHs such as 76 with amide replacement of the usual phosphodiester linkage were synthesized as antisense agents. Judged by melting temperatures and thermodynamic constants some binding properties of DNA and RNA could be mimicked ~41. A library of piperidine-based SPH trimers incorporating monomer 42 (Scheme 6) was tested for glycosidase activity. No inhibition was observed for yeast a-glucosidase and isomaltase, almond P-glucosidase, human placenta a-fucosidase, snail pmannosidase, and E. coli P-galactosidase, possibly because of the small number of hydroxyl groups present. 22.4.4 Conformational Properties In their pioneering work, Kessler and coworkers described the conformational role of CAAs in peptide strands, fixing a certain conformation or inducing turns [7]. Glucosaminuronic acids as dipeptide isosteres constrain the conformation of linear peptides. 1-Aminoglucuronic acids (cf 18, Scheme 4) would be p-turn mimetics, 2,6anhydro-7-amino-7-deoxyheptonic acids (cf: 30, Scheme 5) flexible p-turn mimetics. 2,6-Anhydro-3-amino-3-deoxyheptonic acids ( c j 14, Scheme 3) might serve as yturn mimetics, and hydroxylated pipecolic acid derivatives (cf 27, Scheme 4) can be regarded as homoproline derivatives. Thus, in this view the CAAs are seen as peptide mimetics. The conformation of the oligomers formed from CAAs and D- or Laspartic acid (67, Scheme 9) were, according to calculations, distinct and determined by the interaction of the CAA 3-OH group and the a-carboxylic moiety of the aspartic acid residues (cf: 85, Scheme 13) [62]. The other conformational determinant would be the positioning of the anomeric amide with the carbonyl group avoiding the vicinity to the anomeric oxygen and favoring H-bond formation with the CAA 2-OH group. The resulting two different conformations for the D- and Laspartic acid series would be in keeping with the different biological activity of both series. From the properties of the nucleo-CAAs like 76 and their RNA binding consistent with a Watson-Crick binding model, it follows that preferred conformations were adopted [64]. The SPH pentamer backbone of 83 (Scheme 12) was found to be more rigid, according to force field calculations for hydrated oligosaccharides, than the corresponding oligosaccharide 82, thus explaining the loss of biological activity [56]. The N-acetylneuraminic acid oligomers prepared by Gervay’s group [66] possibly occur in helical conformations according to circular dichroism data and NMR hydrogen-deuterium exchange experiments, the first ordered structure was observed for a tetramer. The C-glycosidic P-D-arabinofuranose oligomer 69 (Scheme 10) and the corresponding hexamer described by Fleet and colleagues were found to form p-turn-
22.4 Amide-Linked Carbohydrate Oliyomers
583
85 For D-aspartic acid: R’ = H, R2 = NH-R For L-aspartic acid: R2 = H, R’ = NH-R
86
BAC
Scheme 13.
type structures stabilized by hydrogen-bonds of neighboring units [ 681. As shown for the analogous terminal amide 86 (Scheme 13), a clear secondary structure was already visible for a trimer [65].The tetramer 87, 3,4-isopropylidene acetalprotected and with regard to the backbone isomeric with 69, and its cyclohexylidene acetal-protected enantiomer had similar p-turn structures [ 581. In contrast, the CI-Darabinofuranose analogs with the C-2/C-5 substituents of the furanose rings in the trans position showed no indication of secondary structure [69]. The conformation of the SPH 70 (Scheme 10) and the tetramer substructures, also with the C-2/C-5 trans substituted tetrahydrofuran monomers, differed from the previous cases, and the NMR data were best interpreted in terms of a left-handed a-helix [58]. In conclusion, it seems that some SPHs as new unnatural oligomers [70] adopt specific compact conformations and can thus be regarded in Gellman’s terminology as ‘foldamers’ [71]. References 1. H. P. Wessel, C. M. Mitchell, C. M. Lobato, G. Schmid, Angew. Chem. Znt. Ed. Engl. 1995, 34, 2712-2713. 2. S. Sabesan, Tetrahedron Lett. 1997, 38, 3121-3130. 3 . K. C. Nicolaou, H. Florke, M. G. Egan, T. Barth, V. A. Estevez, Tetrahedron Lett. 1995, 36, 1775-1778. 4. R. J. Simon, R. S. Kania, R. N. Zuckermann, V. D. Huebner, D. A. Jewell, S. Banville, S. Ng, L. Wang, S. Rosenberg, C. K. Marlowe, D. C. Spellmeyer, R. Tan, A. D. Frankel, D. V. Santi, F. E. Cohen, P. A. Bartlett, Proc. Natl. Acud. Sci. USA 1992, 89, 9367-9371. 5. U. K. Saha, R. Roy, Tetruhedron Lett. 1995, 36, 3635-3638. 6. J. P. McDevitt, P. T. Lansbury Jr., J. Am. Chem. Soc. 1996, 118, 3818-3128.
584
22 Saccharide-Peptide Hybrids
7. E. Graf von Roedern, E. Lohof, G. Hessler, M. Hoffmann, H. Kessler, J. Am. Chem. Soc. 1996,118, 10156-10167. 8. P. S. Ramamoorthy, J. Gervay, J. Org. Chem. 1997, 62, 7801-7805. 9. G. Baschang, Tetrahedron 1989, 45, 633 1-6360. 10. A. R. Williamson, S. Zamenhof, J. Biol. Chem. 1963,238, 2255-2258. 11. S. Hanessian, T. H. Haskell, J. Biol. Chem. 1964,239, 2758-2764. 12. K. Heyns, G. Kiessling, W. Lindenberg, H. Paulsen, M. E. Webster, Chem. Ber. 1959, 92, 2435-2438. 13. J. P. Waltho, D. H. Williams, E. Selva, P. Ferrari, J. Chem. Soc. Perkin Trans. 1 1987, 21032107. 14. R. Schauer, Adv. Carbohydr. Chem. Biochem. 1982, 40, 131. 15. S. Knapp, Chem. Rev. 1995, 95, 1859-1876. 16. K. Isono, K. Asahi, S. Suzuki, J. Am. Chem. SOC. 1969, 91, 7490-7505. 17. W. A. Konig, H. Hahn, R. Rathmann, W. Hass, A. Keckeisen, H. Hagenmaier, C. Bormann, W. Dehler, R. Kurth, H. Zahner, Liebigs Ann. Chem. 1986,407-421. 18. P. Garner, in Atta-ur-Rahman (Ed.): Studies in Natural Product Chemistry, Vol. I , Elsevier, Amsterdam 1988, p. 397-434. 19. G. Casiraghi, L. Colombo, G. Rassu, P. Spanu, J. Org. Chem. 1991, 56, 6523-6527. 20. A. J. Fairbanks, P. Sinay, Synlett 1995, 277-279. 21. S. Knapp, C. Jaramillo, B. Freeman, J. Org. Chem. 1994,59,4800-4804. 22. J. J. Fox, Y. Kuwada, K. A. Watanabe, Tetrahedron Lett. 1968, 57, 6029-6032. 23. F. W. Lichtenthaler, T. Morino, H. M. Menzel, Tetrahedron Lett. 1975, 9, 665-668. 24. K. Heyns, H. Paulsen, Chem. Ber. 1955, 88, 188-195. 25. H. Weidmann, H. K. Zimmerman jr., Justus Liebigs Ann. Chem. 1961, 639, 198-203. 26. C. Muller, E. Kitas, H. P. Wessel, J. Chem. Soc., Chem. Commun. 1995, 2425-2426. 27. R. A. Goodnow Jr., A.-R. Richou, S. Tam, Tetrahedron Lett. 1997, 38, 3195-3198. 28. S. Tokura, Y. Ikeuchi, S.4. Nishimura, N. Nishi, Int. J. Biol. Macromol. 1983, 5, 249. 29. R. W. Myers, Y. C. Lee, Curbohydr. Res. 1984, 132, 61-82. 30. B. Drouillat, B. Kellam, G. Dekany, M. S. Starr, I. Toth, Bioorg. Med. Chem. Lett. 1997, 7, 2241-2250. 31. S.-I. Nishimura, S. Nomura, K. Yamada, J. Chem. Soc., Chem. Commun. 1998, 617-618. 32. D. D. Long, N. L. Hungerford, M. D. Smith, D. E. A. Brittain, D. G. Marquess, T. D. W. Claridge, G. W. J. Fleet, Tetrahedron Lett. 1999, 40, 2195-2198. 33. R. Csuk, H. Honig, J. Nimpf, H. Weidmann, Tetrahedron Lett. 1980,21, 2135-2136. 34. B. P. Bashyal, H.-F. Chow, G. W. J. Fleet, Tetrahedron Lett. 1986, 27, 3205-3208. 35. E.-F. Fuchs, J. Lehmann, Chem. Ber. 1975, 108, 2254-2260. 36. H. S. Overkleeft, S. H. L. Verhelst, E. Pietermann, N. J. Meeuvenoord, M. Overhand, L. H. Cohen, G. A. van der Marel, J. H. van Boom, Tetrahedron Lett. 1999, 40, 4103-4106. 37. E. Graf von Roedern, H. Kessler, Angew. Chem. 1994, 106, 684-686. 38. M. K. Gurjar, A. S. Mainkar, M. Syamala, Tetrahedron Asymm. 1993, 4, 2343-2346. 39. A. Dondoni, M.-C. Scherrmann, A. Marra, J.-L. Delepine, J. Org. Chem. 1994,59, 7517-7520. 40. M. P. Moyer, P. L. Feldman, H. Rapoport, J. Org. Chem. 1985,50, 5223-5230. 41. D. W. Knight, N. Lewis, A. C. Share, Tetrahedron Asymm. 1993, 4, 625-628. 42. E. Byrgesen, J. Nielsen, M. Willert, M. Bols, Tetrahedron Lett. 1997, 38, 5697-5700. 43. P. Bach, A. Lohse, M. Bols, Tetrahedron Lett. 1999, 40, 367-370. 44. E.-F. Fuchs, J. Lehmann, Carbohydr. Res. 1976, 49, 267-273. 45. M. d. G. Garcia Martin, M. Violante de Paz Bafiez, J. A. Galbis Perez, Carbohydr. Res. 1993, 240, 301-305. 46. H. Sumitorno, K. Hashimoto, Macromolecules 1977, 10, 1327-1331. 47. J. Thiem, F. Bachmann, Makromol. Chem. 1991, 192, 2163-2182. 48. J. Thiem, F. Bachmann, Makromol. Chem. 1993, 194, 1035-1057. 49. J. Thiem, F. Bachmann, Trends Polym. Sci. 1994,2,425-432. 50. J. Yoshimura, H. Ando, T. Sato, S. Tsuchida, H. Hashimoto, Bull. Chem. Soc. Jpn. 1976, 49, 2511-2514. 51. Y. Chapleur, Carbohydrate Mimics: Concepts and Methods, Verlag Chemie, Weinheim 1998. 52. H. P. Wessel, Topics Curr. Chem. 1997, 187, 215- 239.
References
585
53. Y. Suhara, J. E. K. Hildreth, Y. Ichikawa, Tetrahedron Lett. 1996, 37, 1575-1578. 54. Y. Suhara, M. Ichikawa, J. E. K. Hildreth, Y. Ichikawa, Tetrahedron Lett. 1996, 37, 25492552. 55. J. Gervay, T. M. Flaherty, C. Nguyen, Tetrahedron Lett. 1997, 38, 1493-1496. 56. C. M. Timmers, J. J. Turner, C. M. Ward, G. A. van der Marel, M. L. C. E. Kouwijzer, P. D. J. Grootenhuis, J. H. van Boom, Chem. Eur. J. 1997,3, 920-929. 57. M. D. Smith, D. D. Long, D. G. Marquess, T. D. Mi. Claridge, G. W. J. Fleet, Chem. Cornmun. 1998,2039-2040. 58. T. D. W. Claridge, D. D. Long, N. L. Hungerford, R. T. Aplin, M. D. Smith, D. G . Marquess, G. W. J. Fleet, Tetrahedron Lett. 1999, 40, 2199-2002. 59. I. McCort, A. Dureault, J.-C. Depezay, Tetrahedron Lett. 1998, 39, 4463-4466. 60. G. Baisch, R. Ohrlein, Bioorg. Med. Chem. 1998, 6, 1673-1782. 61. J. Gervay, P. S. Ramamoorthy, N. N. Mamuya, Tetrahedron 1997, 53, 11039-11048. 62. Y. Suhara, M. Izumi, M. Ichikawa, M. B. Penno, Y. Ichikawa, Tetrahedron Lett. 1997, 38, 7167-71 70. 63. G. Baisch, R. Ohrlein, Carbohydr. Res. 1998, 312, 61-12. 64. R. A. Goodnow Jr., S. Tam, D. L. Pruess, W. W. McCornas, Tetrahedron Lett. 1997,38, 31993202. 65. D. D. Long, M. D. Smith, D. G. Marquess, T. D. W. Claridge, G. W. J. Fleet, Tetrahedron Lett. 1998, 39, 9293-9296. 66. L. Szabo, B. L. Smith, K. D. McReynolds, A. L. Parrill, E. R. Morris, J. Gervay, J. Org. Chem. 1998,63, 1074-1078. 67. I. Abdelmoty, F. Albericio, L. A. Carpino, B. M. Foxman, S. A. Kates, Letters in Peptide Science 1994, 1 , 57. 68. M. D. Smith, T. D. W. Claridge, G. E. Tranter, M. S. P. Sansom, G. W. J. Fleet, Chem. Cornmun. 1998, 2041-2042. 69. M. D. Smith, D. D. Long, A. Martin, D. G. Marquess, T. D. W. Claridge, G. W. J. Fleet, Tetrahedron Lett. 1999,40,2191-2194. 70. M. J. Soth, J. S. Nowick, Curr. Opin. Chem. Bid. 1997, 1, 120-129. 71. S. H. Gellman, Acc. Chem. Res. 1998, 31, 173-180.
Part I Volume 2
I11
Enzymatic Synthesis of Glycosides
Carbohydrates in Chemistry and Biology Editor Beat Ernst, Gerald W. Hart, Pierre Sinay copyright OWILEY-VCH Verlag GmbH, 2000
23 On the Origin of Oligosaccharide SpeciesGlycosyltransferases in Action Dirk H. van den Eijnden
23.1 Introduction Glycoproteins and glycolipids, jointly called glycoconjugates, are macromolecules essential to eukaryotic life. The oligosaccharide chains carried by glycoconjugates often contain biological intelligence which is encoded by the oligosaccharide structure. These chains thus confer specific biological functions on the proteins and lipids carrying them. When embedded in the cellular plasma membrane they decorate the cell surface with specific oligosaccharide structures which can be crucial to cell function. The synthesis of protein- and lipid-linked oligosaccharides proceeds by the sequential addition of monosaccharide building blocks to a growing chain that is covalently linked to the protein or lipid aglycone. In a similar way oligosaccharides present in some secretions (e.g. milk) are built up from a single sugar (glucose). Unlike the processes of protein and nucleic acid synthesis, in which the order of attachment of amino acids and nucleotides is read from a nucleic acid matrix, glycosylation is a non-template-directed process. This enables the formation of branches which are common to oligosaccharides but are not found in nucleic acids and proteins. The enzymes that are responsible for the assembly of the oligosaccharides on glycoconjugates are known as glycosyltransferases (in the early stages of N-glycosylation glycosidases also play a role-see later in this chapter). Being proteins, glycosyltransferases are primary products of their respective genes and, therefore, the oligosaccharide chains formed by their action can be considered as secondary gene products. Glycosyltransferases typically catalyze the following general reaction: nucleoside-P-Psugar
+ acceptor + sugar-acceptor + nucleoside-P-P
In this reaction nucleotideesugars are the donor substrates (for some glycosyltransferases involved in the early stages of protein N-glycosylation a sugarphosphate-dolichol is the donor molecule; see later). In sequential reactions mono-
590
23 On the Origin of Oligosaccharide Species-Glycosyltransferases in Action
saccharides are attached, one at a time, through glycosidic linkages at nonreducing ends and at branching points in a specific order; the sugar-acceptor product of one reaction is the acceptor substrate in the next. In vitro glycosyltransferases generally have optimum activity at a pH of ca 7.0. In most instances Mn2+ ions are required for enzymatic activity (except for sialyltransferases and some p6-N-acetylglucosaminyltransferases which are cation-independent). Because glycosyltransferases are generally membrane-associated proteins, determination of their activity in vitro requires the solubilization of the enzymes with detergents. Some glycosyltransferases, however, occur in a soluble form in body fluids, probably as a result of proteolytic release of the catalytically active part of the enzyme. Glycosyltransferases of eukaryotic organisms have high specificity for the sugar they transfer and the linkage they establish. This has enabled their classification in groups, such as the sialyltransferases which all exclusively utilize CMP-sialic acid as a donor substrate, and the galactosyltransferases which highly prefer UDP-Gal as a donor, with subgroups based on the linkage that is established. In many (but not all) instances the molecular cloning of the cDNAs of these enzymes has provided molecular support for this classification (see Section 23.17, Glycosyltransferase families). Glycosyltransferases are, furthermore, generally specific for the accepting sugar and its anomeric configuration, the linkage by which this sugar is linked to or is substituted by another sugar residue, and the nature of these other residues. Sometimes the entire underlying oligosaccharide structure and possibly its spatial conformation seems to be recognized. It also has been suggested that some glycosyltransferases recognize specific peptide motifs of the polypeptide aglycone, which could be one explanation for site specific glycosylation patterns on glycoproteins with more than one glycosylation site. Very many different glycosidic linkages are known to occur in glycoconjugatelinked and free oligosaccharides. The formation of each linkage is catalyzed by at least one specific glycosyltransferase. As mentioned above, such an enzyme is generally not capable of catalyzing the formation of another linkage-type. A notable exception to the ‘one enzyme-one linkage’ concept [ 11, however, is the Lewis blood group a3/4-fucosyltransferase, which can establish Fuc(a1-4)[Gal( Pl-3)IGlcNAc and Fuc(a1-3)[Gal(P1-4)]GlcNAc linkages in vivo and in vitro [2-41. Also other glycosyltransferases, particularly when the substrate concentrations used are unphysiologically high, can establish more than one linkage by transferring to another acceptor sugar [5] or by transferring a monosaccharide from a different sugardonor substrate (donor promiscuity) [6-111. Even though glycosylation is a non-template directed process, oligosaccharide structures are generally produced with high fidelity. Firstly, the glycosylation potential of a cell (expression levels of the different glycosyltransferases; subcellular localization of the enzymes; supply of donor substrates) determines which pathways potentially can be followed. It should be noted that this potential is not constant but may vary with cell status (normal versus malignant), with the developmental stage of the cell, and with the environment wherein the cell grows (e.g. energy supply). Furthermore the specificities of the glycosyltransferases involved play a major controlling role in the formation of the correct final product structure(s). In such a process, however, it is inevitable that at intersections in the metabolic pathway,
23.2 Protein N-Glycosylation
59 1
where enzymes compete with one another for common intermediate substrate structures, various directions can be taken. In practice this leads to the synthesis of an array of related oligosaccharide structures rather than of one single structure. Such variations in oligosaccharide structure at single glycosylation sites is a common feature of glycoproteins and is known as microheterogeneity. It causes glycoproteins to occur in different glycoforms, which are oligosaccharide variants of the same polypeptide. Many excellent reviews have described the topography, mechanisms, and enzymology of protein and lipid glycosylation [12-25). This chapter gives a concise introduction to the enzymatic properties and molecular relationships of glycosyltransferases with emphasis on protein N-glycosylation and the corresponding pathways of oligosaccharide assembly, in particular the late stages. Where appropriate, however, the glycosyltransferases involved in protein O-glycosylation, biosynthesis of the core of proteoglycans, and the glycosylation of sphingoglycolipids are also discussed.
23.2 Protein N-Glycosylation: Pre-assembly of Oligosaccharide-PPDolichol and en bloc Transfer Protein N-glycosylation is a process that has been highly conserved during evolution. In this process several stages can be distinguished; the early ones are shared by all organisms from yeasts to mammals. The initial steps, in which an oligosaccharide precursor structure is synthesized on a dolichol-diphosphate (Dol-PP) lipid carrier, occur in the endoplasmic reticulum (ER). The most simple picture that complies best with current knowledge [ 12, 15, 181 is that depicted in Figure 1. Several glycosyltransferases, embedded in the membranes of the ER with their catalytic domain facing the cytosol of the cell, transfer GlcNAc-P, GlcNAc, and Man to Dol-P from UDP-GlcNAc and GDP-Man, respectively, to yield a DolPP linked Man~GlcNAc2oligosaccharide. The molecule then flip-flops over the membrane resulting in a luminal exposed oligosaccharide structure. Subsequently four additional Man and three Glc residues yielding Glc3Man~GlcNAc2-PP-Dol are attached by glycosyltransferases that have their catalytic domain facing the lumen. The latter enzymes are unusual in that they utilize Man-P-Do1 and Glc-PDo1 rather than GDP-Man and UDP-Glc as a sugar donor. The Dol-P linked sugars are synthesized from the nucleotide-sugars at the cytosolic face of the ER, whereafter they flip-flop over the membrane to become utilizable for the luminal glycosyltransferases. In the next stage of N-glycosylation this pre-assembled oligosaccharide is transferred en bloc to an Asn-X-Ser/Thr sequon (where X can be any amino acid except Pro or Asp) in the nascent polypeptide chain of a glycoprotein being synthesized at the ribosome (Figures 1 and 2). This co-translational step is catalyzed by the oligosaccharyltransferase (OST) complex which consists of at least three proteins [26, 271 that seem to be intimately associated with membrane-bound ribosomes [ 191.
592
23 On the Origin of Oligosaccharide Species-Glycosyltransferases in Action
Figure 1. Initial steps in protein N-glycosylation. Pre-assembly of oligosaccharide-P-P-Do1 and en bloc transfer to the protein in the endoplasmic reticulum. Dol, dolichol; OST, oligosaccharyltransferase; P, phosphate; W , GlcNAc; 0, Man; A, Glc.
23.3 Trimming of the Polypeptide-Bound Oligosaccharide Once the preformed oligosaccharide is attached to the polypeptide it is extensively trimmed before it is extended with more sugars. Three Glc and four Man residues are removed from the polypeptide-bound oligosaccharide to yield the common intermediate MansGlcNAcz structure (Figure 2). The first Glc (a2-linked) is removed by ER glucosidase I that might already act while the glycosylated polypeptide chain is still being synthesized on the ribosome. The other two Glc residues (that are a3linked) are removed by ER glucosidase I1 (reviewed in [ 19, 231). The catalytic sites of both enzymes face the lumen of the ER in which the glycosylated polypeptide resides after its vectorial translocation over the ER membrane. The first (a2linked) Man may be removed in the ER by ER mannosidase to yield a unique MansGlcNAq structure after which the glycoprotein is translocated to the cisGolgi compartment (Figure 2) [19, 281. Alternatively, the first Man residue is removed after this translocation by Golgi-localized (u2-)mannosidase IA and IB. The specificity of these enzymes differs from that of the ER mannosidase, and they yield the two other possible MansGlcNAcz isomeric structures. Subsequent repeated actions of mannosidase IA and/or IB on either MansGlcNAcz structure results in the aforementioned single intermediate MansGlcNAcl structure [28]. In yet another
23.4 Folu'ing and Quality Control
RER
593
GOLGI CIS
MEDIAL
TRANS
Figure 2. Topography of reactions involved in N-glycosylation in the rough endoplasmic reticulum and the Golgi membranes. Note that the boxed structure remains untouched during the entire process. The pathway leading to a di-antennary disialylated oligosaccharide is shown. Dol, dolichol; OST, oligosaccharyltransferase; P, phosphate; , GlcNAc; 0, Man; A , Glc; 0 , Gal; V , Fuc; 4, NeuAc.
pathway the last Glc and the Man residue to which it is attached are removed as Glc(u1-3)Man in one step by an ER localized endo-mannosidase yielding a Mans GlcNAcz structure that is processed further in the cis-Golgi [29].
23.4 Folding and Quality Control Although the synthesis of the Glc3 Mans GlcNAcz oligosaccharide followed by its degradation to a MansGlcNAcz structure might seem a waste of energy, one important role of the oligosaccharide is that it aids the maturation of the polypeptide chain to a functional protein [30-321. After the removal of the first two Glc residues by ER glucosidase I and I1 the glycosylated polypeptide is released from the ribosome. In the lumen of the ER it then associates with membrane bound calnexin or soluble calreticulin, chaperones which recognize the monoglucosylated oligosaccharide in a lectin like fashion. While transiently bound the polypeptide is as-
594
23 On the Origin of Oligosaccharide Species-Glycosyltransferases in Action
sisted by these chaperones to fold properly and, for oligomeric glycoproteins, to assemble in oligomers. At this stage also disulfide bridges are formed. After subsequent removal of the third Glc from the oligosaccharide by glucosidase I1 the glycoprotein is no longer bound and is released, and the oligosaccharide is further processed on the glycoprotein’s journey via the Golgi to its cellular destination. In case the process of correct folding or assembly fails, however, the deglucosylated polypeptide is retained in the ER where it can be re-glucosylated by a glycoprotein glucosyltransferase specific for improperly folded glycoproteins [ 33, 341. Re-glucosylated proteins can be bound again by calnexin or calreticulin and so have another chance of folding correctly. The glycoprotein glucosyltransferase (together with the chaperones) thus functions in the control mechanism by which only properly folded and assembled glycoproteins can leave the ER.
23.5 Committed Steps in the Formation of Complex-Type Oligosaccharide Chains and Branching After the trimming reactions in the ER and the cis-Golgi the glycoprotein containing the resulting MansGlcNAcz oligosaccharide is translocated to the medial-Golgi compartment (Figures 2 and 3 ) . Here the first committed step in the formation of
Window of action
GlcNAcT 111 GlcNAcT IV & V
tri- and tetra-antennary
Figure 3. Committed steps in the synthesis of complex-type N-linked oligosaccharides. Action of GlcNAcT I on the oligomannoside structure opens the window for several possible reactions as catalyzed by a-mannosidase I1 (Man-ase II), GlcNAcT 11-V, and core-specific ah-FucT. The window is closed by the action of p - Gal T as shown for a di-antennary structure. For key to the symbols see legend to Figure 2.
23.5 Committed Steps in the Formation of Complex-Type Oligosaccharide Chains V
GlcNAcBl
II
GlcNAc~l~SManal
595
I6 6' 111
G l c N A c ~ l 4 M a n ~ l - r 4 G l c N A c ~ l - + 4 G l c N A-Asn c~1
I
GlcNAc~l-.PManulr3
IV
GlcNAcPl
r4
Figure 4. Structural representation of the linkages formed by GlcNAcT ILV. The Roman number refers to the GlcNAcT involved.
complex-type oligosaccharides takes place by the addition of a peripheral GlcNAc residue in ((31-2) linkage to the Man(a1-3)Manp site as catalyzed by GlcNAcT I to form the first branch [13, 17, 201. This GlcNAc functions as a green traffic light for several possible reactions catalyzed by a-mannosidase 11, GlcNAcT 11, 111, IV and V (Figure 4), and N-linked core-specific a6-FucT that catalyzes reactions leading to di-, tri- and tetra-antennary oligosaccharides, hybrid and bisected structures, and core-fucosylated structures [13, 171 (Figure 3). It is at this stage of oligosaccharide processing that there is an extensive regulation at the substrate level. Divergence in the pathway occurs here in particular and structures with varying number of branches (a branch or antenna is build up from a GlcNAc residue as introduced by GlcNAcT I, 11, IV or V) might be formed [ 13,211. Evidently GlcNAcT 11, yielding a di-antennary oligosaccharide, can only act after a-mannosidase I1 has trimmed two additional Man residues from the Man(a16)ManP branch. When GlcNAcT 111 acts before a-mannosidase I1 the oligosaccharide is frozen into a hybrid structure (combining the features of a complex-type structure at the Man(al-3)Manp branch and an oligomannosidic-type structure at the Man(a16)Manp branch) because a-mannosidase I1 is blocked by the bisecting GlcNAc residue attached by GlcNAcT I11 action. When GlcNAcT I11 acts after GlcNAcT I1 a so-called bisected di-antennary oligosaccharide is formed. Action of GlcNAcT IV and V leads to the formation of tri- and tetra-antennary oligosaccharides and these might also become bisected by the subsequent action of GlcNAcT 111. The other route, in which the order of action of these GlcNAcTs is reversed, is impossible because GlcNAcT 11, IV and V do not act on structures containing a bisected GlcNAc residue. A bisecting GlcNAc also inhibits core 1x6-fucosylation. It should be mentioned here that in plants and insects a3-fucosylation of the core GlcNAc might occur as catalyzed by a core-specific a3-FucT [35, 36, 1881. In addition, in plants an 1x2-xylosyltransferase has been described that can introduce an aXyl residue to OH-2 of the (3-Man of the core [37].All these reactions can no longer occur after in a second committed step of complex-type oligosaccharide formation, by P4-galactosyltransferase action, a Gal residue is attached in (81-4) linkage to the GlcNAc((31-2)Man(al-3)ManP branch (to which the first Gal preferentially is in-
596
23 On the Origin of Oligosaccharide Species-Glycosyltransferases in Action
troduced [38, 391) to yield a Gal@-4)GlcNAc (lacNAc) terminus [13, 17, 211. This Gal residue thus functions as a red light and closes the stage of oligosaccharide branching and modification of the core (Figure 3). Occasionally a GalNAc rather to yield a terthan a Gal is introduced by a 1)4-N-acetylgalactosaminyltransferase minal GalNAc(pl-4)GlcNAc ( N ,N’-diacetyllactosediamine, 1acdiNAc) unit [40] (see also Section 23.15-The lacdiNAc Pathway of Complex-Type Oligosaccharide Synthesis).
23.6 Topology of the Reaction Catalyzed by a Typical GlcNAcT All eukaryotic Golgi glycosyltransferases are typically membrane-bound enzymes with a type I1 membrane topology (cytosolic NHz-terminus) as has become apparent from analysis of the deduced amino acid sequences of cloned enzymes [41, 421. GlcNAcT I, 11, 111, IV and V form no exceptions in this respect [43-491. Most glycosyltransferase polypeptides consist of ca 325-425 amino acids and have a very similar domain structure-a short cytoplasmic tail ( ~ 5 - 3 0amino acids) attached to a transmembrane domain (x15-20 amino acids), which functions as a signalanchor sequence by which the enzyme is targeted to and anchored in the Golgi membrane. This is followed by a so-called stem region ( ~ 3 5 - 4 5amino acids) and a large catalytic domain (approximately 300 amino acids, containing the COOH terminus) extending into the lumen of the Golgi (Figure 5). The immature protein-linked oligosaccharide chains are also exposed into the Golgi lumen during the glycoprotein’s traverse through the cellular endo membranes. UDP-GlcNAc is thus utilized inside the Golgi lumen. The synthesis of this substrate by the enzyme UDP-GlcNAc pyrophosphorylase, however, occurs in the cytosol and, as nucleotide-sugars cannot freely diffuse through the Golgi membrane, it has to be transported across this barrier. This transport is facilitated by a specific UDP-GlcNAc transporter by a mechanism in which UDP-GlcNAc is exchanged for UMP that is formed from UDP, the other reaction product of the GlcNAc-transfer reaction [25] (Figure 5). Similar specific transporters for CMPNeuAc, UDP-Gal, GDP-Man, and UDP-GalNAc have also been demonstrated and cloned [25, 501, and aberrant glycosylation patterns are, indeed, observed for cells with defects in transporter activity [25]. As yet, however, it is unclear if and to what extent the import of nucleotide-sugar donor-substrates might be limiting and can thus contribute to the regulation of protein glycosylation.
23.7 Elongation and Termination Reactions in the trans-Golgi By the time the first Gal residue is introduced to the oligosaccharide chain, the glycoprotein has reached the trans-Golgi in which the elongation and termination
23.7 Elongation and Termination Reactions in the trans-Golgi
591
Glycoprotein
-
UTP + m ~ l c ~ ~ c ~ 1 - p UDP--/+ Pyrophosphorylase
PPi
Figure 5. Organization of a typical glycosyltransferase in the Golgi membrane and topology of the reaction catalyzed as exemplified for a GlcNAcT. Note that the nucleotide sugar donor substrate is synthesized in the cytosol. Before it can be utilized by the glycosyltransferase it has to be translocated in exchange for a nucleotide monophosphate into the lumen of the Golgi by a specific transporter. Sugar transfer takes place to the oligosaccharide chain of a glycoprotein during its traverse through the secretory system. TMD, trans-membrane domain.
reactions occur (Figures 2 and 6). As mentioned above, P4-galactosylation disables the action of a-mannosidase 11, GlcNAcT I-V, and N-linked core-specific a6-FucT. In contrast, it is the green traffic light for many glycosyltransferases which all act on an accepting lacNAc unit. The action of these enzymes yields the mature oligosaccharide structures. Here additional diversity (leading to microheterogeneity and different glycoforms of a glycoprotein; see Section 23.1, Introduction) is introduced because these enzymes potentially compete for common Gal@1-4)GlcNAc acceptor sites on the protein-linked oligosaccharides. Six of the most common reactions, as catalyzed by a6-NeuAcT, a3-NeuAcT, a3-GalT, a3-FucT, a2-FucT, and P3-GlcNAcT, respectively, are shown in Figure 6 for a di-antennary glycan as acceptor. a6-NeuAcT, a3-NeuAcT, a3-GalT, and a2-FucT directly cap the Gal of the lacNAc units by introduction of a terminal sugar, whereas a3-FucT adds a fucose to the GlcNAc of these units. Introduction of a P3-GlcNAc residue to a lacNAc unit by P3-GlcNAcT enables a second P4-galactosylation reaction (Figure 6). In this concerted action of P3-GlcNAcT and b4-GalT polylactosaminoglycan extensions are formed which also may be sialylated, fucosylated, or a-galactosylated. These reactions can in principle take place with any terminally exposed lacNAc unit irrespective of whether it is presented on di-, tri- or tetra-antennary, hybrid, or bisected N-linked oligosaccharides or on 0-linked oligosaccharides (see later).
598
23 On the Origin of Oligosaccharide Species-Glycosyltrnnsferases in Action
Window of action
ta
NeuAca2->6 O+
1
Figure 6. Elongation and termination reactions as enabled by the action of p4-GalT. P4-Galactosylation blocks the action of the branching GlcNAcTs, Man-ase I1 and core a6-FucT (c.J Figures 2 and 3). By way of contrast, it opens the window of action for the elongating (polylactosaminoglycan-forming) and terminating enzymes shown. The reactions cannot only occur with the di-antennary substrate shown, but also with tri- and tetra-antennary, bisected, and hybrid structures.
Because of the substrate specificities of each of these enzymes, however, not all of the theoretically possible product structures are formed in nature in detectable amounts. By way of contrast, such structures may be formed in vitro where very active (recombinant) enzyme preparations can be used. In the next paragraphs the specificities of the enzymes are further described.
23.8 Activity with Branched Substrates Not all N-linked oligosaccharides with terminal lacNAc units are acted upon by a6NeuAcT, a3-NeuAcT, a3-GalT, a3-FucT, and p3-GlcNAcT at the same rate. Substrate specificity studies have shown that a6-NeuAcT (bovine colostrum; corresponds to ST6Gal I described below in Section 23.18) much prefers di-antennary substrates and is less active with tri- (having an additional Gal@-4)GlcNAc p4linked at the Man(a1-3)ManD branch as initiated by GlcNAcT IV action), tri'(having an additional Gal@-4)GlcNAc p6-linked at the Man(a1-6)Manp branch as initiated by GlcNAcT V action), tetra- (having both additional G a l @ -
23.8 Activity with Branched Substrates
599
300 h
E
200
h .tl
150
> .c
0 (D
$ .-
100
c (D
-a 50 0 06-NeuAcT 03-NeuAcT 03-GalT
03-FucT B3-GlcNAcT
Figure 7. Relative activities of u6-NeuAcT, a3-NeuAcT, a3-GalT, a3-FucT, and P3-GlcNAcT with structures that form part of branched, N-linked oligosaccharides. The acceptor oligosaccharides are represented by symbolic structures the branch linkages of which correspond to those in the structures shown in Figures 4 and 8.
4)GlcNAc units), and bisected di-(GlcNAcT 111 action) antennary oligosaccharides [51] (Figure 7). By way of contrast, a3-NeuAcT (human placenta; high preference for type-2 chain acceptors; corresponds to ST3Gal IV) prefers the tri-antennary substrate whereas a bisected acceptor is a poorer substrate [52]. Very similar specificity is shown by 1x3-FucT VI from human milk (D. H. Van den Eijnden and W. E. C. M. Schiphorst, unpublished results). a3-GalT (calf thymus) is relatively indifferent to the branching of the oligosaccharide substrates, but also acts at a lower rate on a bisected di-antenna [53]. Finally P3-GlcNAcT (Novikoff tumor cell ascites fluid) has the most unusual behavior of these enzymes, because it strongly prefers structures with a Gal(P1-4)GlcNAc(Pl-6)Man sequence as occurs in tri'- and tetra-antennary oligosaccharides [ 541. Consequently, changes in the activities of GlcNAcT 111, IV and V, yielding a differently branched substrate population for a6-NeuAcT, a3-NeuAcT, a3-GalT, a3-FucT, and p3-GlcNAcT, by themselves will result in quantitative changes in the terminal structures on N-linked oligosaccharides. Increased GlcNAcT I11 activity will result in suppression of the elongation and termination reactions. Elevated activity of GlcNAcT IV will result in a shift from a6-sialylation to a3-sialylation [ 551 and a3-fucosylation (and consequently in increased expression of Lewis' and sialyl-Lewis", see later), whereas increased GlcNAcT V activity will promote polylactosaminoglycan formation and will further suppress a6-sialylation. Elevated activities of the branching GlcNAcTs (111, IV, and V) have been reported [56-601 or have been concluded [61] to occur in cases of malignant transformation. Such changes thus form one cause of the altered terminal
600
23 On the Origin of Oligosuccharide Species- Glycosyltrunsferases in Action
glycosylation of N-linked glycans which is often observed in malignant cells. These effects might be further amplified by changes in the activities of the glycosyltransferases acting on the lacNAc termini [ 581.
23.9 Branch Specificity The different IacNAc termini on branched N-linked oligosaccharides are not acted upon at the same rates by all enzymes. The preference of an enzyme for a lacNAc unit on one particular branch has been termed ‘branch specificity’. A summary of the branch specificities of four common glycosyltransferases acting on Gal(814)GlcNAcp termini is shown in Figure 8. In particular 1x6-NeuAcT shows a strong preference for the Gal( 1-4)GlcNAc( p 12)Man(al-3)Manp branch in di-, tri-, and tetra-antennary oligosaccharides [51, 62, 631. The Gal(pl-4)GlcNAc(pl-2)Man(al-6)Manp branch is a6-sialylated at least 10 times more slowly, whereas the Gal(~1-4)GlcNAc(~l-6)Man(ul-6)Man~ branch as occurs in tri’- and tetra-antennary oligosaccharides is very resistant to a6sialylation. Branch specificity, however, is only displayed by this sialyltransferase when the N-linked substrate contains at least one core GlcNAc residue and it has been proposed that the a6-NeuAcT recognizes this residue in a lectin like fashion aiding the correct positioning of the substrate on the enzyme [62]. By way of conbranch 4-5 trast u3-GalT prefers the Gal(~1-4)GlcNAc(~l-2)Man(al-6)Man~ A. a3-GalT
-D
a6-NeuAcT
-+
Ga Ipl-.4G I c N A c ~ l - D 2 M a n a l bvlanpl4GlcNAc~1-DR
r3 Gal~l-D4GlcNAc~l-r2Manal
B. 83-GlcNAcT
Gal/31-.4GlcNAcpl
-D
a3-GalT/p3-GlcNAcT
+
‘6 Gal~l-~4GlcNAc/31-~2Manal, \r
.36Man,814GlcNAc~l-4 a6-NeuAcT
-D
Gal~1-+4GlcNAc~l-D2Manal
u3-Neu AcT
-s
GaI/31-.4GlcNAc/31
r4
Figure 8. Branch specificities of Gal@-4)GlcNAc-specific enzymes with (A) an N-linked diantennary substrate and (B) a tetra-antennary substrate. The branches to which the enzymes preferentially introduce a sugar residue are indicated by 4. Note that a3-NeuAcT and P3-GlcNAcT do not show branch specificity with the di-antennary structure.
23.10 Essential Requirements for Activity with lacNAc
601
branch in a ditimes more than the Gal(~l-4)GlcNAc(~l-2)Man(al-3)Man~ antennary substrate [64]. The Gal(~1-4)GlcNAc(pl-2)Man(al-6)Man~branch seems to be even more preferred by a3-GalT when it is present in tri- and tetraantennary oligosaccharides [65]. Branch specificity is different yet again for a3NeuAcT (human placenta). Although it shows no preference for either branch in a di-antennary substrate [52] it prefers the Gal(P1-4)GlcNAc(Pl-4)Man(al-3)Man~ branch in tri- and tetra-antennary substrates although this sialyltransferase can also act on all other branches (M. Nemansky, C. H. Hokke and D. H. Van den Eijnden, unpublished results). The typical a3-/a6-sialylation patterns as occur on N-linked glycans [51] therefore seem to be the result of the differential branch specificities of both sialyltransferases. Like a3-NeuAcT, the P3-GlcNAcT does not show branch preference with di-antennary oligosaccharides [ 641. This enzyme, however, highly prefers the Gal( P 1-4)GlcNAc( pl-2)Man(wl-6)Manp and Gal(P1-4)GlcNAc( Pl 6)Man(al-6)Manp branches equally well to the other branches of tri’- and tetraantennary oligosaccharide structures [ 541.Because of the strongly increased activity of P3-GlcNAcT on such substrates (Figure 7) polylactosaminoglycan chain formation is highly favored on such oligosaccharides at the branches on the Man(a16)ManP unit. This further emphasizes the important role of GlcNAcT V (which initiates the formation of the Gal(P1-4)GlcNAc(~l-6)Man(al-6)Man~branch) in this process. -
23.10 Essential Requirements for Activity with LacNAc It will be clear from the previous paragraphs that the activity of the glycosyltransferases with each of the lacNAc units on branched N-linked oligosaccharide substrates can be strongly influenced by the structural environment (number and nature of neighboring branches, branch location) of the oligosaccharide substrate. In addition, studies with deoxygenated, substituted and otherwise modified IacNAc derivatives as acceptors for a6-NeuAcT, w3-NeuAcT (rat liver; preference for type 1 acceptors but also acting on type 2 structures; corresponds to ST3Gal I11 described below in Section 23.18, Siulyltransferase Family), a3-FucT and a3-GalT have revealed which modifications of the lacNAc acceptor site are allowed and which abolish the acceptor property for each of these enzymes [ 5 , 39, 66-72]. Obviously the OH group to which a sugar is to be attached by the glycosyltransferase is essential and should be unsubstituted. The further requirements for activity explain why some enzymes can still act after another glycosyltransferase has introduced a sugar to the lacNAc unit (e.g. a3-fucosylation is still possible after a3-sialylation), whereas other enzymes are mutually exclusive (e.g. 1x6-sialylation precludes a3fucosylation vice versa). On the other hand not all product structures of reactions that are possible in vitro are found in nature (e.g. no a6-sialylated form of the blood group H type 2 oligosaccharide has been reported even though a6-NeuAcT can act on Fuc(al-2)Gal(Pl-4)GlcNAc). A summary of the requirements for activity of these enzymes with lacNAc is given in Figure 9.
602
23 On the Origin of Oligosaccharide Species-Glycosyltvansferases in Action
no aGal
no Fuc
NHAc
fmo* m -
OH
OH
OH no Neu,Ac fFuc no atial or NHAc r10PGlcNAc
-
OH
OH
f Fuc
no Fuc no NHAc
a6-sialyltransferase
f Fuc
a3-sial yltransferase
Hoeo no SO; no Fuc
HO
fNeuAc
aGal
.O=OH
OH +Fuc
or N H A ~
NHAc
f
so;
f PGlcNAc
a3-fucosyltransferase
OH
f no Fuc no NHAc
a3-galactosyltransferase
Figure 9. Summary of the structural requirements of a6-NeuAcT, a3-NeuAcT, a3-FucT and a3GalT for activity with lacNAc and its derivatives. Essentially required OH groups are indicated by an arrow (site of sugar attachment) and by a circle. The latter OH groups are required for enzyme binding and catalysis (key polar groups). OH groups that can be deoxygenated without complete loss of activity but cannot be substituted by another sugar are indicated by a square. Other OH groups may be modified (deoxygenated, substituted, and/or replaced by an N-acetyl group) with only limited effect on the activity of the enzyme. All enzymes accept the replacement of the NHAc group of the GlcNAc residue by an OH moiety but are (much) less active with the resulting compound (lactose).
In particular a3-FucT seems to be the enzyme that is most easily satisfied, which explains why in metabolic routes a3-fucosylation is generally a late step. It clearly seems that each of the enzymes recognizes a lacNAc acceptor site in a unique way, suggesting that these enzymes do not share a common lacNAc recognition sequence motif. Indeed it has not yet been possible to identify such a motif in the deduced amino acid sequences of cloned lacNAc-specific enzymes.
23.11 Further Terminal Reactions in Complex-Type Oligosaccharide Synthesis In addition to the glycosyltransferases mentioned in the previous paragraphs several other enzymes are known that can act on lacNAc units but catalyze less common reactions. Among these are a p3-GlcAT [73], a 3’-sulfotransferase (to Gal) [74], and
23.12 Specijic ModGcations of Polylactosaminoglycans
603
a 6-sulfotransferase (to GlcNAc) [75], a P6-GlcNAcT [76], and a P3-GalNAcT [77] (Table 1). Furthermore, an a4-GalT acting on the lacNAc terminus of lacto-N-neotetraosylceramide [ 781 has been reported. Very recently a novel a4-GlcNAcT was cloned that preferentially acts on lacNAc present on O-linked core 2 [79]. After a6sialylation or a3-fucosylation of lacNAc generally no further modifications are possible. The only exception known so far is the action of schistosomal a2-fucosyltransferase on the Lewis’ structure to form Gal(P1-4)[Fuc(al-2)Fuc(al-3)]GlcNAcP-R (difucosyllacNAc) [ 801. Action of one of the other enzymes, however, often enables additional reactions to occur. This is particularly true of a2-fucosylation or a3-sialylation (Table I). The first reaction yields the H-type 2 antigen that not only can be converted into the LewisY determinant by the action of a3-FucT7 but also into the blood group A or B antigenic structures by the action of the Aenzyme (1x3-GalNAcT)and B-enzyme (a3-GalT), respectively [24, 8 11. a3-Sialylation in turn yields the structure that is intermediate in the synthesis of the sialylLewis’ determinant by a3-FucT action, or polysialic acid through the action of one of the two known polysialyltransferases [82], or the Sda antigen by the action of a P4-GalNAcT [83] (Table 1). For reasons of completeness the reactions catalyzed by three different sulfotransferases are also included in the figure. On the basis of the sequences of the two sulfotransferases of this group that have been cloned so far it seems that these enzymes have a domain structure that strongly resembles that of a Golgi glycosyltransferase [75, 841 (c.$ Figure 5).
23.12 Specific Modifications of Polylactosaminoglycans
The terminal lacNAc unit on polylactosaminoglycan chains can be capped like any other terminal lacNAc unit by the action of the glycosyltransferases described in the previous paragraphs, albeit that some of these enzymes act only at low rates on these glycans (e.g. a6-NeuAcT [55]).In addition to these reactions, however, several specific reactions can occur in which sugars are introduced to internal Gal or GlcNAc residues. Linear polylactosaminoglycan chains as synthesized by the concerted action of P3-GlcNAcT and B4-GalT (Figure 6) show blood group i-activity [24, 811. They may be converted to branched, blood group I-active structures by the action of a P6-GlcNAcT and subsequent B4-galactosylation yielding a Gal(P14)GlcNAc(al-3)[Gal( B1-4)GlcNAc( Pl-6)IGal branching point [85, 861. Two different type of branching P6-GlcNAcTs have been described, one of which acts only on penultimate Gal residues capped by a single B3-linked sugar (GlcNAc or Gal) (‘predistally acting’) [87, 881, the other acting on any Gal residue along the linear chain except the terminal or penultimate (‘centrally acting’) [89, 901. In a recent study, however, a recombinant B6-GlcNAcT was described which has both these activities [91]. Earlier a P6-GlcNAcT acting on terminal Gal residues (‘terminally acting’) has been reported [76]. A P3-GlcNAcT acting on resulting
Sda Lewis'
a-galactosyl-Lewisx
ti-type 2
HNK-1
Gal/31-.4[Fucal -ZFucul-.31GlcNAc/3-R
Galul-.3Ga1j1-.4[Fucu1-b3]GlcNAcj-R
Fucal-2Gal/31-4[Fucal-.31GlcNAc/3-R Fucul-.2 [GalNAcal -31Gala1-4G IcNAcB-R Fucal-.2[Galal-b3]Gal/3l-b4GlcNAc/3-R S04--3GlcA/31-3Gal/31+4G Ic NAcp-R S04--3Gal/31-.4IFucal-.31GlcNAc~-R (NeuAco2-3) ~Gal~1-.4[Fucal-.31[SO~-6]GlcNAc/3-R
? ? ?
7
Gal/314[Fucal-.3lGlcNAc/3-R
Galo1-.3Gal/31+4G IcN Acp-R
Fucul-2Gal/31-.4GlcNAc/3-R
GlcA/31-.BGal/31-.4GlcNAcf?-R
SO~-3Gal/31-.4GlcNAc/3-R
Gal/314[SO~-GIGlcNAc/3-R
GlcNAcfll -.GGal/31-4GlcNAc/3-R
GalNAc/31+3Ga1/31 -4GlcNAcB-R
Gala1-+4Gal/3l-b4GlcNAc/3-R
GIcNAcal--4Gal/31+4GlcNAc/3-R
LewisY blood group B
blood group A
PI
6-sulfo-(sialyl-)LewisX
3'-sulfo-Lewis'
-b
difucosyllacNAc
polysialic acid
NeuAca2-.3[GalNAc/31-.41Ga1/314GlcNAc/3-R
+
sialyl-Lewis'
NeuAca2-.3Gal/31-b4GlcNAc/3-R
(NeuAca2-.8),NeuAca2-3Gal/31-.4GlcNAc~-R
no further reaction
NeuAca2+6Gal/3l-b4GlcNAc/3-R
Name of (antigenic) structure
NeuAca2~3Gal/31-.4[Fucal-.31GlcNAc/3-R
Final structure
Primary product structure
Table 1. Modifications of lacNAc as shown by primary product structures and further terminal reactions in complex-type oligosaccharide synthesis. After the introduction of a sugar to lacNAc further additions are often, but not always, possible.
23.12 Specific Modijcations of Polylactosaminoylycans
605
-
G-GN-G-GN-G-GN LAG
a3-FT
G-GN-G-GNG-GN-
LeX
a3-ST
a3-FT
SA-GGN-G-GN-G-GN-
\
G-GN-G-GN-G-GNF F
, /
J’-sialyl-LAG,
/
i n t e T l dimeric-LeX a3-ST I
-
SAG-GN-G-G N-G-GN
k 7 F 4
-
G-GN-G-GN-G-GN F F F
sialyl-LeX
F
L
.$
-
SA-G-G N-G -GN-G-GN F F
frimeric-LeX
VIM-2/internal dimeric-LeX
/
\ a3-FT
4
\r SA-G-GN-G-GN-G-GN F F F
-
sialyl-trimeric-Le“
Figure 10. Cooperative action of a3-NeuAcT (a3-ST, dashed arrow), a3-FucT IV (myeloid enzyme; u3-FT, thin solid arrow), and a3-FucT VII (w3-FT, leukocyte enzyme; bold solid arrow) in the synthesis of sialyl-oligomericLewisXand related structures on polylactosaminoglycan chains. F, Fuc; G, Gal; GN, GlcNAc; LAG, polylactosaminoglycan; SA, NeuAc.
GlcNAc(p1-6)Galpl-R to yield a GlcNAc(al-3)[GlcNAc( p1-6)]GalpI-R branching point, however, has not yet been described. (See also Section 23.23, p6-Nacetylylucosaminyltransferase family). Another specific modification of polylactosaminoglycan chains is the introduction of a3-Fuc residues to non-terminal GlcNAcs along the chain to yield a (sialyl-) oligomeric-Lewis” structure. In leukocytes the formation of this structure, which is a ligand for E- and P-selectin, is catalyzed by two different a3-FucTs, the myeloid FucT (FucT IV) and the leukocyte FucT (FucT VII) [92, 931 (Figure 10) (see also Section 23.20, a3/4-Fucosyltransferase Family below). p-GlcNAc cannot be introduced to position C-6 of Gal residues that form part of the Lewis’ structure [90]. In turn, as the OH at C-6 of Gal in lacNAc is a key polar group for a3-FucTs [ 691, a3-fucosylation of GlcNAc residues underlying blood group 1 branching points is impossible. It thus seems that the formation of oligomeric LewisX and of blood group I branching points are competing processes. Branched polylactosaminoglycans can, therefore, only carry LewisXgroups at lacNAc units that are not engaged in a branch [94]. Similar to the P6-GlcNAcTs, different a3-FucTs show differential preferences for the penultimate and internal acceptor sites along the polylactosaminoglycan acceptor chain (“site specificity”). This specificity is further detailed under a3/4-Fucosyltransferase family described below.
606
23 On the Origin of Oligosaccharide Species-Glycosyltransferases in Action
23.13 The Invariable Core of N-linked Oligosaccharide Chains, and Site- and Protein-Specific Processing
Mature N-linked oligosaccharides all have the same basic core consisting of three Man and two GlcNAc residues. This core structure is already present in the preformed dolicholLPP-linked oligosaccharide precursor and remains untouched throughout the entire processing after its en bloc transfer to the polypeptide chain (Figure 2). Nevertheless, the oligosaccharide chains on different glycoproteins synthesized in one particular tissue or cell type are often structurally different. Apparently, the protein-linked chains can be processed to different extents. When, for some reason, GlcNAcT I fails to act or when not all a2-linked Man residues are removed by a-mannosidase IA or IB, the oligosaccharide will remain as an oligomannosidic-type structure. Such a chain can occur at one Asn of a polypeptide even though the oligosaccharides at other Asn residues of the same glycoprotein have been processed to complex-type structures. Many examples of glycoproteins with this characteristic are known (e.g. tissue plasminogen activator [95]; glycodelin [96]). A differential accessibility of the various oligosaccharide chains to the processing enzymes as dictated by the polypeptide environment of the individual chains has been proposed to account for this feature [97]. However, the variations in structure at a single glycosylation site (microheterogeneity) rather seem to be determined stochastically. In other instances the N-linked oligosaccharides at different Asn residues differ in their amount of branching (e.g. human a1-acid glycoprotein [98]) or in the presence or absence of a bisecting GlcNAc residue (human myeloma IgGl [99]). A hypothetical explanation of the latter differences might be that the polypeptide can interact with the oligosaccharide in a way that it is locked in a conformational state that is not acted upon by certain GlcNAcTs (c.J: [991). Finally, two classes of glycoprotein have been described the N-linked chains of which have a specific character because of the recognition of a peptide motif by a sugar-transferring enzyme. The first class is formed by the lysosomal enzymes. When aZlinked Man residues are still present in the oligosaccharide the structure is phosphorylated by the sequential action of an N-acetylglucosamine phosphotransferase and a phosphodiesterase resulting in the formation of Man 6-P determinants [loo] (Figure 2). This structural element mediates the targeting of the lysosomal enzyme to the lysosome through its interaction with one of the two Man 6-P receptors [ 1001. Only lysosomal enzymes become phosphorylated because the N-acetylglucosamine phosphotransferase specifically recognizes a peptide sequence that is uniquely present in these enzymes. The second class comprises the pituitary glycohormones which are rich in complex-type glycans based on GalNAc(014)GlcNAc (lacdiNAc) rather than Gal(fl1-4)GlcNAc (lacNAc). It has been proposed that the pathway is directed toward the synthesis of such oligosaccharide chains by a hormone-specific N-acetylgalactosaminyltransferase that recognizes a peptide determinant present in the polypeptide of these hormones [ 1011.
23.15 The IacdiNAc Pathway of Complex-Type Oligosaccharide Synthesis
601
23.14 Synthesis of Type 1 (Gal(p1-3)GlcNAcp-R) versus Type 2 (Gal(p1-4)GlcNAcP-R) Chains Most complex-type chains are based on lacNAc and are referred to as type 2 chains. In some instances, however, protein-linked chains are based on lacto-N-biose (Gal(pl-3)GlcNAc) and are referred to as type-1 chains. Notable examples of glycoproteins carrying such chains are bovine fetuin [ 1021 and rat a1-acid glycoprotein [ 1031. Although glycoproteins with this type of chain are rather rare, a major fraction of human milk oligosaccharides is based on the type-1 chain compound lacto[104]. For a long time the only N-tetraose (Gal(~1-3)GlcNAc(a1-3)Gal(~1-4)Glc) p3-GalT involved in the synthesis of type-1 chains was that described in porcine trachea [105]; this activity was not reported in tissues such as mammary gland. Recently the cloning of four human GlcNAcp-R-specific p3-GalTs [ 106-1091 and the mouse orthologs of three of these [110] has been reported. Indeed these 03GalTs seem to have a rather restricted tissue expression. Like lacNAc termini, type-1 structures may be fucosylated by a2-FucT and a4FucT (Lewis enzyme, FucT 111) to yield the H-type-l and the Lewisa structures, respectively [24, 811. In turn the H-type-1 structure can be converted into a Lewisb, or a blood group A or B active compound, by the action of a4-FucT, and blood group A- (a3-GalNAcT) or B-enzyme (a3-GalT), respectively [24, 8 I]. Alternatively, type-1 structures can be disialylated by the concerted action of a a3-NeuAcT and a a6-NeuAcT to yield the terminal NeuAc(a2-3)Gal(pl-3)[ NeuAc(a2-6)]GlcNAc structure [ 111, 1 121.
23.15 The LacdiNAc Pathway of Complex-Type Oligosaccharide Synthesis As another alternative to type-2 chain complex-type oligosaccharide formation a GalNAc can be introduced to accepting GlcNAc residues yielding a GalNAc(p14)GlcNAc (lacdiNAc) unit [40]. In particular bovine milk glycoproteins carry oligosaccharide chains which are rich in such units, but they also occur on many other glycoproteins including several of human origin (reviewed in Ref. [40]). As mentioned above it has been proposed that a glycoprotein hormone-specific 84GalNAcT exists which specifically recognizes a peptide determinant present in the polypeptide chain of these glycoproteins [ 1011. In schistosomes [ 113, 1141, snail [115], and bovine mammary gland [116], however, a P4-GalNAcT has been described which is hormone-non-specific and strongly resembles P4-GalT in acceptor properties. It has been proposed that this 04-GalNAcT controls the lacdiNAc pathway of complex-type oligosaccharide synthesis [40]. Several enzymes that act on lacNAc can also act on lacdiNAc, in conformity with their essential requirements for activity described above. a6-NeuAcT and a3-
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23 On the Origin of Oligosaccharide Species- Glycosyltransferases in Action
FucT can catalyze the formation of NeuAc(a2-6)GalNAc( 01-4)GlcNAc [66] and GalNAc(~l-4)[Fuc(al-3)]GlcNAc (lacdiNAc variant of Lewis") [67], respectively. By way of contrast a3-NeuAcT and a3-GalT do not act on GalNAc(P1-4)GlcNAc [ 1171 and P3-GlcNAcT only acts with very low activity [40]. Furthermore a sulfotransferase catalyzing the transfer of sulfate to OH-4 of GalNAc has been identified [118], as has a p3-GalT catalyzing the formation of Gal~l-GalNAc(~l-4)G1cNAc [119].
23.16 Protein 0-Glycosylation In contrast with N-glycosylation, formation of 0-linked chains as occurs on mucins is initiated by the addition of a single a-GalNAc residue (rather than of a preformed oligosaccharide) to the OH-group of a Ser or Thr in the polypeptide chain. It is believed that this step occurs in the cis-Golgi [22].Additions to this GalNAc residue of Gal in (P1-3)-, GlcNAc in (pl-3)- and (p1-6)-, and GalNAc in (al-3)-linkage might subsequently occur in the medial-Golgi which leads to different core structures (Figure 11). These reactions are all catalyzed by glycosyltransferases specific for 0-linked chains. Also, direct addition of NeuAc to GalNAc and to the core 1 disaccharide (Gal@-3)GalNAc) can occur as catalyzed by NeuAcTs that are highly specific for these 0-linked sugars (Figure 11, right). In the trans-Golgi terminal GlcNAc residues on the different core structures can be further acted upon by P4-GalT yielding 0-linked complex-type chains. Polylactosaminoglycans can also be formed on these chains by the concerted action of P3-GlcNAcT and P4-GalT. The chains can be completed by addition of NeuAc, Fuc and sulfate residues as catalyzed by the same sialyltransferases, fucosyltransferases, and sulfotransferases that act in the synthesis of N-linked chains. Although not all glycosyltransferases acting on lacNAc in Nlinked chains (see Figure 6) act with the same efficiency on lacNAc in O-linked chains (e.g. a6-NeuAcT does not act well on Gal(~1-4)GlcNAc(~1-6)[Gal(~l-3)]GalNAca-0-R) elongation and termination of the complex-type 0-linked structures follows in principle the same pathways as for N-linked chains and are catalyzed by the same enzymes that act in the terminal stages of N-glycosylation. Thus on 0-linked chains blood group determinants such as (sialyl-)Lewis",H(O), A, B, Sda, i, I, and type-1 chain-based H, Lewisa and Lewisb can be formed in an analogous way [22].
23.17 Glycosyltransferase Families Before the days of molecular cloning of glycosyltransferases the classification of these enzymes was based on common nucleotide-sugar donor substrate usage with sub-classification based on the linkage formed and the acceptor sugar used.
23.17 Glycosyltransferase Families
609
GOLGI MEDIAL GalNAca-3GalNAca-R core 5
GlcNAc p-3GalNAca-R core 3
CIS
TRANS
R (= Ser/Thr-polypeptide)
Gal p-3GalNAca-RF core 1
Galp-3GalNAca-R
GlcNAcp-3GalNAca-R core 4
NeuAc a-3Galp-3GalNAca-R
GlcNAcp 6 GlcNAcp-3Galp-3GalNAca-R
6 GlcNAcp-3GalP-3GalNAca-R
GlcNAcp 6 NeuAc a-3Galp-3GalNAca-R
Figure 11. Major pathways in 0-linked o~igosdcchdridesynthesis. The reactions leading to the sialylated and the different core structures are depicted. Once a GlcNAc residue is attached it can be acted upon by P4-GalT to form a lacNAc unit, which can be further modified as outlined in Figures 6 and 10, and Table 1.
Thus for a long time families of sialyltransferases, fucosyltransferases, galactosyltransferases, etc. have been distinguished. During the past 14 years the cloning of the cDNAs of over 50 glycosyltransferases in eukaryotes has been achieved, (A glance at the web site www.vei.co.uk/tgn/gt-guide.htm.gives an impression of the vast number of glycosyltransferases that have been cloned. Note, however, that the list has not been updated recently.) The structural information obtained not only led to the insight that all of these enzymes share a common domain structure (Figure 5), but also has provided a rationale for the aforementioned classification system. On the basis of sequence similarities at least 11 different glycosyltransferase families can now be identified (Table 2). Members within a family have extensive sequence identity at the amino acid level, which is often confined to specific boxes, and have one or more enzymatic properties in common. In the following paragraphs these glycosyltransferase families are discussed in more detail.
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23 On the Origin of Oligosaccharide Species-Glycosyltransferases in Action
Table 2. Glycosyltransferase families. Transferdse family
Linkage-type established
Sialyl
17 a3-/a6-/a8-NeuAc to Gal(NAc)/NeuAc 2 a2-Fuc to Gal a 3 - l a 4 - F ~to Glc(NAc) 6
a2-Fucosyl a3/4-Fucosyl
Number of Notes enzymes with sequence similarity
a3-Galactosyl/Na3-Gal(NAc) to Acetylgalactosaminyl Gal(NAc)
4
p4-NAcetylglucosaminyl
P4-GlcNAc to Man
2
P6-NAcetylglucosaminyl
P6-GlcNAc to Gal(NAc)
3
a-GalNAc to Ser/Thr a-NAcetylgalactosaminyl P4-GalNAc to P4-NAcetylgalactosaminyl [NeuAca3]Gal fi4-Galactosyl P4-Gal/p4-GlcNAc to Glc(NAc)/Xyl
7
P3-Galactosyl
P3-Gal to GlcNAc or GalNAc
5
P3-Glucuronyl
P3-GlcA to Gal
3
2
8
All contain sialyl motif L and S H- and Se-enzyme FucT 111-VII and IX; a3-fucosyl motif A- and (pseudo) Benzyme, Forssman glycolipid synthase GlcNAcT IV is the only N-glycan branching GlcNAcT known so far that is not a “stand alone” 0-linked core 2 and 4 synthases and Ienzyme (GlcNAcT-V is NOT a member) Enzymes have different peptide specificities Sda- and GM2-synthase a-lactalbumin-sensitive and insensitive p4GalTs and chitobiosynthase; all share several P4-galactosyl motifs Type 1 chain synthases and ganglioside GM I -synthase (Core 1 synthase is NOT a member) Involved in HNK-1 epitope and proteoglycan core synthesis
23.18 Sialyltransferase Family All members of the sialyltransferase family share a highly conserved so called ‘sialyl motif’ (designated L for large) in their primary amino acid sequence; this motif has been found to participate in the binding of the common donor substrate CMPNeuAc [120]. In addition to this sialyl motif L a smaller more C-terminally located
23.19 a2-Fucosyltransferase Family
6 11
common sequence motif has been found; this is designated sialyl motif S (S for small) [121]. Once these motifs had been identified they were exploited to clone novel sialyltransferases by homology and today the cDNAs of seventeen of such enzymes have been identified (reviewed in Refs. [ 1221 and [ 1231). A unique feature of this family is that it comprises enzymes that establish different linkages ((a2-3)-, (a2-6)- or (a2-8)-) and use different acceptor sugars (Gal, GalNAc, or NeuAc). This is quite different from, for instance, the galactosyltransferases where enzymes with different linkage and acceptor specificity have very little sequence similarity and consequently are grouped in separate families (see below). Members of the sialyltransferase family are now commonly sub-classified on the basis of the linkage they establish and the accepting sugar [123]. There are six a3NeuAcTs that act on terminal Gal residues (ST3Gal I-VI). Best substrate structures for these enzymes are ST3Gal I, Gal(f3-3)GalNAca (protein linked); ST3Gal 11, Gal(plL3)GalNAcp (lipid linked); ST3Gal 111, Gal(p1-3)GlcNAcp; ST3Gal IV, Gal(~1-4)GlcNAc~ (protein linked); ST3Gal V, Gal(P1-4)Glcpl-Cer, and ST3Gal VI, Gal(~1-4)GlcNAc~ (lipid or protein linked), respectively [ 122, 124-1261. Only one a6-NeuAcT occurs (ST6Gal I); this specifically acts on Galp-4GlcNAcp (Fig. 6, Table 1) and GalNAcp-4GlcNAcp [66] but not on type 1 chains or O-linked aGalNAc and core 1. Five a6-NeuAcTs (ST6GalNAc I-V) have been cloned that use a GalNAc residue in different structures as accepting sugar (see also Fig. 11). ST6GalNAc I acts on O-linked GalNAca-, Gal(pl-3)GalNAca, and NeuAc(a23)Gal@-3)GalNAca- but requires a protein aglycone; ST6GalNAc I1 acts on Galpl-3GalNAca, and NeuAca(2-3)Galp1-3GalNAca- but not on GalNAca; ST6GalNAc 111, -1V and -V all three act on O-linked as well as lipid linked NeuAca(2-3)Gal(Pl-3)GalNAca/p [ 122, 127-129, 1891. The latter three enzymes tolerate aromatic aglycons, but do not act on non-sialylated substrate structures. ST6GalNAc I11 has no pronounced preference for either acceptor structure, but ST6GalNAc IV prefers O-linked acceptor chains, while ST6GaINAc V acts with preference on lipid linked substrates. In contrast to ST6GalNAc I-IV, which seem to be involved in the sialylation of O-glycans, ST6GalNAc V has been suggested to function in the synthesis of ganglioside GD1,. Five a8-NeuAcTs (ST8Sia I-V) are known of which ST8Sia I1 and IV are involved in the synthesis of protein-linked polysialic acid chains, whereas ST8Sia I, I11 and V seem to function in the synthesis of NeuAc(a2-8)NeuAca and NeuAc(a2-8)NeuAc(a2-8)NeuAca sequences occurring on gangliosides such as GD3, GTl,, GT3, and GQlb [122].
23.19 a2-Fucosyltransferase Family Two human a2-FucTs have been cloned [ 130, 1311. One enzyme, occurring in serum and plasma, is the product of the H gene and controls the synthesis of the blood group H-antigen (Fuc(al-2)Galp), which is the precursor of the A- and B-antigens, on erythrocytes. The other a2-FucT occurs in milk, saliva, and other secretions, and is the product of the Secretor (Se) gene. It determines the expression of soluble H(and A- and B-) antigen in secretions and the Lewisb (Fuc(al-2)Gal(~1-3)[Fuc(al4)IGlcNAc) blood group antigens on red cells [24, 811. The enzymes have 68%
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23 On the Origin of Oligosuccharide Species-Giycosyltrunsferuses in Action
identity at the amino acid level. They both catalyze the formation of a Fuc(u12)Galp linkage, but have different acceptor specificities. Whereas the H enzyme highly prefers type-2 chain acceptors and efficiently acts on Calp-phenyl, the secretor enzyme has a preference for type-1 chain-based acceptors and for Gal(p13)GalNAc [ 131- 1331. The latter enzyme also has much lower affinity for the donor substrate GDP-Fuc than the H enzyme [131, 1331. In rabbit three a2-FucTs occur; one resembles the human H enzyme in molecular and enzymatic properties, the others are molecularly similar to, and have the enzymatic characteristics of, the secretor enzyme [ 1341.
23.20 a3/4-Fucosyltransferase Family Within the a3/4-fucosyltransferase family six different members have been identified. They are numbered in the order they were cloned FucT 111-VII and IX. Human FucT 111 (Lewis enzyme), V and VI (liver/plasma enzyme) are highly related enzymes with >90% identity at the amino acid level in their catalytic domain; their genes are all located on human chromosome 19 [135]. By contrast, FucT IV (myeloid enzyme, chromosome 11) and FucT VII (leukocyte enzyme, chromosome 9) have a more distal relationship to each other and to the other members of this family [135, 1361. Recently a novel member, FucT IX, which has highest homology to FucT IV, was cloned from a mouse library [ 1371. Recently the human ortholog of FucT IX was described [ 1381. A highly conserved common motif consisting of some 10-15 amino acids in the primary structures of these enzymes could be identified upon the cloning of a bacterial (Helicobacter pylori) a3-FucT [ 139, 1401. The members of this transferase family all utilize GDP-Fuc as a common donor but it remains to be established whether this ‘u3-fucosyl motif’ is somehow involved in the binding of this nucleotide-sugar. All members of this family use a substituted GlcNAc or Glc as accepting sugar and all but one establish a Fuc(al-3)-linkage. The exception is formed by FucT I11 which preferentially acts on type 1 acceptors yielding the Lewisa structure in which the Fuc is linked (al-4) (Gal(~1-3)[Fuc(al-4)]GlcNAc), whereas the other a3-FucTs exclusively act on type-2 chain-based acceptors yielding LewisX blood group determinant-based structures. FucT I11 can, however, also support the synthesis of LewisXby establishing a Fuc(ul-3)-linkage both in vitro and in vivo [2-41. This enzyme is still the only exception to the one linkage-one enzyme concept mentioned in the introduction. Recently it was reported that a single amino acid in the hypervariable stem domain of FucT I11 determines the preference of this enzyme for type-1 structures [ 1411. Preferred substrate structures for the members of this family are: FucT 111, Gal@-3)GlcNAcp-R; FucT IV, Gal(p1-4)GlcNAcpR; FucT V and VI, both Gal(P1-4)GlcNAcP-R and NeuAc(a2-3)Gal(p1-4)GlcNAcp-R; FucT VII, NeuAc(a2-3)Gal( p 1-4)GlcNAcp-R, and FucT IX, Gal(p1-4)GlcNAcp-R, respectively [69, 92, 93, 137, 1421. FucT 111-VI also can act with varying efficiencies on lactose (Gal(p1-4)Glc) and H-type-2 acceptors such as Fuc(al-2)Gal( p1-4)Glc(NAcp-R). With oligomers of lacNAc (polylactosaminoglycans), wherein Fuc may be introduced to penultimate and to internal GlcNAc
23.22 ~6-N-Acetyl~lucosuminyltvclnsferuse Family
6 13
residues, a3-FucTs show “site specificity”. Whereas FucT IV highly prefers internal GlcNAc residues, FucT 1X acts preferentially on the penultimate GlcNAc [92, 1901. FucT VII acts specifically on penultimate GlcNAc residues too, but requires that the lacNAc unit comprising this GlcNAc carries a terminal (a2-3)-linked NeuAc residue [92, 931 (Fig. 10). By contrast FucT VI (from human milk) can introduce an a3-Fuc to any GlcNAc in such acceptors whether the chain is a3-sialylated or not [143, 1441.
23.21 a3-Galactosyl/N-Acetylgalactosaminyltransferase (Histo-Blood Group ABO) Family This family, consisting of four members, is heterogeneous in that two member enzymes preferentially utilize UDP-Gal whereas the two others use UDP-GalNAc. Furthermore, one GalNAcT (blood group A transferase) and one GalT (blood group B transferase) act exclusively on substrates with Fuc(a1-2)GalP- termini (blood group H structure) to yield the blood group A and B epitopes, respectively [7, 1451 (Table 1). By contrast the other GalT (‘pseudo B-enzyme’, for which the enzymatic properties of a3-GalT have been described in detail above) [146, 1471 acts exclusively on non-fucosylated structures, preferentially on Gal( pl-4)GlcNAc, whereas the other GalNAcT acts on P-linked GalNAc in globoside (GalNAc(P13)Gal(al-4)Gal(~l-4)GlcPl-Cer) to yield the Forssman glycolipid (GalNAc(a13)GalNAc(p1-3)Gal(al-4)Gal( B1-4)Glcpl -Cer) [ 1481. The single common enzymatic property of these enzymes is that they all establish a (al-3)-linkage. Nevertheless the primary structures of these enzymes have extensive sequence similarities. This is particularly true for the human blood group A and B transferases which differ in four amino acids only [7]. It has long been known that for these enzymes sugar-donor promiscuity is relatively high [ 1491. By mutating each of the four amino acids in these blood group transferases it has been shown that two of these amino acids are crucial in determining the nucleotide-sugar specificity [7].
23.22 P4-N-Acetylglucosaminyltransferase(IV)Family The individual GlcNAcTs involved in the branching of N-linked glycans (Fig. 4) do not generally seem to belong to a glycosyltransferase family (see Glycosyltransferuses standing alone below). An exception is human GlcNAcT IV that has been described to occur in two isoforms, which show 62% identity at the amino acid level [191]. So far it is not clear whether these two forms have different specificities.
23.23 P6-N-AcetylglucosaminyltransferaseFamily This family comprises three members; all occur in man. The enzyme that was cloned first was identified as an 0-linked core 2 synthase [150] ( c . j Figure 11). It
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23 On the Origin of Oligosaccharide Species-Glycosyltransferases in Action
acts on Gal(pl-3)GalNAca-pNP, but not on GlcNAc(p1-3)GalNAca-pNP or structures that form part of linear polylactosaminoglycans [91, 1511. A structurally related glycosyltransferase has been shown to control the synthesis of blood group I active branching points in polylactosaminoglycans [ 1521. In vitro this enzyme has been reported to act on acceptors that form part of linear polylactosaminoglycans, either as a centrally acting p6-GlcNAcT [90] or a centrally and predistally acting enzyme [91], but not on Gal(p1-3)GalNAca-pNP or GlcNAc(P1-3)GalNAcapNP. A third member of this family was found to act preferentially on Gal(p1-3)GalNAca-pNP, but also on GlcNAc(pl-3)GalNAca-pNP and thus also has 0linked core 4 synthase activity [91, 1511. A mouse homolog of the human core 2 synthase seems to be involved in the synthesis of a globo-type sphingoglycolipid core structure (GlcNAc(pl-6)[Gal(pl3)]GalNAc(~l-3)Gal(a1-4)Gal(~1-4)Glc~l-Cer) [ 1531, suggesting that core 2 synthase might have activity with acceptors having Gal(p1-3)GalNAca or Gal(p13)GalNAcp termini. This family might contain additional, not yet cloned enzymes which act with preference on penultimate (predistal) or terminal Gal residues (see also Section 23.12, Specific Modzjications of’Polylactosaminoyl~~cans).
23.24 Polypeptide N-Acetylgalactosaminyltransferase Family This family contains seven members cloned from human, rat, mouse, or bovine libraries [ 149-155, 192-1941. These enzymes (ppGalNAcTs), which catalyze the initiating step in 0-glycosylation (c.f Figure I 1), have overlapping yet distinctly different peptide-acceptor specificities [ 1 1, 156- 1581. With most peptide substrates ppGalNAcT 1 seems to be the most potent enzyme in terms of rate. Different relative rates, however, have been found for these enzyme with various peptides and some peptides are acted upon by one enzyme but not by others, and vice versa. In general all enzymes seem to prefer Thr residues to Ser in most peptide substrates. ppGalNAcT 1 and 2 introduce GalNAc residues to two Thr and one Ser position in a peptide (DTRPAPGSTAPPAHGVTSAP, points of attachment in italics) which is a repeat of MUCI. Rates for ppGalNAcT 1-3 are, however, different for each of these attachment sites [ 111. With another peptide substrate (PTTTPLK, repeat of MUC2) it seemed that the number of GalNAc residues that can be incorporated by ppGalNAcT 1-3 varies from one to three and that the order of attachment is also different for each enzyme [159]. ppGalNAcT 4 and ppGalNAcT 5 seem to act on a more limited range of peptides than ppGalNAcT 1-3 [157]. On the other hand ppGalNAcT 4 seems to complement ppGalNAcT 1-3 in that it acts on a Thr and a Ser site in TAPPAHGVTSAPDTRPAPGSTAPPA (repeat of MUC1) [ 1581. ppGalNAcT 6 resembles ppGalNAcT 3 in acceptor preferences [ 1921. Interestingly, ppGalNAcT 7 shows exclusive specificity for partially GalNAc glycosylated substrate peptides resulting from prior action of other ppGalNAcTs [ 193, 1941. This shows that the initiating steps in 0-glycosylation occur in a coordinate fashion by different ppGalNAcTs. So far all ppGalNAcTs but one are specific for UDP-GalNAc as a donor substrate. Only ppGalNAcT 2 can also efficiently utilize UDP-Gal [ 1 11. Although the
23.25 ~4-Gal~ctosyltransjerase Family
6 15
overall sequence similarity of the enzymes is ca 45% [155], all contain a segment of 41 amino acids that is highly conserved in all ppGalNAcTs [160]. Interestingly, a box within this segment shows strong similarity with a conserved box found in the P4-galactosyltransferase family. It has been suggested that this segment is involved in UDP-sugar binding [ 1601.
23.25 P4-N-AcetylgalactosaminyltransferaseFamily Of this family one human and one murine member have been identified [83, 1611. Little information is available on the specificities of the recombinant forms of these GalNAcTs, but it has been demonstrated that the murine enzyme acts on 3'sialyllacNAc (but not on lacNAc) to yield the Sd" blood group-active structure GalNAc(P1-4)[NeuAc(u2-3)]Gal( pl-4)GlcNAc [83]. Transfection studies using the cDNA of the other enzyme showed that the human GalNAcT can support the synthesis of ganglioside GM2 from GM3, and GD2 from GD3 [161].Whether these enzymes have overlapping acceptor specificities is not known, but they share the common property of acting on a Gal residue bearing a sialic acid at its OH-3 and establishing a GalNAc(P1-4)[ NeuAc(tr2-3)IGal linkage.
23.26 P4-Galactosyltransferase Family This family consists of eight enzymes, seven vertebrate and one molluscan. All establish a (01-4) linkage and although the sequence similarity among them might be lower than 40%, so far all vertebrate members are true P4-GalTs acting on GlcNAc, Glc, or Xyl. The numbering of the P4-GalTs has been confusing. Originally they were numbered in order of their cloning, the system commonly used in other glycosyltransferase families (e.g. sialyltransferases and a3-fucosyltransferases). Nowadays they are numbered according to their similarity to P4-GalT I [162], the first mammalian glycosyltransferase ever cloned [ 163-1 651. Of all members P4-GalT I has by far the highest intrinsic activity. It can act on virtually any terminally exposed P-GlcNAc to form a Gal( plL4)GlcNAc unit (see also Section 23.5, Committed Steps in the Formation of Complex-Type Oligosaccharide Chains and Branching). Free GlcNAc and P-GlcNAc linked to an aromatic or fatty aglycone are excellent acceptors [166, 1671. Also GlcNAc in oligosaccharides, whether related to N-protein (GlcNAc linked to Man) or 0-protein (GlcNAc linked to GalNAc) linked glycans, or contained in a polylactosaminoglycan chain (GlcNAc linked to Gal), is an efficient acceptor site for P4-GalT I [38, 39, 85, 167-1691. P4-GalT I can, furthermore, act on GlcNAc(P1-3)Gal(~l4)GlcPl-Cer to yield Gal(~1-4)GlcNAc(~1-3)Gal(~l-4)Glc~l-Cer (paragloboside) [166, 1671. P4-GalT I thus can function in N-protein, 0-protein, and lipid glycosylation. For over 30 years it has been known that in the lactating mammary gland this enzyme can combine with the milk protein a-lactalbumin (a-LA) to form the lactose
6 16
23 On the Origin of Oligosaccharide Species- Glycosyltransferases in Action
synthase complex [170, 1711. a-LA stimulates (34-GalT I to act on Glc yielding lactose, at the same time inhibiting the action on GlcNAc. This enzyme, therefore, also serves a specific function in lactose synthesis. (34-GalT I1 and (34-GalT I11 have acceptor properties which resemble those of (34-GalT I but act much less efficiently [166]. Whereas (34-GalT I1 is similarly responsive to a-LA as (34-GalT I, (34-GalT I11 is not, however, induced by a-LA to act on Glc, and activity toward GlcNAc is not inhibited by this modifier protein [166]. (34-GalT IV is a poorly acting enzyme and no activity could be detected with glycoprotein acceptors [ 1721. It has, however, notable activity with glycolipid acceptors and can support the synthesis of paragloboside. a-LA does not significantly induce the enzyme to act on Glc, but rather stimulates activity toward free GlcNAc [172]. (34-GalT V, like (34-GalT I, can act on a variety of acceptors albeit at a relatively low rate [167, 1731. The enzyme is not induced by a-LA to act on Glc, but a-LA inhibits its action on free GlcNAc. The highest activity is shown toward acceptors containing a GlcNAc((3 1-6)GalNAca structural element, suggesting that this enzyme predominantly functions in the galactosylation of O-linked core 2- and core 6based structures [167]. A similar function has been ascribed to P4-GalT IV [174], but this contradictory finding is probably a consequence of erroneous numbering of the clones resulting from the confusion in the numbering system mentioned above. (34-GalT VI has been identified as a lactosylceramide synthase [ 1751. Whether it acts on substrates other than glucosylceramide has not been determined. Finally, the (34-GalT involved in the first step of the synthesis of the GlcA(P1-3)Gal((313)Gal((31-4)Xyl core region of proteoglycans has recently been cloned and seems to be another member of this family 1176, 1951. It is proposed to refer to this enzyme as (34-GalTVII. The only member of this family found in invertebrates has acceptor specificity resembling that of (34-GalT I. In stead of Gal, however, it transfers GlcNAc from the corresponding nucleotide sugar to form a GlcNAc((31-4)GlcNAc ( N ,N’diacetylchitobiose) unit and therefore is a (34-GlcNAcT [177, 1781. It has no structural relationship to any other GlcNAcT yet cloned. The enzyme is insensitive to aLA. A mutant of this (34-GlcNAcT, in which two out of three amino acid repeats are removed, shows acquired donor promiscuity toward UDP-GalNAc coupled to an overall higher kinetic efficiency [9]. Donor promiscuity toward UDP-GalNAc is also shown by bovine milk (34-GalT ((34-GalT I) [8]. An interesting common property of (34-GalT I, (34-GalT V, and (34-GlcNAcT is that their activity is highest with acceptors containing a ((31-6)-linked GlcNAc [85, 167, 173, 1781. With branched acceptors such as GlcNAc((3l-6)[GlcNAc((3l-3)]Gal ((34-GalT I [85], P4-GlcNAcT [ 1781) and GlcNAc((3l-6)[GlcNAc((31-2)]Man(al-6)Man(3-R ((34-GalT I 11691) they act with preference on the ((31-6)-linked GlcNAc. Despite the small amount of overall sequence similarity, several stretches of amino acids have been highly conserved in all members (‘P4-galactosyl motifs’) 1172, 175, 1791. Interestingly one conserved stretch is very similar to a box conserved in the sequences of the pp-GalNAcTs [ 1601. The amino acids in this box might, therefore, be involved in UDP-sugar binding, but apparently do not determine donor sugar specificity. It should, furthermore, be noted that there is very little sequence similarity between members of the a3-galactosyltransferase, (34-
23.28 Glycosyltransferases Standing Alone
6 17
galactosyltransferase, and 83-galactosyltransferase families and hence it seems that UDP-Gal is recognized in different ways within each of these families. Potential other members of the P4-galactosyltransferase family are the 04GalNAcTs that occur in snail [180] and bovine mammary gland [116]. These enzymes not only strikingly resemble P4-GalT I in acceptor requirements, but also are both induced by a-LA to act on Glc yielding the lactose analog GalNAc(pl-4)Glc, suggesting that they are molecularly related to the P4-GalTs. Attempts to clone the P4-GalNAcT have, however, failed so far.
23.27 P3-Galactosyltransferase Family This family comprises five different enzymes; all utilize UDP-Gal as donor substrate and all establish a Gal(PlL3)-linkage [ 106-1 101. Whereas four enzymes (P3-GalT 1-111 and V) act on P-linked GlcNAc yielding a type-1 disaccharide unit, P3-GalT IV acts on P-linked GalNAc as in ganglioside GM2 and thus rather functions in ganglioside biosynthesis. Only slight differences in acceptor specificities have so far been observed for the type-1 chain synthases, but it has been suggested that P3-GalT V is specifically involved in elongation of the O-linked core 3 [109]. It has been anticipated that the 03-GalT responsible for the synthesis of O-linked core 2 (Gal(~l-3)GalNAca-Ser/Thr) (Fig. 11) was also a member of this family. The recent cloning of this enzyme, however, revealed that it shows no homology with any other galactosyltransferase in the databases [ 1961.
23.28 P3-Glucuronyltransferase Family This family comprises enzymes which transfer GlcA from UDP-GlcA in P3-linkage to 0-galactosides. They are involved in the synthesis of either the precursor of the occurring on glycoHNK-1 epitope (S04--3GlcA( ~1-3)Gal(~1-4)GlcNAc~-R) proteins and glycolipids, or the GlcA(~l-3)Gal(~l-3)Gal(~l-4)Xyl core structure of proteoglycans. Two rat GlcATs have been cloned, GlcAT-P [181] and GlcATD [182]; these are 50% identical with each other at the amino acid level. Whereas GlcAT-P seems to act preferentially on protein-linked type-2 chains [ 1831, GlcATD has a broader specificity and acts on both type-1 and type-2 chains whether protein- or lipid-linked [182]. The third member of this family (GlcAT-I) has been cloned from a human library [184]. This enzyme specifically acts on G a l @ 3)Gal(P1-4)Xyl to form the proteoglycan core tetrasaccharide sequence, but does not act on Gal@-4)GlcNAcP-R.
23.29 Glycosyltransferases Standing Alone Although most glycosyltransferases can be grouped in a family, several important enzymes so far cannot. Well known examples are the GlcNAcTs involved in N-
6 18
23 On the Origin of Oligosaccharide Species-Glycosyltrunsferuses in Action
glycan branching (with the exception of GlcNAcT IV, see p4-N-acetylglucosaminyltransferase ( I V ) family). The cloning of these enzymes has been described: GlcNAcT I [43, 441, GlcNAcT I1 [45], GlcNAcT 111[46], and GlcNAcT V [48, 491. These enzymes are molecularly unrelated, either to each other or to any other cloned GlcNAcT. Also the a6-FucT is involved in N-glycan core fucosylation is an enzyme that does not belong to a family [185]. Finally the cloning of two p3GlcNAcTs, both of which might be involved in polylactosaminoglycan synthesis, has revealed that these enzymes are not related to each other [86, 1861. It can, however, not be excluded that in the future molecular relatives of each of these enzymes will be discovered.
23.29 Concluding Remarks In this chapter the pathways leading to the various oligosaccharide structures occurring on soluble and cell-surface-bound glycoconjugates have been described, and the specificities of the glycosyltransferases which control the glycosylation reactions have been discussed. As a result of the molecular cloning of a vast number of these enzymes it has become evident that many can be grouped into families. It also has become clear that the same reaction can be catalyzed by related, yet different enzymes. Tissue-specific expression of these enzymes is, therefore, a way in which oligosaccharide biosynthesis might be fine-tuned. In addition to glycosyltransferases many other enzymes are involved in the process of glycosylation (processing glycosidases involved in the early stages of Nglycosylation; transporters carrying the nucleotide sugars across the intracellular membranes; pyrophosphorylases catalyzing the synthesis of these nucleotide sugars; enzymes of the intermediary carbohydrate metabolism, and epimerases, mutases, transaminases, acetylases, dehydrogenases, decarboxylases, dehydratases, etc., yielding the various precursor sugars) and the number of these ‘glycosylation enzymes’ might easily exceed 500. Within a few years the entire human genome will have been sequenced and the functional genes will become known. Some 3-5% of these genes can be estimated to be somehow involved in the process of glycosylation [ 1871. It will be a challenge for the future to identify these ‘glycosylation genes’ and to find the function of their individual protein products.
Note added in proof Very recently two additional glycosyltransferases were cloned. The first one is a sialyltransferase (ST6GalNAc VI) that acts on gangliosides but not on glycoproteins. It seems to function in the synthesis of ganglioside GDla, GQlba and GTlaa (T. Okajima, H.-H. Chen, H. Ito, M. Kiso, T. Tai, K. Furukawa, T. Urano and K. Furukawa, J. Biol. Chem., 2000, 275, 6717-6723). The other enzyme is an additional B6-GlcNAcT that seems to function in the synthesis of the 0-linked core
References
6 19
2 structure (T. Schwientek, J.-C. Yeh, S. B. Levery, B. Keck, G. Merkx, A. Geurts van Kessel, M. Fukuda and H. Clausen, J. Biol. Chem., 2000,275, 11106-1 11 13). Consequently the number of cloned sialyltransferases counts 18 and of cloned p6GlcNAcTs 4. References 1. A. Hagopian and E. H. Eylar, Arch. Biochem. Biophys., 1968, 128, 422-433. 2. J. F. Kukowska Latallo, R. D. Larsen, R. P. Nair and J. B. Lowe, Genes & Dev., 1990, 4, 1288- 1303. 3. J. B. Lowe, J. F. Kukowska Latallo, R. P. Nair, R. D. Larsen, R. M. Marks, B. A. Macher, R. J. Kelly and L. K. Ernst, J. Biol. Chem., 1991,266, 17467-17477. 4. E. Grabenhorst, M. Nimtz, J. Costa and H. S. Conradt, J. Biol. Chem., 1998, 273; 3098530994. 5. C . H. Hokke, J. G. Van der Ven, J. P. Kamerling and J. F. G. Vliegenthart, Glycoconjugate J., 1993, 10, 82-90. 6. A. D. Yates, P. Greenwell and W. M. Watkins, Biochem. Soc. Trans., 1983, 11, 300-301. 7. F. Yamamoto and S. Hakomori, J. B i d Chem., 1990,265, 19257-19262. 8. M. M. Palcic and 0 . Hindsgaul, Glycohiology, 1991, I , 205-209. 9. H. Bakker, A. van Tetering, M. Agterberg, A. B. Smit, D. H. Van den Eijnden and I. van Die, J. Biol. Chem., 1997,272, 18580-18585. 10. H. Kitagawa, H. Shimakawa and K. Sugahara, J. Biol. Chem., 1999,274, 13933-13937. 11. H. H. Wandall, H. Hassan, E. Mirgorodskaya, A. K. Kristensen, P. Roepstorff, E. P. Bennett, P. A. Nielsen, M. A. Hollingsworth, J. Burchell, J. Taylorpapadimitriou and H. Clausen, J. Biol. Chem., 1997,272, 23503-235 14. 12. R. Kornfeld and S. Kornfeld, Annu. Rev. Biochem., 1985, 54, 631-664. 13. H. Schachter, Glycobiology, 1991, I , 453-461. 14. S. C. Basu, Glycobiology, 1991, 1 , 469-475. 15. C. Abeijon and C. B. Hirschberg, TIBS, 1992, 17, 32-36. 16. G. van Echten and K. Sandhoff, J. Biol. Chem., 1993,268, 5341-5344. 17. Schachter H., in: Molecular Glycobiology, eds. M. Fukuda and 0. Hindsgaul (IRL Press, Oxford, 1994) p. 88. 18. F. W. Hemming in Glycoproteins (J. Montreuil, H. Schachter and J. F. G. Vliegenthart, eds.), Elsevier, Amsterdam, 1995, pp. 127-143. 19. A. Verbert in Glycoproteins (J. Montreuil, H. Schachter and J. F. G. Vliegenthart, eds.), Elsevier, Amsterdam, 1995, pp. 145-1 52. 20. H. Schachter in Glycoproteins (J. Montreuil, H. Schachter and J. F. G. Vliegenthart, eds.), Elsevier, Amsterdam, 1995, pp. 153-199. 21. H. Schachter in Glycoproteins (J. Montreuil, H. Schachter and J. F. G. Vliegenthart, eds.), Elsevier, Amsterdam, 1995, pp. 28 1-286. 22. I. Brockhausen, in Glycoproteins (J. Montreuil, H. Schachter and J. F. G. Vliegenthart, eds.), Elsevier, Amsterdam, 1995, pp. 201-259. 23. J. Roth, in Glycoproteins (J. Montreuil, H. Schachter and J. F. G. Vliegenthart, eds.), Elsevier, Amsterdam, 1995, pp. 287-312. 24. W. M. Watkins in Glycoproteins (J. Montreuil, H. Schachter and J. F. G. Vliegenthart, eds.), Elsevier, Amsterdam, 1995, pp. 313-339. 25. C. B. Hirschberg, P. W. Robbins and C. Abeijon, Annu. Rev. Biochem., 1998, 67, 49-69. 26. D. J. Kelleher, G. Greibich and R. Gilmore, Cell, 1992, 69, 55-65. 27. D. J. Kelleher and R. Gilmore, J. Biol. Chem., 1994,269, 12908-12917. 28. A. Lal, P. Pang, S. Kalelkar, P. A. Romero, A. Herscovics and K. W. Moreman, Glycobiology, 1998, 8, R 4. 29. S. Weng and R. G. Spiro, Glycobiology, 1996, 6, 861-868. 30. W. J. Ou, P. H. Cameron, D. Y . Thomas and J. J. Bergeron, Nature, 1993, 364, 771-776. 31. C. Hammond, I. Braakman and A. Helenius, Proc. Natl. Acad. Sci. USA, 1994,9I, 913-917. 32. C. Hammond and A. Helenius, Curr. Opin. Cell Biol., 1995, 7, 523-529.
620
23 On the Origin of Oligosuccharide Species-GlycosyltrLinsferuses in Action
33. C. Labriola, J. J. Cazzulo and A. J. Parodi, J. Cell B i d , 1995, 130, 771-779. 34. M. Sousa and A. J. Parodi, EMBO J., 1995, 14,4196-4203. 35. E. Staudacher, F. Altmann, J. Glossl, L. Marz, H. Schachter, J. P. Kamerling, K. Hird and J. F. G. Vliegenthart, Eur. J. Biochem., 1991, 199, 745-751. 36. H. Leiter, J. Mucha, E. Staudacher, R. Grimm, J. Glossl and F. Altmann, J. Biol. Chem., 1999,274, 21830--21839. 37. Y. C. Zeng, G. Bannon, V. H. Thomas, K. Rice, R. Drake and A. Elbein, J. Biol. Chem., 1997,272, 31340-31347. 38. M. R. Paquet, S. Narasimhan, H. Schachter and M. A. Moscarello, J. Bid. Chem., 1984,259, 4716-4721. 39. W. M. Blanken, A. van Vliet and D. H. Van den Eijnden, J. Biol. Chem., 1984,259, 1513115135. 40. D. H. Van den Eijnden, H. Bakker, A. P. Neeleman, 1. M. Van den Nieuwenhof and I. van Die, Biochem. Soc. Truns., 1997, 25, 887-893. 41. J. C. Paulson and K. J. Colley, J. Biol. Clzem., 1989, 264, 17615- 17618. 42. D. H. Joziasse, Glycobiology, 1992, 2, 271-277. 43. R. Kumar, J. Yang, R. D. Larsen and P. Stanley, Proc. Nutl. A c d . Sri. U S A , 1990,87, 99489952. 44. M. Sarkar, E. Hull, Y. Nishikawa, R. J. Simpson, R. L. Moritz, R. Dunn and H. Schachter, Proc. Nutl. Acud. Sci. USA, 1991, 88, 234-238. 45. G. A. F. Dagostaro, A. Zingoni, R. L. Moritz, R. J. Simpson, H. Schachter and B. Bendiak, J. Biol. Chem., 1995,270, 15211-15221. 46. A. Nishikawa, Y. Ihara, M. Hatakeyama, K. Kangawa and N. Taniguchi, J. Biol. Chem., 1992,267, 18199-18204. 47. M. T. Minowa, S. Oguri, A. Yoshida, T. Hard, A. Iwamatsu, H. Ikenaga and M. Takeuchi, J. Biol. Chem., 1998,273, 11556-11562. 48. M. G. Shoreibah, G. S. Perng, B. Adler, J. Weinstein, R. Basu, R. Cupples, D. Wen, J. K. Browne, P. Buckhaults, N. Fregien and M. Pierce, J. Biol. Chem., 1993, 268, 15381-15385. 49. H. Saito, A. Nishikawa, J. Gu, Y. Ihara, H. Soejima, Y. Wada, C. Sekiya, N. Niikawa and N. Taniguchi, Biochem. Biophys. Rex Comrnun., 1994, 198, 318-327. 50. L. Puglielli, E. C. Mandon, D. M. Rancour, A. K. Menon and C. 9 . Hirschberg, J. Bid. Chem.. 1999.274.4414-4479. 51. D. H.’Joziasse, W. E. C. M. Schiphorst, D. H. Van den Eijnden, J. A. Van Kuik, H. van Halbeek and J. F. G. Vliegenthart, J. Biol. Chem., 1987; 262, 2025-2033. 52., M. Nemansky and D. H. Van den Eijnden, Gl~vcoconjugateJ., 1993, 10, 99-108. 53. W. M. Blanken and D. H. Van den Eijnden, J. Biol. Chem.: 1985,260, 12927-12934. 54. D. H. Van den Eijnden, A. H. L. Koenderman and W. E. C. M. Schiphorst, J. Biol. Chem., 1988,263, 12461- 12471. 55. M. Nemansky, W. E. C. M. Schiphorst and D. H. Van den Eijnden, FEBS Lett., 1995, 363, 280-284. 56. K. Yamashita, Y. Tachibana, T. Ohkura and A. Kobata, J. Bid. Chem., 1985,260,3963-3969. 57. S. Narasimhan, H. Schachter and S. Rajalakshmi, J. Biol. Chem., 1988, 263, 1273-1281. 58. E. W. Easton, J. G. Bolscher and D. H. Van den Eijnden, J. Bid. Chem., 1991, 266, 2167421680. 59. E. W. Easton, I. Blokland, A. A. Geldof, B. R. Rao and D. H. Van den Eijnden, FEBS Lett., 1992,308, 46-49. 60. M. Pierce, P. Buckhaults, L. Chen and N. Fregien, Clycoconjugute J . , 1997, 14, 623-630. 61. A. Kobata, Glycoconjugute J.? 1998, 15, 323-331. 62. D. H. Joziasse, W. E. C. M. Schiphorst, D. H. Van den Eijnden, J. A. Van Kuik, H. van Halbeek and J. F. G. Vliegenthart, J. Biol. Chem., 1985,260, 714-719. 63. M. Nemansky, H. T. Edzes, R. A. Wijnands and D. H. Van den Eijnden, Glycobiology, 1992, 2, 109-117. 64. D. H. Van den Eijnden, D. H. Joziasse, A. H. L. Koenderman, W. M. Blanken, W. E. C. M. Schiphorst and P. L. Koppen, in Proc. VIIIth Int. Symp. Glycoconjugates. (E. A. Davidson, J. C. Williams and N. M. Di Ferrante, eds.), Praeger, New York, 1985, Vol. I, pp. 285-286. 65. M. J. Elices and I. J. Goldstein, J. Biol. Chem., 1989,264, 1375-1380. 66. M. Nemansky and D. H. Van den Eijnden, Biochem. J., 1992,287, 31 1-316.
References
621
67. A. A. Bergwerff, J. A. Van Kuik, W. E. C. M. Schiphorst, C. A. M. Koeleman, D. H. Van den Eijnden, J. P. Kamerling and J. F. G. Vliegenthart, FEBS Lett., 1993, 334, 133-138. 68. K. B. Wlasichuk, M. A. Kashem, P. V. Nikrad, P. Bird, C. Jiang and A. P. Venot, J. Biol. Cheni., 1993,268, 13971-13977. 69. T. De Vries, C. A. Srnka, M. M. Palcic, S. J. Swiedler, D. H. Van den Eijnden and B. A. Macher, J. Biol. Chem., 1995,270, 8712-8722. 70. K. Sujino, C. Malet, 0. Hindsgaul and M. M. Palcic, Curbohydr. Rex, 1997, 305, 483-489. 71. J. Rabina, J. Natunen, R. Niemela, H. Salminen, K. Ilves, 0. Aitio, H. Maaheimo, J. Helin and 0. Renkonen, Curbohydr. Res., 1997,305,491-499. 72. C. L. M. Stults, B. A. Macher, R. Bhatti, 0. P. Srivastava and 0. Hindsgaul, Glycobiology, 1999, 9, 661-668. 73. S. Oka, K. Terayama, C. Kawashima and T. Kawasaki, J. Biol. Cheni., 1992, 267, 2271122714. 74. Y. Kato and R. G. Spiro, J. Bid. Chem., 1989, 264, 3364-3371. 75. K. Uchimura, H. Muramatsu, T. Kaname, H. Ogawa, T. Yamakawa, Q. W. Fan, C. Mitsuoka, R. Kannagi, 0. Habuchi, I. Yokoyama, K. Yamamura, T. Ozaki, A. Nakagawara, K. Kadomatsu and T. Muramatsu, J. Biochem. Tokyo, 1998, 124, 670-678. 76. D. H. Van den Eijnden, H. Winterwerp, P. Smeeman and W. E. C. M. Schiphorst, J. Bid. Chem., 1983,258, 3435-3437. 77. A. Takeya, 0. Hosomi, N. Shimoda and S. Yazawa, J. Biochern. Tokyo, 1992, 112; 389-395. 78. P. Bailly, F. Piller, B. Gillard, A. Veyrieres, D. Marcus and J. P. Cartron, Curbohydr. Rex, 1992,228, 277-287. 79. J. Nakayama, J.-C. Yeh, A. K. Misra, S. Ito, T. Katsuyama and M. Fukuda, Proc. Nut/. Acud. Sci. USA, 1999, 96, 8991 -8996. 80. C. H. Hokke, A. P. Neeleman, C. A. M. Koeleman and D. H. Van den Eijnden, Glycobiology, 1998,8, 393-406. 81. W. M. Watkins, P. Greenwell, A. D. Yates and P. H. Johnson, Biochimie, 1988, 70, 1597-161 1. 82. K. Angata, M. Suzuki and M. Fukuda, J. Biol. Chem.. 1998, 273, 28524-28532. 83. P. L. Smith and J. B. Lowe, J. Biol. C%em.,1994, 269, 15162--15171. 84. H. Bakker, I. Friedmann, S. Oka, T. Kawasaki, N. Nifantev, M. Schachner and N. Mantei, J. Biol. Chenz., 1997, 272, 29942L29946. 85. W. M. Blanken, G. J. M. Hooghwinkel and D. H. Van den Eijnden, Eur. J. Biochem., 1982, 127, 547-552. 86. K. Sasaki, K. Kuratamiura, M. Ujita, K. Angata, S. Nakagawa, S. Sekine, T. Nishi and M. Fukuda, Proc. Nutl. Acud Sci. USA. 1997, 94, 14294-14299. 87. F. Piller, J. P. Cartron, A. Maranduba, A. Veyrieres, Y. Leroy and B. Fournet, J. Bid. Chrm., 1984,259. 13385-13390. 88. A. H. L. Koenderman, P. L. Koppen and D. H. Van den Eijnden, Eur. J. Biochem., 1987,166, 199--208. 89. Y. Sakamoto, T. Taguchi, Y. Tano, T. Ogawa, A. Leppanen, M. Kinnunen, 0. Aitio, P. Parmanne, 0. Renkonen and N. Taniguchi, J. Biol. Chem., 1998,273, 27625-27632. 90. P. Mattila, H. Salminen, L. Hirvas, J. Niittymaki, H. Salo, R. Niemela, M. Fukuda, 0. Renkonen and R. Renkonen, J. Bid. C/2ein., 1998, 273, 27633-27639. 91. J. C. Yeh, E. Ong and M. Fukuda, J. Bid. Chem., 1999,274, 3215-3221. 92. R. Niemela, J. Natunen, M. L. Majuri, H. Maaheimo, J. Helin, J. B. Lowe, 0. Renkonen and R. Renkonen, J. Biol. Chrm., 1998, 273, 4021-4026. 93. C. J. Britten, D. H. Van den Eijnden, W. McDowell, V. A. Kelly, S. J. Witham, M. R. Edbrooke, M. I. Bird, T. De Vries and N. Smithers, Glycobiology, 1998, 8, 321-327. 94. H. Salminen, K. Ahokas, R. Niemela, L. Penttila, H. Maaheimo, J. Helin, C. E. Costello and 0. Renkonen, FEBS Lett., 1997, 419, 220-226. 95. G. Pfeiffer, K. H. Strube, M. Schmidt and R. Geyer, Eur. J. Biochem., 1994,219, 331L348. 96. A. Dell, H. R. Morris, R. L. Easton, M. Panico, M. Patankar, S. Oehninger, R. Koistinen, H. Koistinen, M. Seppala and G. F. Clark, J. Biol. Chem., 1995,270, 24116-24126. 97. R. T. Camphausen, H.-A. Yu and D. A. Cumming, in Glycuproteins (J. Montreuil, H. Schachter and J. F. G. Vliegenthart, eds.), Elsevier, Amsterdam, 1995, pp. 391-414. 98. M. J. Treuheit, C. E. Costello and H. B. Halsall, Biochem. J., 1992, 283, 105-1 12. 99. D. A. Cumming, Glycobiology, 1991, I , 115-130.
622
23 On the Origin of Oligosaccharide Species-Glycosyltransferases in Action
100. S. Kornfeld, FASEB J., 1987, I , 462-468. 101. S. M. Manzella, L. V. Hooper and J. U. Baenziger, J. Biol. Chem., 1996, 271, 12117-12120. 102. E. D. Green, G. Adelt, J. U. Baenziger, S. Wilson and H. van Halbeek, J. Biol. Chem., 1988, 263, 18253-18268. 103. H. Yoshima, A. Matsumoto, T. Mizuochi, T. Kawasaki and A. Kobata, J. Biol. Chem., 1981, 256, 8476-8484. 104. A. Kobata, Methods Enzymol., 1972,28, 262- 27 1. 105. B. T. Sheares and D. M. Carlson, J. Biol. Chem., 1983,258, 9893-9898. 106. F. Kolbinger, M. B. Streiff and A. G. Katopodis, J. Biol. Chem., 1998,273, 433-440. 107. M. Amado, R. Almeida, F. Carneiro, S. B. Levery, E. H. Holmes, M. Nomoto, M. A. Hollingsworth, H. Hassan, T. Schwientek, P. A. Nielsen, E. P. Bennett and H. Clausen, J. Biol. Chem., 1998, 273, 12770--12778. 108. S. Isshiki, A. Togayachi, T. Kudo, S. Nishihara, M. Watanabe, T. Kubota, M. Kitajima, N. Shiraishi, K. Sasaki, T. Andoh and H. Narimatsu, J. Biol. Chem., 1999,274, 12499-12507. 109. D. Zhou, E. G. Berger and T. Hennet, Eur. J. Biochem., 1999,263, 571-576. 110. T. Hennet, A. Dinter, P. Kuhnert, T. S. Mattu, P. M. Rudd and E. G. Berger, J. Biol. Chem., 1998, 273, 58-65. 11I . J. C. Paulson, J. Weinstein and S. de Souza, Eur. J. Biochem., 1984, 140, 523-530. 112. H. T. de Heij, P. L. Koppen and D. H. Van den Eijnden, Carbohydr. Rex, 1986,149, 85-99. 113. A. P. Neeleman, W. P. W. Van der Knaap and D. H. Van den Eijnden, Glycobiology, 1994,4, 64 1-65 1. 114. J. Srivatsan, D. F. Smith and R. D. Cummings, J. Parasitol., 1994, 80, 884-890. 115. H. Mulder, B. A. Spronk, H. Schachter, A. P. Neeleman, D. H. Van den Eijnden, M. De Jong-Brink, J. P. Kamerling and J. F. G. Vliegenthart, Eur. J. Biochem., 1995,227, 175-185. 116. I. M. Van den Nieuwenhof, W. E. C. M. Schiphorst, I. van Die and D. H. Van den Eijnden, Glycobiology, 1999, 9, 115-123. 117. D. H. Van den Eijnden, A. P. Neeleman, W. P. W. Van der Knaap, H. Bakker, M. Agterberg and I. van Die, Biochem. Soc. Trans., 1995,23, 175-179. 118. T. P. Skelton, L. V. Hooper, V. Srivastava, 0. Hindsgaul and J. U. Baenziger, J. Biol. Chem., 1991,266, 17142- 17150. 119. H. Mulder, H. Schachter, M. de Jong Brink, J. G. Van der Ven, J. P. Kamerling and J. F. G. Vliegenthart, Eur. J. Biochem., 1991, 201, 459--465. 120. A. K. Datta and J. C. Paulson, J. Bid. Chem., 1995, 270, 1497-1500. 121. K. Drickamer, Glycobiology, 1993, 3. 2-3. 122. S. Tsuji, J. Biochem. Tokyo, 1996, 120, 1-13. 123. S. Tsuji, A. K. Datta and J. C. Paulson, Glycobiology, 1996, 6, R 5-R 7. 124. A. Ishii, M. Ohta, Y. Watanabe, K. Matsuda, K. Ishiyama, K. Sakoe, M. Nakamura, J. Inokuchi, Y. Sanai and M. Saito, J. Biol. Chem., 1998, 273, 31652-31655. 125. M. Kono, S. Takashima, H. Liu, M. Inoue, N. Kojima, Y . C. Lee, T. Hamamoto and S. Tsuji, Biochem. Biophys. Res. Commun., 1998,253, 170-175. 126. T. Okajima, S. Fukumoto, H. Miyazaki, H. Ishida, M. Kiso, K. Furukawa and T. Urano, J. Biol. Chem., 1999,274, 11479-11486. 127. M. L. E. Bergh and D. H. Van den Eijnden, Eur. J. Biochem., 1983,136, 113-118. 128. E. R. Sjoberg, H. Kitagawa, J. Glushka, H. van Halbeek and J. C. Paulson, J. Biol. Chem., 1996,271, 7450-7459. 129. Y. C. Lee, M. Kaufmannn, S. Kitazume-Kawaguchi, M. Kono, S. Takashima, N. Kurosawa, H. Liu, H. Pircher and S. Tsujii, J. Biol. Chem., 1999, 274, 11958-11967. 130. V. P. Rajan, R. D. Larsen, S. Ajmera, L. K. Ernst and J. B. Lowe, J. Biol. Chem., 1989,264, 11158-1 1167. 131. R. J. Kelly, S. Rouquier, D. Giorgi, G. G. Lennon and J. B. Lowe, J. Biol. Chem., 1995,270, 4640-4649. 132. T. Kumazaki and A. Yoshida, Proc. Nail. Acad. Sci. USA, 1984,81, 4193-4197. 133. A. Sarnesto, T. Kohlin, 0. Hindsgaul, J. Thurin and M. Blaszczyk-Thurin, J. Biol. Chem., 1992,267,2737-2744. 134. S. Hitoshi, S. Kusunoki, I. Kanazawa and S. Tsuji, J. Bid. Chem., 1996, 271, 16975- 16981. 135. B. W. Weston, P. L. Smith, R. J. Kelly and J. B. Lowe, J. Biol. Chern., 1992, 267, 24575.24584.
References
623
136. K. Sasaki, K. Kurata, K. Funayama, M. Nagata, E. Watanabe, S. Ohta. N. Hanai and T. Nishi, J. Bid. Chem., 1994,269, 14730-14737. 137. T. Kudo, Y. Ikehara, A. Togayachi, M. Kaneko, T. Hiraga, K. Sasaki and H. Narimatsu, J. Biol. Chem., 1998,273, 26729-26738. 138. M. Kaneko, T. Kudo, H. Iwasaki, Y. Ikehara, S. Nishihara, S. Nakagawa, K. Sasaki, T. Shiina, H. Inoko, N. Saitou and H. Narimatsu, FEBS Lett., 1999, 452, 237-242. 139. S. L. Martin, M. R. Edbrooke, T. C. Hodgman, D. H. Van den Eijnden and M. I. Bird, J. Biol. Chem.; 1997,272, 21349-21356. 140. Z. M. Ge, N. W. C. Chan, M. M. Palcic and D. E. Taylor, J. Biol. Chem., 1997,272, 2135721363. 141. F. Dupuy, J. M. Petit, R. Mollicone, R. Oriol, R. Julien and A. Maftah, J. Biol. Chem., 1999, 274, 12257-12262. 142. T. De Vries, M . P. Palcic, P. S. Schoenmakers, D. H. Van den Eijndeu and D. H. Joziasse, Glycobiology, 1997, 7, 921-927. 143. T. De Vries, T. Norberg, H. Lonn and D. H. Van den Eijnden, Eur. J. Biochem., 1993,216, 769-717. 144. T. De Vries and D. H. Van den Eijnden, Biochemistry, 1994, 33, 9937-9944. 145. F. Yamamoto, J. Marken, T. Tsuji, T. White, H. Clausen and S. Hakomori, J. Bid. Chem., 1990,265, 1146-1151. 146. D. H. Joziasse, J. H. Shaper, D. H. Van den Eijnden, A. J. Van Tunen and N. L. Shaper, J. Biol. Chem., 1989,264, 14290-14297. 147. R. D. Larsen, V. P. Rajan, M. M. Ruff, J. Kukowska Latallo, R. D. Cummings and J. B. Lowe, Proc. Natl. Acad. Sci. USA, 1989,86, 8227-8231. 148. D. B. Haslam and J. U. Baenziger, Proc. Nutl. Acad. Sci. USA, 1996, 93, 10697-10702. 149. A. D. Yates, P. Greenwell and W. M. Watkins, Biochem. Soc. Trans., 1983, 11, 300-301. 150. M. F. A. Bierhuizen and M. Fukuda, Proc. Nutl. Acud. Sci. USA, 1992, 89, 9326-9330. 151. T. Schwientek, M. Nomoto, S. B. Levery, G. Merkx, A. G. Van Kessel, E. P. Bennett, M. A. Hollingsworth and H. Clausen, J. Bid. Chem., 1999, 274,4504-4512. 152. M. F. A. Bierhuizen, M. G. Mattei and M. Fukuda, Genes & Dev., 1993, 7, 468-478. 153. M. Sekine, K. Nara and A. Suzuki, J. Biol. Chem., 1997,272,27246-27252. 154. F. K. Hagen, B. Van Wuyckhuyse and L. A. Tabak, J. Bid. Chem., 1993: 268, 18960-18965. 155. E. P. Bennett, H. Hassan and H. Clausen, J. Biol. Chern., 1996,271, 17006-17012. 156. F. K. Hagen, K. G. Tenhagen, T. M. Beres, M. M. Balys, B. C. Vanwuyckhuyse and L. A. Tabak, J. Biol. Chem., 1997,272, 13843-13848. 157. B. G. Tenhagen, F. K. Hagen, M. M. Balys, T. M. Beres, B. Vanwuyckhuyse and L. A. Tabak, J. Biol. Chem., 1998,273, 27749-27754. 158. E. P. Bennett, H. Hassan, U. Mandel, E. Mirgorodskaya, P. Roepstorff, J. Burchell, J. Taylorpapadimitriou, M. A. Hollingsworth, G . Merkx, A. G. Van Kessel, H. Eiberg, R. Steffensen and H. Clausen, J. Biol. Chem., 1998,273, 30472-30481. 159. S. Iida, H. Takeuchi, H. Hassan, H. Clausen and T. Irimura, FEBS Lett., 1999, 449, 230-234. 160. F. K. Hagen, B. Hazes, R. Raffo, D. deSa and L. A. Tabak, J. Biol. Chem., 1999,274, 67976803. 161. Y. Nagata, S. Yamashiro, 3. Yodoi, K. 0. Lloyd, H. Shiku and K. Furukawa, J. Bid. Chem., 1992,267, 12082-12089. 162. N. W. Lo, J. H. Shaper, J. Pevsner and N. L. Shaper, Glycobiology, 1998,8: 517-526. 163. N. L. Shaper, J. H. Shaper, J. L. Meuth, J. L. Fox, H. Chang, I. R. Kirsch and G. F. Hollis, Proc. Nutl. Acad. Sci. USA, 1986, 83, 1573-1577. 164. H. Narimatsu, S. Sinha, K. Brew, H. Okayama and P. K. Qasba, Proc. Natl. Acad. Sci. USA, 1986, 83,4720-4724. 165. H. E. Appert, T. J. Rutherford, G. E. Tarr, J. S. Wiest, N. R. Thomford and D. J. McCorquodale, Biochem. Biophys. Res. Commun., 1986, 139, 163-168. 166. R. Almeida, M. Amado, L. David, S. B. Levery, E. H. Holmes, G. Merkx, A. G. Van Kessel, E. Rygaard, H. Hassan, E. Bennett and H. Clausen, J. Biol. Chem., 1997,272, 31979-31991. 167. I. van Die, A. van Tetering, W. E. C. M. Schiphorst, T. Sato, K. Furukawa and D. H. Van den Eijndeu, FEBS Lett., 1999, 450, 52-56. 168. D. H. Van den Eijnden, W. E. C. M. Schiphorst and E. G. Berger, Biochim. Biophys. Acta, 1983, 755, 32-39.
624
23 On the Origin of’ Oligosaccharide Species- Glycosyltransjerases in Action
169. M. Ujita, J. McAuliffe, 0. Hindsgaul, K. Sasaki, M. N. Fukuda and M. Fukuda, J. Biol. Chem., 1999,274, 16717-16726. 170. U. Brodbeck, W. L. Denton, N. Tanahashi and K. E. Ebner, J. Biol. Chem., 1967,242, 13911397. 171. K. Brew, T. C. Vanaman and R. L. Hill, Proc. Nutl. Acad. Sci. USA, 1968, 59,491-497. 172. T. Schwientek, R. Almeida, S. B. Levery, E. H. Holmes, E. Bennett and H. Clausen, J. Biol. Chem., 1998,273, 29331-29340. 173. T. Sato, K. Furukawa, H. Bakker, D. H. Van den Eijnden and I. van Die, Proc. Natl. Acud, Sci. USA, 1998, 95, 472-477. 174. M. Ujita, J. McAuliffe, T. Schwientek, R. Almeida, 0. Hindsgad, H. Clausen and M. Fukuda, J. Biol. Chem., 1998, 273, 34843-34849. 175. T. Nomura, M. Takizawa, J. Aoki, H. Arai, K. Inoue, E. Wakisaka, N. Yoshizuka, G. Imokawa, N. Dohmae, K. Takio, M. Hattori and N. Matsuo, J. Biol. Chem., 1998, 273, 13570-13577. 176. T. Okajima, K. Yoshida, T. Kondo and K. Furukawa, J. Biol. Chem., 1999, 274, 2291522918. 177. H. Bakker, M. Agterberg, A. van Tetering, C. A. M. Koeleman, D. H. Van den Eijnden and I. van Die, J. Bid. Chem., 1994,269, 30326-30333. 178. H. Bakker, P. S. Schoenmakers, C. A. M. Koeleman, D. H. Joziasse, 1. van Die and D. H. Van den Eijnden, Glycobiology, 1997, 7, 539-548. 179. I. van Die, H. Bakker and D. H. Van den Eijnden, Glycobiology, 1997, 7, R 5-R 8. 180. A. P. Neeleman and D. H. Van den Eijnden, Proc. Nutl. Acud. Sci. USA, 1996, 93, 1011110116. 181. K. Terayama, S. Oka, T. Seiki, Y. Miki, A. Nakamura, Y. Kozutsumi, K. Takio and T. Kawasaki, Proc. Nutl. Acad. Sci. USA, 1997, 94, 6093-6098. 182. Y. Shimoda, Y. Tajima, T. Nagase, K. Harii, N. Osumi and Y. Sanai, J. Biol. Chem., 1999, 274, 17115-17122. 183. K. Terayama, T. Seiki, A. Nakamura, K. Matsumori, S. Ohta, S. Oka, M. Sugita and T. Kawasaki, J. Biol. Chem., 1998,273, 30295-30300. 184. H. Kitagawa, Y. Tone, J. Tamura, K. W. Neumann, T. Ogawa, S. Oka, T. Kawasaki and K. Sugahara, J. Biol. Chem., 1998,273, 6615-6618. 185. S. Yanagidani, N. Uozumi, Y. Ihara, E. Miyoshi, N. Yamaguchi and N. Taniguchi, J. Biochem. Tokyo, 1997, 121, 626-632. 186. D. P. Zhou, A. Dinter, R. G. Gallego, J. P. Kamerling, J. F. G. Vliegenthart, E. G. Berger and T. Hennet, Proc. Nutl. Acad. Sci. USA, 1999, 96, 406-41 I . 187. D. H. Van den Eijnden and D. H. Joziasse, Curr. Opin. Struct. B i d , 1993, 3, 71 1 -721. 188. A. Van Tetering, W. E. C. M. Schiphorst, D. H. Van den Eijnden and I. Van Die, FEBS Lett.. 1999, 461, 311-314. 189. Y. Ikehara, N. Shimizu, M. Kono, S. Nishihara, H. Nakanishi, T. Kitamura, H. Narimatsa, S. Tsuji and M. Tatematsu, FEBS Lett., 1999, 463, 92-96. 190. S. Nishihara, H. Iwasaki, M. Kaneko, A. Tawada, M. Ito and H. Narimatsu, FEBS Lett., 1999, 462, 289-294. 191. A. Yoshida, M. T. Minowa, S. Takamatsu, T. Hara, H. Ikenaga and M. Takeuchi, Glycoconjugate J., 1998, 15, l 115-1 123. 192. E. P. Bennet, H. Hassan, U. Mandel, M. A. Hollingsworth, N. Akisawa, Y. Ikematsu, G. Merkx, A. Geurts van Kessel, S. Olofsson and H. Clausen, J. Biol. Chem., 1999, 274, 2536225370. 193. K. G. Ten Hagen, D. Tetaert, F. K. Hagen, C. Richet, T. M. Beres, J. Gagnon, M. M. Balys, B. VanWuyckhuyse, G. S. Bedi, P. Degand and L. A. Tabak, J. Bid. Chem., 1999, 274, 27867-27874. 194. E. P. Bennet, H. Hassan, M. A. Hollingsworth and H. Clausen, FEBS Lett., 1999, 460, 226230. 195. R. Almeida, S. B. Levery, U. Mandel, H. Kresse, T. Schwientek, E. P. Bennett and H. Clausen, J. Biol. Chem., 1999,274, 26165-26171. 196. J. Tongzhong, K. W. Bremer, A. D’Souza, R. D. Cummings and W. M. Canfield, Glycobiology, 1999, 9, 1123.
Carbohydrates in Chemistry and Biology Editor Beat Ernst, Gerald W. Hart, Pierre Sinay copyright OWILEY-VCH Verlag GmbH, 2000
24 Synthesis of Sugar Nucleotides Reinhold Ohrlein
24.1 Introduction Recent progress in molecular glycobiology has revealed the important biological roles of numerous glycoconjugates [ 11. In particular cell-surface carbohydrates play key roles in cellular communication processes via selective adhesion phenomena [2, 31. They guide e.g. leukocyte extravasation [4], the homing of lymphocytes and adhesion of myeloid cells to activated platelets and the endothelium [5-7]. Besides these interactions carbohydrates are involved in fertilization events, cell growth, and parasitic infections of viruses and bacteria 181. A very recent finding is the involvement of certain cell surface a-galactosides in hyperacute rejection reactions; this is of pharmaceutical interest in organ transplantation 191. This makes sugars worthwhile targets for pharmaceutical applications [ 10, 1 11. Although chemical synthesis of oligosaccharides has reached a mature stage [ 12- 141 the overall synthetic sequences are still lengthy and cumbersome [ 151. Biologically active carbohydrates are, moreover, composed of various different monosaccharide units and are often only active if presented in a polyvalent or multiantennary array to their respective receptors [ 16, 171. These multiantennary heteropolymers, in particular, have resisted efficient chemical synthesis. Recently biocatalysts have been found to be valuable tools for complementing the classical synthetic approach [ 181. Nature has invented several ways of assembling complex oligosaccharides [ 11. The Leloir pathway is one of the preferred routes of mammalian systems. The Leloir enzymes-the glycosyl transferases-transfer a monosaccharide unit from a nucleotide-activated donor substrate to a growing oligosaccharide chain with rigorous regio- and stereoselectivity [19] (see Scheme 1). Exclusively one of the many OH groups of the acceptor is glycosylated, in either an a or p mode! Neither the donor nor the acceptor sugar needs protection from the highly homofunctional reactants-often OH groups only are present on the sugars. Thus, selective protecting group manipulations, a preponderant task in the classical, chemical oligosaccharide assemblage can be reduced to an absolute minimum.
626
24 Synthesis of Sugar Nucleotides
(Ho)n
DONOR
ACCEPTOR E.C.2.4....I
SIDE-PRODUCT
J. transferase
(HO),/
Scheme 1. Leloir glycosylation pathway.
The availability of the glycosyltransferases and the activated donor substrates is, however, a prerequisite for the application of the biocatalytic methodology. Initially the transferases were isolated from body fluids such as human and bovine milk, e. g. p( 1-4)galactosyl transferase [20, 211, or blood, e.g. a(1-3)fucosyl transferase [22],or a variety of animal tissues such as mouse kidney, e.g. the core-I1 N-acetylglucosaminyl transferase [23], and rat liver, e.g. a(2-3)- and a(2-6)sialyl transferases [24, 251, to mention just a few. Modern cloning and expression techniques make an ever growing number of glycosyl transferases available to the glycochemist [26-281. Astonishingly, only eight nucleotide-activated donors are used by mammalian cells (see Scheme 2) [29]. In principal all are commercially available, although on a small scale only. The high prices of the natural donor substrates and the unavailability of non-natural derivatives of those donors is the reason why several chemical and biomimetic pathways have been probed to increase access to these crucial compounds [29, 301. The ensuing sections will summarize the most recent attempts to synthesize these donors, with emphasis on non-natural derivatives.
24.2 Synthesis of Sugar Nucleotides 24.2.1 Chemical Synthesis
UDP-Activated Donors All the UDP-activated donors (Scheme 2) are a-pyrophosphate sugars and thus are inherently prone to degradation in acidic media, leading to monosaccharide units and uridine diphosphate [31].This lability must always be taken into account at the outset of an efficient synthetic strategy. In addition the separation of the highly polar target compounds from side-products in the reaction mixture necessitates the
24.2 Synthesis of Sugar Nucleotides HocOH
0
627
0
HocOH
. i !
[UDP-Galj
'OH
HO'
OH HO
O
H
@GziiiE]HO'
OH
q 'OH
0
0
II
0-P-0-P-0 AcH
[GzG]
HO
OH
OH
OH
Ho
Scheme 2. Nucleotide-activated donor sugars.
strict adherence to sophisticated purification protocols. Generally, overall yields are poor. General approaches towards UDP-activated sugars are outlined in Scheme 3. The key intermediates in the synthesis of nucleotide a-diphosphates are the asugar phosphates. The MacDonald procedure [ 321 proved to be a good protocol for generation of numerous a-glycosyl phosphates (see Scheme 3 ) . They are obtained in
628
24 Synthesis of Sugar Nucleotides /OH
OH Man
OH P' OH
e;p
H
OAc
N
RO
2) 1) Bn,P(O,)OH H,/Pd/C
OAc
3)NaOMe
OH QOj-OH
Ro
NHAc OH
R = Ac, perAc-GlcNAc
OH
R = H,GlcNAc
I'
OH
RrY
O
H 0 ManAc O \ l - O H OH
HC OH
W
ACHN~o O,l1 OH GalNAc P' OH
-. .
0
HO
OH
Scheme 3. Formation of nucleotide a-diphosphate sugars; Y: leaving group.
ca 30-40% yield by fusion of an alp mixture of the peracetylated hexopyranosides with phosphoric acid, then base-catalyzed deacetylation. The low yields give rise to the assumption of the concomitant formation of the undesired fi isomers. It has, however: been claimed that these hydrolyze more readily in the deacetylation step and can thus be removed completely [32, 331. u-Glucose-1-phosphate has also been prepared in 87% yield directly from glucose and phosphoric acid in the presence of propylene oxide [341. Alternative routes for the synthesis of u-sugar- 1 -phosphates started from protected sugars with a free anomeric OH group which is deprotonated in a first step and subsequently reacted with diphenyl chlorophosphate [35]. The authors found variable ratios of a / f i mixtures depending, e.g., on the base used, the reaction temperature, and the structure of the reacted hexoses. Hydrogenation over PtOl and treatment with base gave the unprotected sugar phosphates. Although the uphosphates of N-acetylglucosamine and also chitobiose could be obtained from the corresponding oxazoline precursors (Scheme 3), the a-sugar-I-dibenzylphosphate intermediates proved quite unstable [36]. Accordingly, the even more unstable a-
24.2 Synthesis of Sugar Nucleotides , OAc OAc *OAc A%’eA:O c OAc
OAc
629
1)NH31MeO!
OAc
2) phosphite
0-i-H 1
-
0
.OH
0
Scheme 4. Synthesis of a-cellobiosyl phosphate.
mannosyl-1-phosphate was obtained in 61% yield [37]. Yet another approach towards a-sugar-1-phosphates of mannose, galactose, glucose, and cellobiose was reported by Luu and coworkers; this is exemplified in Scheme 4 for a-cellobiosyl phosphate [38]. The peracetylated sugars were first treated with ammonia in methanol to remove the anomeric acetate and the resulting 1-OH sugars were subsequently phosphitylated. Only a-phosphites were obtained; in the cellobiose synthesis illustrated the final a-phosphate could be isolated in ca 50% yield after iodine oxidation and deacetylation. Besides the a-phosphates of the naturally occurring sugars (Scheme 2) a number of a-sugar-1-phosphates of non-natural derivatives have been prepared by the above phosphorylation procedures. These sugar phosphates are reacted with activated nucleotide phosphates as is exemplified in Scheme 3 to form the desired UDP sugars. Usually the commercial uridine 5’-monophosphomorpholidate has been used (Y = morpholine). We found the in situ activation of the uridine monophosphate by carbonyldiimidazole to be superior [39] (Y = imidazole). Some recent examples of the preparation of non-natural UDP- and dTDPactivated sugars by use of this general pathway (Scheme 3) are compiled in Table 1. The literature listed should be consulted because the individual experimental details can vary. The simultaneous replacement of uridine by deoxythymidine and galactose by a number of deoxy sugars to give non-natural donor substrates has also been reported [50].Partially protected a-sugar-1-phosphates were coupled to morpholidateactivated deoxythymidine-5-phosphateand subsequently deprotected in the presence of lithium hydroxide to give the corresponding dTDP sugars (see Table 1). CMP-Activated Sugars
The stereochemical preparation of CMP-sialic acid is quite a synthetic challenge. This is because of the absence of neighboring group participation to assist the
630
24 Synthesis of Sugar Nucleotides
Table 1. Yields and scales for the coupling of sugar phosphates to nucleotide phosphates Sugar
Couple %
Scale (mg)
17*
Couple Yo
Scale (ms)
14
60*
69
10*
15
82*
82
14*
12
10*
17
11*
17
22*
72
19*
85
13*
12
*
-
25"
18
32*
33
67*
90
71*
70
18*
-
66*
33
71*
35
80*
40
44**
42
34**
33
21**
36
37**
33
-
* UDP sugar ** dTDP sugar.
Ref.
Sugar
Ref.
24.2 Synthesis of Sugar Nucleotides pathway A OAc
I I
pathway B
ox
l
DAc
63 1
AcO
OMe
1 I
OAc
/
I
43 - 82%
couple
I
I
46.50%
I
47 - 61%
I
oxidize
0
OAc
I
I 1I
68%
4
couple
I
deprolect
I ‘NAO
~
HO
I I R = NHAc, OAc, NHC(O)CH,Ac, NHCBz R = NHAc. OH, NHC(O)CH,OH. NHCBz
I 1 I
OH
HO
R = NHAc, OAc, NHC(O)CH,Ac. NHCBz R
= NHAc, NHC(O)CH,OH
Scheme 5. Synthesis of CMP-sialic acid and derivatives.
stereoselective phosphorylation of the tertiary anomeric center. Two strategies have been followed recently to prepare the parent CMP-sialic acid and some derivatives thereof. Pathway A relies on the coupling of a protected cytidine phosphoramidite with protected sialic acid [51, 521. The resulting phosphite is subsequently oxidized with a t-butyl hydroperoxide solution and finally deprotected (see Scheme 5). Next to the natural CMP-sialic acid, CMP-ketodeoxyoctulonic acid and the CMP-Nglycolylsialic acid were obtained by this protocol, albeit in the low mg-range! Pathway B starts from dialkyl sialyl phosphites [53, 541 (see Scheme 5). In this case the sialyl phosphite group is directly replaced by the N/O-acetylated cytidine phosphate with retention of configuration. Application of strategy B also made accessible nucleotide monophosphosialic acid derivatives with the cytidine replaced by other pyrimidine and purine bases, although in modest overall yields only [55]. By following pathway B a CMP‘disialyl’ derivative could be synthesized. This compound has a second sialic acid unit a-linked to the 8-OH group of the CMP-activated sialic acid [56].
632
24 Synthesis of Sugar Nucleotides
GDP-Activated Donors D-Mannose and L-fucose are related biogenetically and are transferred from their corresponding guanosine diphosphate-activated donors [ 571 by the corresponding transferases. GDP-Man constitutes an a-phosphate whereas in GDP-Fuc the diphosphate bridge is P-linked to the fucose unit. The synthesis of GDP-Man was accomplished analogously as described for UDP-Gal (see Scheme 3). In this case an a-mannosyl phosphate [58] is coupled with a GMP-morpholidate in the presence of lH-tetrazole [59] to give GDP-Man. Coupling yields vary from 40-76% (scale: 13-147 mg). Also GDP-3-N-acetyl-3deoxy-a-D-mannose was obtained in 35% (46 mg) yield according the morpholidate procedure [601. Alternatively, the coupling of a phosphinothioate-activated GMP with amannosyl phosphates has also been successful [61] (see Scheme 6).
,o HOrP BnO
"1
0 0.11,OBn
P
HO
'OH
Ny
OBn
'r
i
5, GDP-Man
Scheme 6. Synthesis of GDP-man and congeners. 1) NaH, CCl,CN, 81%; 2) HOP(O)(OBn)z, 99%; 3) Na, liquid NH3 then BaC12, 60%; 4) BuZP(S)Br, B q N , THF, 61%; 5 ) AgOAc, pyr, 40%.
24.2 Synthesis of Sugar Nucleotides
633
2,3,4,6-Tetra-O-benzyl-1-hydroxymannose is first deprotonated and then treated with trichloroacetonitrile to give the trichloroacetamidate intermediate which is subsequently reacted with dibenzylphosphoric acid to give the a-perbenzylated phosphate ester. All benzyl groups could be removed by sodium in liquid ammonia. The resulting a-mannosyl phosphate is subsequently treated with the guanosine 5'monophosphate dibutyl phosphinothioic anhydride in the presence of silver acetate to form the desired GDP-Man. The necessary thiophosphoryl-activated anhydride is obtained from GMP and commercial dibutylthiophosphoryl bromide. The thiophosphoryl approach was also employed for the synthesis of GDP-Gal, GDPGlcNAc and GDP-Xyl (see Scheme 6). GDP-Fuc is a particularly delicate compound. This is reflected by the numerous papers describing various, albeit low-yielding synthetic approaches. The early investigations [62-641 were plagued by the inefficient and cumbersome access to the p-fucosyl- 1-phosphate (Scheme 7). The most reliable, fully chemical preparations have been reported by Hindsgaul [65] and Whitesides [66]. The full synthetic pathway is outlined in Scheme 7. Commercial L-fucose is first peracetylated and subsequently treated with conc. hydrobromic acid to give the a-fucosyl bromide in quantitative yield. The bromide can be stored at -20°C for several months as a solid. In the presence of silver carbonate the bromide is reacted with dibenzyl phosphate. This comphosphate to give the dibenzyl 2,3,4-tri-O-acetyl-a-~-fucosyl pound is completely deprotected to form the labile p-fucosyl phosphate which is stirred with morpholidate-activated guanosine-5'-monophosphateat room temperature for a prolonged time. The pure GDP-Fuc is obtained from this reaction mixture by repeated passages over Dowex and Sephadex columns. Thus gramamounts of GDP-Fuc were prepared. According the synthetic pathway depicted in Scheme 7, a series of non-natural GDP-deoxyfucoses have been prepared successfully (compare Table 2). Hindsgaul and coworkers [67, 681 also reported the synthesis of GDP-Fuc derivatives which bear alkyl chains, aminoalkyl residues capable of being linked to fluorescent labels, and oligosaccharide moieties on the fucose C-6. Surprisingly, those GDP-'fucose' derivatives are accepted by certain fucosyl transferases [67, 691.
Scheme 7. 1) AczO, pyr, 100%; 2) 30% HBr in AcOH, CH2C12, 100%; 3) HOP(O)(OBn)zxNEt,, Ag2C03, -78"C, CH2C12, 72%; 4) H2/Pd/C, MeOH, 100%; 5) NaOMe, MeOH, 92%; 6) GMPmorpholidate, pyr, 27-43%.
634
24 Synthesis of Sugar Nucleotides
Table 2. Successful preparations of non-natural GDP-deoxyfucoses. Position
Residue
Ref.
X = 0, unless otherwise stated. Always only one residue is altered, the remaining residues are either OH for R,, Rb, R, or CH3 for Rd. Spacer = fluorescent label added to spacer H2N(CH2)2S(CH2)20CH2-,CSHII-,C ~ H I S - .
The critical intermediate in the outlined synthesis is the p-fucosyl phosphate, which can hardly be separated cleanly from the concomitant a isomer [65, 701. The ensuing coupling to the morpholidate-activated GMP proceeds sluggishly and is always accompanied by epimerization and hydrolysis. This makes the synthesis a low-yielding process and impedes the isolation of the pure GDP-fucose from this reaction mixture. Comments
The first hurdle which must be overcome in the chemical synthesis of sugar nucleotides is the preparation of anomerically pure sugar phosphates. In sialic acid there is no neighboring group which can control the stereoselective phosphorylation of the C-2 center to form the desired p-phosphates selectively. Optimization of protecting groups and phosphorylation protocols led to modest improvements only of the alp ratios. This problem is not yet solved satisfactorily. In GDP-Fuc and derivatives the anomeric phosphate group is P-linked to the C 1 atom. Peracetate protection of the fucose enables the quantitative synthesis of the a-bromide intermediate which is converted to the desired p-phosphate almost exclusively (see Scheme 7). The UDP-activated sugars and GDP-Man are a-phosphates and their chemical synthesis poses greater problems. Although various protecting group combinations and phosphorylation protocols were investigated, none led to the exclusive formation of the desired a-phosphates (see Schemes 3 and 6). The purification of those compounds is further hampered by their notorious instability. The ensuing step is pyrophosphate formation, generally a very sluggish reaction, even if one of the two phosphate groups-usually that of the nucleotide-is activated by a good leaving group like morpholine. The diphosphate coupling reaction
24.2 Synthesis of Sugar Nucleotides
635
often takes up to 3-5 days at room temperature and is still not complete. An increase in temperature to promote the reaction is not advisable because of the lability of the reaction products. The introduction of in situ activation of one phosphate group by tetrazoles or imidazoles has only led to exceptional improvements in the coupling yields (see Scheme 7). The long reaction time is generally accompanied by decomposition of the sugar phosphates. Thus the reaction mixture often contains a number of polar compounds which are difficult to separate from the desired sugar nucleotides. This cuts down overall yields. A few sugars only could be obtained in a satisfactory yield (see Table 1). For these reasons the full chemical preparation of nucleotide donor sugars is restricted to the laboratory scale only. Large-scale production still needs much developmental effort.
24.2.2 Chemo-Enzymatic Synthesis The delicate nature of the activated donor sugars and the problems encountered with their chemical synthesis prompted glycochemists to investigate preparative alternatives mimicking the in viuo synthesis [30, 71, 721. The activated donors are obtained either by a combined chemo-enzymatic or a fully enzymatic approach. The individual approach depends on the availability of the required biocatalysts. Although some donor sugars could even be generated in situ via multi-enzyme systems before enzymatic transfer [73], this route seems limited to natural donors only; it will be discussed in more detail in Section 24.3. Uridine Diphosphate-Activated Donor Sugars
Whitesides and coworkers extensively studied various enzymatic routes offered by nature to prepare UDP-activated donors on a gram scale (see Scheme 8) [74, 751. It is apparent that the key intermediates are again the a-sugar- 1-phosphates. Galactose and 2-aminogalactose were phosphorylated directly at the anomeric centers in the presence of galactokinase and ATP [75]. The resulting a-sugar phosphates were only crudely purified and converted to the desired UDP-sugars in a multi-enzyme cycle in the presence of UTP, UDP-Glc, UDP-glucose pyrophosphorylase (E.C. 2.7.7.9), pyrophosphatase (E.C. 3.6.1.l), and galactose-l-phosphate uridyl transferase (E.C. 2.7.7.12). This last enzyme transfers a UMP moiety from UDP-Glc on to the phosphate group of galactose and 2-amino-2-deoxygalactose to produce the corresponding UDP-sugar (see Scheme 8) and glucose-1-phosphate, which is recycled by the other enzymes to form again the UDP-Glc cosubstrate with UTP as the phosphate source. With glucose or glucosamine [75] a phosphate group is first transferred from ATP on to the 6-OH group by a hexokinase. The 6-phosphate sugars are then rearranged with either a phosphoglucomutase or a N-acetylglucosamine phosphomutase, respectively, to give the a-phosphates of glucose and N-acetylglucosamine. Glucose-lphosphate is subsequently incubated with UTP and UDP-glucose pyrophosphorylase, and a pyrophosphatase to decompose the pyrophosphate side-product to give
636
24 Synthesis of Sugar Nucleotides
H?,
OH
no:
"OH
O:+ -OH
5)
HO HOfioi HO
0 OH
- P-
OH
3)0 H'0Oho$:Z$ HO
7)
NH*
HO
NHAc
AcHN 0
9 OH -7OH
,
I
,
,I
[ UDP-GlcNAc]
no.
on
Scheme 8. 1) galactokinase, ATP; 2) galactose-l-phosphate uridyltransferase, UDP-glucose, UTP, pyrophosphatase; 3) hexokinase, ATP, pyrophosphatase; 4) phosphoglucomutase; 5 ) UDP-glucose pyrophosphorylase, UTP, pyrophosphatase; 6) UDP-glucose dehydrogenase; 7) N-acetoxysuccinimide; 8) N-acetylglucosamine phosphomutase; 9) UDP-N-acetylglucosamine pyrophosphorylase, UTP, pyrophosphatase; 10) if R = OH then UDP-glucose-4-epimerase; 1 1) UDP-N-acetylglucose4-epimerase.
UDP-Glc. UDP-GlcNAc is obtained via an analogous incubation with UDP-Nacetylglucosamine pyrophosphorylase and UTP, and a pyrophosphatase. UDP-glucuronic acid is synthesized by oxidation of the 6-OH group of UDPglucose with UDP-glucose dehydrogenase and NAD+ as a hydrogen acceptor. The process works most efficiently if the generated NADHt is immediately re-oxidized e.g. with L-lactic acid dehydrogenase and pyruvic acid [74]. UDP-glucose and UDP-N-acetylglucosamine can also be converted in situ into UDP-galactose or UDP-N-acetylgalactosamine, and vice versa, by treatment with commercial UDP-glucose epimerase (E.C. 5.1.3.2) or UDP-N-acetylglucosaminyl epimerase (E.C. 5.1.3.7), respectively. These enzymes convert the equatorial OH groups of glucose and N-acetylglucosamine, into axial 4-OH groups by an oxidation-reduction sequence. The equilibria, however, lie far on the side of the starting gluco isomers. The application of the epimerization reaction is, therefore, useful only when the generated UDP-Gal and UDP-GalNAc are consumed immediately, e.g. by a successive transferase reaction. The enzymatic and semi-enzymatic routes towards UDP-sugars have been nicely compiled by Heidlas et al. [29]. Many other enzymes and enzyme sources are listed
24.2 Synthesis o j Sugar Nucleotides
637
by Elling [71]. Although a vast variety of mammalian and non-mammalian sugar nucleotides might be accessible in this manner, most of the enzymes or enzyme systems are not generally available and have not been probed on a preparative scale. There are, however, a limited number of reports concerning the enzymatic synthesis of non-natural UDP-sugars such as UDP-2F-galactose [76]. Also some uridine analogs, e.g. 5-fluorouridine 5’-monophosphate, have also been coupled to give the corresponding ‘UDP-galactose derivatives. This reaction was catalyzed by the microorganism C. sauitoana in 74% yield on a 250 mg scale [77]. The synthetic routes exactly follow the route outlined for the preparation of UDP-Gal (Scheme 8). A direct microbial conversion of glucose and UMP to UDP-Glc has also been explored [78] as has the production of a-galactosyl phosphate from bacteria [79]. Hollow fiber reactor technology has also been investigated for the production of UDP-glucosamine and UDP-GlcNAc [SO]. The principles of this preparation are similar to those outlined in Scheme 8. In a first step the sugars are phosphorylated at the 6-OH group by a hexokinase and UTP, which is regenerated in situ from phosphoenol pyruvate in the presence of a phosphokinase. The 6-phosphate is subsequently rearranged to the sugar-l-phosphate in the same reactor. In a second step the a-anomeric phosphate is treated with UTP and UDP-glucose pyrophosphorylase to give UDP-glucosamine. The amino group can be acetylated with acetic anhydride to give UDP-GlcNAc. CMP-Activated Sugars Following the biosynthetic pathway has proved to be the most efficient means of preparing CMP-sialic acid and some derivatives thereof [29, 30, 711. Glc-NAc is epimerized in a first step to give Man-NAc which is condensed with pyruvic acid in the presence of sialic acid aldolase to produce sialic acid (Scheme 9). The sialic acid is subsequently converted enzymatically to CMP-sialic acid by CMP-sialic acid synthetase. The required CTP can be produced in situ from CMP and phosphoenolpyruvate as the ultimate phosphate source via several phosphate-transfer reactions catalyzed by phosphokinases (see box in Scheme 9). During the coupling of sialic acid to CTP a pyrophosphate is again released. This side-product must be decomposed by a phosphatase to shift the equilibrium toward the product. The whole synthesis has been scaled up to a multi-gram production of pure CMP-sialic acid [81] by use of a synthetase cloned and overexpressed in E. coli. Recently, the sequence has been further improved by a Japanese group who employed a deacetylase to remove selectively any non-epimerized glcNAc which inhibits the ensuing aldolase-reaction [82]. The sialic acid synthetase could also be employed successfully to prepare a series of non-natural CMP-Sia analogs [83] (see Schemelo). The 9-amino derivative was used to attach a fluorescenyl photolabel [83]. All the donor substrates listed in the table have been probed on various a(2-6) and a(2-3) sialyl transferases [ 841. An important activated donor sugar of Gram-positive bacteria is CMP-ketodeoxyoctulosonic acid (3-deoxy-~-mannooctulosonicacid). This compound and its
638
24 Synthesis of Suyur Nucleotides
N"2
HO
H AcHN
O
HO
'
M
__ 2)
O
AcH
OH
CTP
HO
OH HO'
t
0
Scheme 9. 1) aldolase; 2) sialic acid synthetase; 3 ) pyrophosphatase; 4) phosphokinase; 5 ) deacylase.
Scheme 10. Synthesis of non-natural CMP-'Sia' derivatives with sialic acid synthetase. y
2
R
HO'
OH
R
R'
R
R'
OH
24.2 Synthesis of Sugar Nucleotides
.
R=OH,F
HO'
639
.
'OH
Scheme 11. 1) CMP-Kdo-synthetase, CTP.
V
R = OH, N, AcHN-
HO-
OH
Scheme 12. 1) Mannose phosphomutase; 2) GDP -mannose pyrophosphorylase, GTP, pyrophosphatase.
5-fluoro congener (see Scheme 11) have been prepared recently on a preparative scale by use of cloned and overexpressed microbial CMP-Kdo-synthetase (E.C. 2.7.7.38) [85]. GDP-Activated sugars The in vivo synthesis of GDP-mannose is equivalent to that of the UDP-sugars [29, 301 (see Scheme 12). Mannose- 1-phosphate, prepared from mannose-6-phosphate either by a phosphomutase reaction or chemically [61], is converted to GDPmannose by a GDP-mannose pyrophosphorylase in the presence of GTP [86]. Also in this case a pyrophosphate side-product is generated which must be decomposed by a pyrophosphatase to shift the equilibrium to the desired GDP-Man. GDP-3azido- and GDP-3-N-acetylmannose have also been obtained successfully by following this pathway [60]. Highly efficient access to GDP-fucose and several non-natural analogs is gained by the combined application of chemical and enzymological methods (Scheme 13). A series of peracetylated fucose analogs have been prepared by the chemical route described for the peracetylated p-fucosyl phosphate (Scheme 7) [87]. Two decisive improvements have been introduced with regard to the fully chemical synthesis of GDP-Fuc (Scheme 13). Firstly, the acetylated 'fucosyl' phosphates used for the coupling reaction are more stable than the unprotected variety and they are far more soluble in the coupling solvent than the unprotected ones. This speeds up the diphosphate formation without any detectable, concomitant anomerization of
640
24 Synthesis of Sugar Nucleotides 0
R R 2 F g f ’ ‘-OH ‘OH Ac OoAc
2 ’1 ) R 2
p 0 , ’
AcOoAC
NH2 HO
OH
R1 0
~
R2
__ OH NH, F OH OH OH
%
overall CH, CH, CH, H CH,OH CH,OH
89 50
80 77 70 46*
Scheme 13. 1) a: DMF, GMPxNBu3 and carbonyldiimidazole, b: DOWEX-H+; 2) H20, pH = 7, calf intestine alkaline phosphatase; 3) H20, pH = 6.8, acetylesterase, 0.1 M NaOH; *4-epi-OH.
the ‘fucosyl’ phosphates. In addition the resulting product mixture is subsequently treated with calf intestine alkaline phosphatase to remove any unreacted monophosphate selectively. This facilitates the isolation of the protected GDP-sugars. They are simply precipitated from this mixture by the addition of ethanol. In a subsequent step the desired unprotected donor-sugars are obtained under very mild conditions via incubation with commercial acetylesterase and final ethanol precipitation. The overall sequence benefits from the absence of any chromatographic purification step, high overall yields, and broad versatility. Thus the fucose moiety has been replaced by D-arabinose, L-glucose and L-galactose or 2-amino- and 2fluorofucose (Scheme 13). The procedure could be applied likewise to prepare adenosine diphosphate-, xanthosine diphosphate-, and inosine diphosphate-fucose 1871. Investigation of the human FX protein has recently revealed its remarkable involvement in the in uiuo synthesis of GDP-Fuc from GDP-Man. The purified enzyme had combined epimerase and NADPH-reductase activity, converting GDP4-keto-6-deoxymannose into GDP-Fuc [ 881. The very same reaction sequence is also achieved by use of a crude extract from A. aerogenes, and has been used to prepare 14C-labeledGDP-Fuc [89]. Another microbial production of GDP-Fuc has been reported by a Japanese group 1901. GDP-Man obtained by fermentation was converted to GDP-Fuc by A. rudiobacter. A crude extract from hog submaxillary glands was also found to convert fucose directly into GDP-Fuc by a two-step enzymatic process (see Scheme 14) [911. The protein extract probably contained both a fucokinase to generate fucose-1phosphate and GDP-fucose pyrophosphorylase. This same analytical protocol was later on applied by chemists to synthesize small amounts of GDP-Fuc [92].
641
24.3 In situ Generation of Sugar Nucleotides
Fuc
1) /7T-
ATP
Fuc-p-phosphate
*)
7-7
GDP-Fuc
GTP ADP
PYrophosphate
Scheme 14. 1) fucokinase; 2) GDP-fucose pyrophosphorylase; 3) pyrophosphatase.
Comments
In comparison with the fully chemical synthesis of nucleotide-activated donor sugars the chemo-enzymatic or enzymatic approaches are distinguished by fewer protecting group manipulations and high chemo- and stereoselectivity. This simplifies purification protocols significantly. These features reduce the number of synthetic steps and increase overall yields. The fully enzymatic preparation of sugar nucleotides, however, requires the availability of a series of enzymes. In addition, their mutual interactions presupposes exact knowledge of their physiological properties by the user; this, in particular, makes chemists hesitate to use biocatalysts. Not all the involved enzymes may tolerate altered, non-natural substrates. A combined chemo-enzymatic approach therefore offers the possibility of combining the advantages of the chemical synthesis and those of the enzymatic preparation, e. g. to prepare non-natural nucleotide activated substrates.
24.3 In situ Generation of Sugar Nucleotides Several papers describe enzymatic glycosylations with in situ generation of the required activated donor sugars; these are impressively compiled in Refs. [30] and [73]. Especially impressive is the synthesis of the trisaccharide Siau(2-6)Galp( 14)GlcNAcOH with the in situ generation of CMP-sialic acid from GlcNAc (compare Scheme 9) and UDP-galactose from glucose (compare Scheme 8) coupled with the enzymatic transfer of those sugars. In this one-pot reaction a total of nine enzymes has been applied successfully [93]! The synthesis of the linear B-trisaccharide Gala(1-3)Galu( 1-4)GlcNAcpOR, an important human xenoantigen [94], has also been tackled by a multi-enzyme onepot procedure, albeit only on a microscale (see Scheme15) [95]. Especially noteworthy is the use of sucrose, Glccx(l-2)Fru, as the ultimate source of UDP-Gal. In a first step sucrose synthase from rice cleaves the disaccharide and forms UDP-Glc in the presence of UDP. UDP-Glc is subsequently epimerized at the 4-position by UDP-galactose epimerase to give the required UDP-Gal. This donor substrate is first used by p( 1-4)galactosyl transferase to produce the Nacetyllactosamine intermediate and then by a(1-3)galactosyl transferase to gal-
642
24 Synthesis of Sugar Nucleotides
\
Glca(I-2)Fru Glca(I-2)Fru
A GlCNAcpI-OR
\
UDP-Gal UDP
A
I
I),
3)
UDP-Glc
Galp(1-4)GlcNAcpl-OR
Scheme 15. 1) sucrose synthase, UTP; 2) UDP-glucose-4’-epimerase; 3) ferase; 4) a( 1-3)galactosyl transferase.
p( 1-4)galactosyl
trans-
actosylate the intermediate disaccharide at the 3-OH group of the previously introduced galactose unit. The released UDP is recycled again. Neither the p(14)galactosyl transferase nor the a( 1-3)galactosyl transferase attacks the sucrose. The in situ generation of GDP-Fuc from p-fucose-1-phosphate coupled with the transfer of the fucose moiety on to N-acetyllactosamine has also been probed on an analytical scale [96]. In the presence of GTP P-fucose-1-phosphate is converted to GDP-Fuc by GDP-fucose pyrophosphorylase. GDP-Fuc is then used by an a(13)fucosyl transferase to transfer the fucose moiety on to N-acetyllactosamine. The released GDP is recycled and phosphorylated to give GTP by a phosphokinase with phosphoenol pyruvate as phosphate source. The pyrophosphate produced in the complete cycle is decomposed by pyrophosphatase (see Scheme 16). There is only one report of the transfer of a non-natural galactose derivative (see Scheme 17) [97]. 2-Deoxyglucose-6-phosphate, which is itself produced by a hexokinase reaction, is first equilibrated to a-2-deoxyglucose-1-phosphate by a phosphoglucomutase. In the presence of UTP and UDP-glucose pyrophosphatase this sugar is converted to UDP-2-deoxyglucose which is subsequently epimerized to UDP-2-deoxygalactose by galactose epimerase. The resulting UDP-2-deoxygalactose is then transferred by a p(l-4)galactosyl transferase on to the 4-OH group of N-acetylglucosamine to give the deoxylactosamine derivative. The released UDP is re-phosphorylated to UTP in the presence of phosphoenolpyruvate by pyruvate kinase to close the reaction cycle. Thus milligram amounts of 2’-deoxy-Nacetyllactosamine were synthesized in ca 40% yield. Comments
It goes without saying that the most elegant oligosaccharide synthesis is achieved by enzymatic assemblage of sugars starting from simple, unprotected monosaccharide building blocks and harvesting the desired oligosaccharide. This can be achieved by use of cyclic enzyme systems which activate and transfer the corresponding mono-
24.3 In situ Generation of Sugar Nucleotides
643
9 ~
H,C
H
HOoH 1) GTP
Scheme 16. 1) GDP-fucose pyrophosphorylase; 2) a( 1-3)fucosyl transferase; 3) phosphokinase; 4) pyrophosphatase. OH O,'P-OH P
H i o S O H
2)
I
0 O-prOH OH UTP,+
Scheme 17. 1) phosphoglucomutase; 2) UDP-glucose pyrophosphorylase; 3) UDP-galactose-4'epimerase; 4) j3( 1-4)galactosyl transferase; 5) phosphokinase.
644
24 Synthesis of Sugur Nucleotides
saccharides to form the desired product. No sugar phosphates and sugar nucleotides have to be isolated. In contrast, the released nucleotide diphosphate is re-used by the enzymes to start a new activation cycle (see Scheme 17). This task can also be achieved by use of microorganisms-the so called wholecell synthesis-provided the microorganisms do not metabolize the product oligosaccharide. The practicability of such cyclic multi-enzyme preparations has been confirmed, e. g. for the synthesis of N-acetyllactosamine or the tetrasaccharide, sialyl Lewis', containing four different monosaccharide units. There have also been a limited number of reports showing that slightly altered natural sugars can be introduced into such cyclic multi-enzyme systems to generate non-natural oligosaccharides. Carbohydrates with major non-natural elements will, however, still have to be prepared chemically in the near future.
24.4 Outlook The steady progress in cloning and expression techniques on the one hand and improvements in cell cultivation and fermentation procedures on the other hand will make an increasing number of enzymes available to the carbohydrate chemist. Tailor-made biocatalysts which will convert non-natural substrates also will be provided by these biotechnological methods. Additionally, screenings of microorganisms will increase access to rare and unknown sugars. Thus the preparation of natural and non-natural oligosaccharides will become cheaper. This will lead to the identification of a growing number of therapeutically active glycoconjugates and consequently an increased demand for sugars by the pharmaceutical industry. There are already suppliers who offer kilogram amounts of activated natural donor sugars for enzymatic synthesis on request (Boehringer, Wandrey, Yamasa Corp. Japan). Provided the required sugar transferases are available, combinatorial techniques will be probed in enzymatic oligosaccharide synthesis. This will give easy and rapid access to oligosaccharides and will promote studies of oligosaccharideprotein interactions. Nonetheless, chemistry will not be totally and abruptly banned from the carbohydrate field, although the era of lengthy and sophisticated assemblage of oligosaccharides by purely chemical means will wane.
References 1. 0. Hindsgaul and M. Fukuda, Molecular Glycobiology, Oxford University Press 1994. 2. A. Varki, Glycobiology 1993, 3, 97. 3 . R. A. Dwek, Chem. Rev. 1996, 96, 683. 4. P. Sears, C.-H. Wong, Chem. Commun. 1998, 1161. 5. F. R. Carbone, P. A. Gleesen, Glycobiology 1997, 7, 725. 6. C. R. Bertozzi, Chem. & Biol. 1995, 2, 703. 7. L. A. Lasky, Ann. Rev. Biochem. 1995, 64, 113.
References
645
8. A series of papers on parasites in Science 1994,264, 1857-1886. 9. K. Gustafsson, K. Strahan, A. Preece, Immunol. Rev. 1994, 141, 59. 10. J . M. McAuliffe, 0. Hindsgaul, Chem. & Ind. 1997: 3, 70. 11. Z. J. Witczak, Curr. Med. Chem. 1995, I , 392. 12. Compare preceding chapters of this volume. 13. S. Hanessian, Preparative Carbohydrate Chemistry, Marcel Dekker 1997. 14. F. Barresi, 0. Hindsgaul, Modern Synth. Methods 1995, 283. 15. F. Barresi, 0. Hindsgaul, Curbohydr. Chern. 1995, 14, 1043. 16. T. Feizi, Curr. Opin. Struct. Biol. 1993, 3. 701. 17. Y. Nagai, Pure & Appl. Chem. 1997, 69, 1893. 18. H. J. M. Gijsen, L. Qiao, W. Fitz, C.-H. Wong, Chem. Rev. 1996, 96, 443. 19. S. C. Crawley, M. M. Palcic, Modern Methods Carbohydr. Synth. 1996, 492. 20. P. H. Johnson, W. M. Watkins, Glycocojugate J. 1992, 9, 241. 21. H. E. Appert, T. J. Rutherford, G. E. Tarr, N. R. Thomford, J. McCorquindale, Biochem. Biophys. Res. Conzmun. 1986, 138, 224. 22. A. Sarnesto, T. Kohlin, 0. Hindsgaul, K. Vogele, M. Blaszczyk-Thurin, J. Thurin, J. Biol. Chem. 1992,267, 2745. 23. R. Ohrlein, 0. Hindsgaul, M. M. Palcic, Carbolzydr. Res. 1993, 244, 149. 24. L. J. Melkerson-Watson, L. C. Sweeley, J. Biol. Chern. 1991, 266, 4448. 25. J. C. Paulson, J. L. Rearick, R. L. Hill, J. Biol. Chem. 1977,252, 2363. 26. M. C. Field, L. J. Wainwright, Glycobiology 1995, 5, 463. 27. M. Fukuda, M. F. A. Bierhuizen, J. Nakayama, Gl.vcobiology 1996, 6, 683. 28. J. B. Lowe, Sem. Cell Biol. 1991, 2, 289. 29. J. E. Heidlas, K. W. Williams G. M. Whitesides, Acc. Chem. Res. 1992,25, 307. 30. C.-H. Wong, R. L. Halcomb, Y. Ichikawa, T. Kajimoto, Angew. Chem. Int. Ed. Engl. 1995,34, 412, 521. 31. N . Kochetkov, V. N. Shibaev, Adv. Carhohydr. Cliem. Biochem. 1973,28, 307. 32. D. L. MacDonald, Methods Enzymol. 1966, 8, 121. 33. C. D. Warren, R. W. Jeanloz, Biochemistry 1973, 12, 5031. 34. S. B. Tzokov, I. T. Devedjiev, E. K. Bratovanova, D. Petkov, Angew. Chem. Int. Ed. Engl. 1994, 33, 2302. 35. S. Sabesan, S. Neira, Carbohydr. Res. 1992,223, 169. 36. J. Lee, J. K. Coward, J. Org. Chem. 1992, 57, 4126. 37. T. Yamazaki, C. D. Warren, A. Herscovics. R. W. Jeanloz, Carbohydr. Res. 1980, 79, C9-CI2. 38. L. Knerr, X. Pannecoucke, B. Luu, Tetrahedron Lett. 1998, 39, 273. 39. F. Cramer, H. Neunhoeffer, K. H. Scheit, G. Schneider, J. Tennigkeit, Angew. Chem. 1962, 74, 387. 40. M.-C. Chapeau, P. A. Frey, J. Org. Chem. 1994, 59, 6994. 41. A. Burton, P. Wyatt, G. J. Boons, J. Chem. Soc. Perkin Trans. I1997, 16, 2375. 42. T. Endo, Y. Kajihara, H. Kodama, H. Hashimoto, Bioorg. Med. Chem. 1996, 4 , 1939. 43. Y. Kajihara, T. Endo, H. Ogasawara, H. Kodama, H. Hashimoto, Carbohydr. Res. 1995, 269, 273. 44. G. Srivastava, 0. Hindsgaul, M. M. Palcic, Carbohydr. Res. 1993, 245, 137. 45. H. Yuasa, 0. Hindsgaul, M. M. Palcic, J. Am. Cheni. Soc. 1992, 114, 5891. 46. H. Yuasa, 0. Hindsgaul, M. M. Palcic, Can. J. Chem. 1995, 73, 2190. 47. L. Thomas, S. A. Abbas, K. L. Abbas, Carbohydr. Res. 1988,184, 77. 48. 0. Tsuruta, G. Shinohara, H. Yuasa, H. Hashimoto, Bioorg. Med. Chem. Lett. 1997, 7, 2523. 49. T. Yamazaki, C. D. Warren, A. Herscovic, R. W. Jeanloz, Can. J. Chem. 1981, 59, 2247. 50. S. Wiemann, W. Klafie, Liebigs Ann. Chem. 1995, 1779. 51. M. D. Chappell, R. L. Halcomb, Terrahedron 1997, 53, 11109. 52. S. Makino, Y. Ueno, M. Ichikawa, Y. Hayakawa, T. Hata, Tetrahedron Lett. 1993, 34, 2775. 53. T. J. Martin, R. R. Schmidt, Tetrahedron Lett. 1992, 33, 6123. 54. T. Yoshino, R. R. Schmidt, Carhohydr. Lett. 1995, 1, 329. 55. T. J. Martin, H. Braun, R. R. Schmidt, Bioorg. Med. Chem. 1994,2, 1203. 56. Y. Kajihara, T. Ebata, K. Koseki, H. Kodama, H. Matsushita, H. Hashimoto, J. Org. Chem. 1995, 60, 5732.
646 57. 58. 59. 60. 61. 62. 63. 64.
24 Synthesis of Sugar Nucleotides
V. Ginsburg, J. Biol. Chem. 1961,236, 2389. com. available from SIGMA, CH-9741 Buchs, Switzerland, P.O. 260. V. Wittmann, C.-H. Wong, J. Org. Chem. 1997, 62, 2144. W. Klafie, Carbohydr. Res. 1995,266, 285. J. E. Pallanca, N. J. Turner, Chem. Soc. Perkin Trans. I 1993, 301 7. H. A. Nunez, J. O’Connor, P. L. Rosevar, R. Baker, Can. J. Chem. 1981,59, 2086. R. R. Schmidt, B. Wegmann, K.-H. Jung, Liehigs Ann. Chem. 1991, 121. B. M. Heskamp, H. J. G. Broxtermann, G. A. van der Marel, H. van Boom, J. Carbohydr. Chem. 1996, 15, 61 1. 65. U. B. Gokhale, 0. Hindsgaul, M. M. Palcic, Can. J. Chem. 1990, 68, 1063. 66. K. Adelhorst, G. M. Whitesides, Curbohydr. Rex 1993, 242, 69. 67. C. Hallgren, 0. Hindsgaul, J. Carbohydr. Chem. 1995, 14, 453. 68. C. Vogel, C. Bergmann, A.-J. Ott, T. K. Lindhorst, J. Thiem, W. V. Dahlhoff, C. Hallgren, M. M. Palcic, 0. Hindsgaul, Liebiqs Ann. Chem. 1997, 601. 69. G. Srivastava, K. J. Kaur, 0. Hindsgaul, M. M. Palcic, J. Biol. Chem. 1992, 267, 22356. 70. Y. Ichikawa, M. M. Sim, C.-H. Wong, J. Org. Chem. 1992, 57, 2943. 71. L. Elling, Adu. Biochem. Eng. & Biotrchnol. 1997, 58, 91. 72. S. David, C. Auge, C. Gautheron, Adv. Carbohydr. Chem. Biochem. 1991. 49, 175. 73. Y. Ichikawa, R. Wang, C.-H. Wong, Methods Enzymol. 1994, 247, 107. 74. E. J. Toone, E. S. Simon, G. M. Whitesides, J. Org. Chem. 1991, 56, 5603. 75. J. E. Heidlas, W. J. Lees, G. M. Whitesides, J. Org. Chem. 1992, 57, 146, 152. 76. T. Hayashi, B. W. Murray, R. Wang, C.-H. Wong, Bioorg. Med. Chem. 1997, 5, 497. 77. K.4. Fujita, A. Matsukdwa, K. Shibata, T. Tanaka, M. Taniguchi, S. Oi, Curbohydr. Res. 1994,265, 299. 78. J. H. KO, H . 3 . Shin, Y. S. Kim, D.-S. Lee, C.-H. Kim, Appl. Biochem. Biotechnol. 1996, 60, 41. 79. B. Nidetzky, A. Weinhausel, R. GrieBner, K. D. Kulbe, J. Curbohydr. Chem. 1995, 14, 1017. 80. P. A. Ropp, P.-W. Cheng, Anal. Biochem. 1990, 187, 104. 81. M. Kittelmann, T. Klein, U. Kragl, C. Wandrey, 0. Ghisalba, Appl. Microbiol. Biotechnol. 1995, 44, 59. 82. A. Kuboki, H. Okazaki, T. Sugai, H. Ohta, Tetrahedron 1997, 53, 2387. 83. R. Brossmer, H. J. Gross, Methods Enzymol. 1994, 247, 153. 84. H. J. Gross, R. Brossmer, Glycoconjugate J. 1995, 739. 85. T. Sugai, C.-H. Lin, G.-J. Shen, C.-H. Wong, Bioorg. Med Chem. 1995, 3, 313. 86. E. S. Simon, S.,,Grabowski, G. M. Whitesides, J. Ovg. Chem. 1990, 55, 1834. 87. G. Baisch, R. Ohrlein, Bioorg. Med. Chem. 1997, 5, 383. 88. M. Tonetti, L. Sturla, A. Bisso, U. Benatti, A. De Flora, J. Biol. Chem. 1996,271, 27274. 89. V. Ginsburg, Methods Enzymol. 1966, 8, 293. 90. K. Yamamoto, T. Maruyama, H. Kumagai, T. Tochkura, T. Seno, H. Yamaguchi, Agric. Biol. Chem. 1984, 48, 823. 91. R. Prohaska, H. Schenkel-Brunner, Anal. Biochem. 1975, 69, 536. 92. R. Stiller, J. Thiem, Liebigs Ann. Chem. 1992, 467. 93. Y. Ichikawa, J. L.-C. Liu, G.-J. Shen, C.-H. Wong, J. Am. Chem. Soc. 1991, 113, 6300. 94. U. Galili, K. Swanson, Proc. Natl. Acad. Sci. USA 1988, 7401. 95. C. H. Hokke, A. Zervose, L. Elling, D. H. Joziasse, D. H. van den Eijnden, Glycoconjugate J. 1996, 13, 687. 96. Y. Ichikawa, Y.-C. Lin, D. P. Dumas, G.-J. Shen, E. Garcia-Junceda, M. A. Williams, R. Bayer, C. Ketcham, L. E. Walker, J. E. Paulson, C.-H. Wong, J. Am. Chem. Soc. 1992, 114, 9283. 97. J. Thiem, T. Wiemann, Angew. Chem Znt. Ed. Engl. 1991, 30, 1163.
Carbohydrates in Chemistry and Biology Editor Beat Ernst, Gerald W. Hart, Pierre Sinay copyright OWILEY-VCH Verlag GmbH, 2000
25 Enzymatic Glycosylations with Glycosyltransferases Ossi Renkonen
25.1 Introduction Samples of pure molecular species of protein- and lipid-bound oligosaccharideg are needed for studies of their biological functions. These functions, in turn, are mediated by interactions between oligosaccharides and other biomolecules. Successful studies of these interactions require the use of naturally occurring compounds in the pure form and often also the use of non-natural analogs of protein- and lipid-bound oligosaccharides. The multitude of possible isomeric oligosaccharide structures has hampered the development of glycobiology [ 1) because isolation of pure samples from naturally occurring mixtures of oligosaccharide isomers has been a difficult task. To avoid this difficulty some researchers rely on enzyme-assisted in vitro synthesis of oligosaccharides. The major branch of this approach is based on Leloir’s description of biosynthesis of glycosidic bonds [2]. In Leloir-type of reactions, a monosaccharide unit is enzymatically transferred from a sugar nucleotide to a saccharide acceptor. The reactions proceed stereoselectively with unprotected acceptors, produce one glycosidic linkage in each step, and are applicable to a number of acceptors. A particular advantage of the Leloir type synthesis lies in the availability of several glycosyltransferases in recombinant form; the number of commercially available recombinant glycosyltransferases is in rapid growth and the number of enzymes cloned in research laboratories increases even more rapidly [3]. Another booster is the rapidly improving availability of sugar nucleotides required for the enzymatic synthesis of oligosaccharides (cf Chapter 24, by R. Ohrlein, in this book). Regeneration of sugar nucleotides during the transferase reactions shows promise in large scale enzymatic oligosaccharide synthesis (cf the chapter by C.-H. Wong in this book). This chapter will describe several Leloir-type transferase reactions that have been used for stepwise construction of a oligosaccharides comprising five different monosaccharides, GlcNAc, GalNAc, Gal, Fuc, and NeuSAc. This is a very limited approach among the myriads of possible oligosaccharides that can be potentially
648
25 Enzymatic Glycosylations with Glycosyltransferases
constructed from the few hundreds of different monosaccharides expressed in Nature. The limitations are remarkable even when considering the number of reported glycosyltransferase-catalyzed reactions. The examples actually selected for discussion represent steps to synthetic oligosaccharides with particularly interesting biomedical properties. Some of the final products are potent in vitro inhibitors of murine sperm-to-egg adhesion, and antagonists of mammalian leukocyte adhesion to activated endothelium. The examples discussed will show that relatively easily synthesized glycans can have considerable contraceptive and anti-inflammatory potential. To compensate for the limitations of this material, the reader is referred to the biosynthetic chapters of this book, and to recent reviews published elsewhere on the general theme, including those of Sears and Wong [3] and Van den Steen et al. [4].
25.2 In nitro Synthesis of the Core Region of 0-Glycans Many plasma membrane proteins contain domains of mucin type with clustered 0-linked glycans; the peptide cores within these clusters adopt stiff and extended conformations, representing rods from which the saccharides protrude [ 51. 25.2.1 Initialization of 0-Glycan Biosynthesis The addition of the first sugar in mucin-type 0-glycosylation is directed by the family of UDP-Ga1NAc:polypeptide N-acetylgalactosaminyltransferases (EC 2.4.1.41) (ppGaNTases). This activity has been partially purified from several sources [6]. More recently, several isoforms of the mammalian ppGaNTase family have been cloned and functionally expressed [7-91. The acceptor specificities of the individual enzymes are mostly broad and overlapping, yet distinct. The large number of the transferases is probably responsible for the lack of a general consensus sequence for mucin type 0-glycosylation. An algorithm for predicting 0-glycosylation sites in mammalian proteins is available, however [ 101. 25.2.2 Synthesis of Core 1 In vitro synthesis of 0-glycan Core 1 (Gal(D1-3)GalNAc) by UDP-Ga1:GalNAcR P3-Gal-transferase (EC 2.4.1.122) from pig submaxillary glands was described almost thirty years ago by Roseman and colleagues [ 111. Porcine submaxillary gland microsomes catalyzed the transfer of galactose from UDP-[ ''C]galactose to sialidase-treated ovine submaxillary mucin, which carries single a-linked GalNAc residues in clustered arrays on serine and threonine residues. As much as 3.4 pmol ['4C]galactose was transferred to the protein in an experiment involving 10 pmol multivalent mucin acceptor, 20 pmol UDP-[ 14C]Galand 23 mg microsomal pro-
25.2 In vitro Synthesis of the Core Region of 0-Glycans
649
tein. Treatment with P-galactosidase from C. perfringens, liberated [ ''C]galactose from the mucin product, and its treatment with 0.05 M KOH + 1 M NaBH4 released a reduced oligosaccharide. The latter gave [ 14C]galactoseand galactosaminitol in a molar ratio of 1.0:0.75 upon acid hydrolysis. Accordingly, it represented [ ''C]GalpGalNAc,,d. The product was chromatographically and electrophoretically identical with authentic Gal(p 1-3)GalNAc,d obtained from gangliosides by a known procedure; other positional isomers were not available for comparison and could not be excluded, however. This pioneering piece of work illuminates many features characteristic of enzymeassisted synthesis of oligosaccharides. Firstly, it is possible to obtain apparently homogeneous saccharide products by using unprotected acceptors, radiolabeled sugar nucleotides and even very crude enzymes. Secondly, product characterization is a difficult and a crucially important task. Later work by Medicino et al. [ 121 and Cheng and Bona 1131 on purified Core 1 galactosyltransferase from tracheal mucosa established the formation of the Gal( P 1-3)GalNAc linkage firmly; permethylation and periodate oxidation were used in the product analysis. A family of homologous P3-galactosyltransferase genes encoding enzymes that transfer to GlcNAc and to GalNAc has been identified recently 1141, but the Core 1 Gal transferase has not yet been cloned. 25.2.3 Synthesis of Core 2
In vitro synthesis of 0-glycan Core 2 [Gal(~l-3)[GlcNAc(~l-6)]GalNAc] by UDP-GlcNAc:Gal( p1-3)GalNAc-R (GlcNAc to GalNAc) p6-GlcNAc-transferase activity of dog submaxillary gland microsomes was reported by Williams and Schachter [ 151. The enzyme is conveniently abbreviated as C2GnT. Preliminary identification of the mucin product of this branch-generating reaction became possible when [ 14C]Gal(P 1-3)GalNAc-[ovine submaxillary mucin] was synthesized in vitro (see above) and was used as acceptor in the C2GnT reaction. The resulting mixture of acceptor- and product-oligosaccharides was released from the protein by reductive p-elimination, was methylated, and was finally hydrolyzed. Only 2,3,4,6tetra-@methyl-[ ''C]galactose was found in the hydrolysate, implying that the new GlcNAc unit had been transferred to the GalNAc residue of the mucin acceptor. Later, Williams et al. performed preparative experiments using Gal(p1-3)GalNAc-a-0-benzyl as the acceptor, generating a few hundred nanomoles of the trisaccharide product [ 161. With this amount of material they could apply methylation analysis and NMR spectroscopy for product characterization, establishing that the canine submaxillary microsomes had catalyzed the formation of the GlcNAc(pl6)GalNAc linkage. At least two enzyme isoforms are involved in the biosynthesis of Core 2; C2GnT of L-type is restricted to Core 2 synthesis and C2GnT of M-type synthesizes Core 2, Core 4 (see below) and GlcNAc( P1-3)[GlcNAc( p1-6)IGal-OR which is known as blood group I antigen (see below) [17]. A cDNA has been isolated that encodes the C2GnT of the L-type [18]. Also the C2GnT of the M-type has been cloned, and the recombinant forms of the two distinct C2GnTs have been used for in vitro synthesis of Core 2 0-benzyl glycosides
650
25 Enzymatic Glycosylations with Glycosyltransferases
[19]. In our laboratory the analogous transformation has been performed by use of hog gastric mucosal microsomes, which catalyzed the conversion of the free Core 1 disaccharide Gal@-3)GalNAc into Gal(~l-3)[GlcNAc(~1-6)]GalNAc [20]. Functionally active molecules of P-selectin glycoprotein ligand- 1 (PSGL- 1) are not expressed in wild type Chinese hamster ovary (CHO) cells, but are expressed in CHO cells transfected with the cDNA encoding L-type C2GnT [21]. This implies that the physiological counter-receptor of P-selectin has to carry 0-glycans of Core 2-type in order to be recognized by P-selectin.
25.2.4 Synthesis of Core 3 and Core 4 Brockhausen et al. [22] described in vitro synthesis of 0-glycan Core 3 (GlcNAc(P13)GalNAc) and Core 4 [GlcNAc(Pl-3)[GlcNAc( pl-6)]GalNAc] by UDP-GlcNAc: GalNAc-R P3-GlcNAc-transferase and UDP-GlcNAc:GlcNAc( Pl-3)GalNAc-R (GlcNAc to GalNAc) P6-GlcNAc-transferase, respectively. The enzymes are conveniently abbreviated as C3GnT and C4GnT. GalNAc-a-phenyl and GalNAc[ovine submaxillary mucin] served as acceptors in the reactions studied, and the mucin products (0.26-0.43 pmol) were characterized by liberating the oligosaccharides by reductive p-elimination, followed by NMR spectroscopy and methylation analysis of the liberated alditols. Later experiments have shown that C3GnT activity is high in human colonic and respiratory tissues and in salivary glands from several species. This enzyme has, however, proven difficult to solubilize by detergents and is unstable in the solubilized form, which has hampered its further study [23]. Recently, C4GnT activity was demonstrated in the recombinant form of C2GnT of M-type [ 191.
25.2.5 In vitvo Extension of Core 1 Glycans Mucin type 0-glycans carry often single or multiple N-acetyllactosamine (LacNAc) units. Brockhausen et al. [24] showed that pig gastric mucosal microsomes, upon incubation with UDP-[ ''C]GlcNAc and Gal@-3)GalNAcal-OR (where R is an appropriate mucin or antifreeze glycoprotein), are able to generate [ ''C]GlcNAc(Pl3) Gal@-3)GalNAcal-OR. The transfer of [ ''C]GlcNAc amounted to 1.65 pmol. The resulting [ ''C]glycans were released from the protein by reductive a-elimination, and HPLC was used to isolate altogether 100 nmol pure ['4C]GlcNAc((313)Gal(~I-3)GalNAcr,~. Methylation analysis and NMR spectroscopy were used to establish its structure. This P3-GlcNAc transferase is a distinct initiator of polylactosamine chain biosynthesis on Core 1, but it is unlikely to support the further growth of the chain. The data of Brockhausen et al. [25] showed that pig gastric mucosal microsomes fail to transfer GlcNAc to Gal@1-4)GlcNAc. The trisaccharide sequence of Core 1 type is most likely P4-galactosylated at the non-reducing end in vitro by a number of Gal-transferases and further extended in the polylactosamine mode (see below).
25.3 Enzymatic in vitro Synthesis o j Polylactosamine Backbones
65 1
25.2.6 In vitvo Extension of Core 2 Glycans
The growth of Core 2 structures resembles that of Core 1 saccharides, but can occur both at the Gal and the GlcNAc residues. Brockhausen et al. showed that pig gastric mucosa contains P3-GlcNAc transferase activity, which converts Core 2 into GlcNAc(~1-3)Gal(~1-3)[GlcNAc(~1-6)]GalNAcal-R, where R is the polypeptide backbone of either a mucin or antifreeze glycoprotein [24]. It is likely that this reaction is catalysed by the same enzyme that elongates Core 1 disaccharide. Core 2 trisaccharide in unconjugated form was quantitatively galactosylated by incubation with UDP-Gal and P4GalT I from bovine milk (EC 2.4.1.90), which is commercially available at reasonable prices [20]. The resulting tetrasaccharide Gal(~l-4)GlcNAc(~l-6)[Gal(~l-3)]GalNAc was isolated in pure form by gel filtration on Bio-Gel P-2 and characterized by NMR. P4Galactosylation of Core 2 is catalyzed even more effectively by P4GalT-IV than by P4GalT-I [26]. The P1,3-GlcNAc transferase activity (iGnT) of human serum is known to transfer to Gal(P1-4)GlcNAc, Gal(Pl-4)Glc, their simple glycosides as well as with Gal(~1-4)GlcNAc(~1-3)Gal(~l-4)Glc [27-291, but it works poorly with Gal(P13)GalNAc [28]. Put together, the acceptor-data on the P1,3-GlcNAc transferases of human serum and pig gastric mucosal microsomes seem to be distinct and complementary in their substrate specificities, such that the serum enzyme probably extends the 6-linked arm and the pig stomach mucosal enzyme extends the 3-linked arm of the tetrasaccharide Gal(~1-3)[Gal(~l-4)GlcNAc(~l-6)]GalNAc. 25.2.7 Extension of Core 3 and Core 4 Glycans Extension of Core 3 and Core 4 glycans is known to occur (4, 301, but in vitro syntheses of the extended species have not been described.
25.3 Enzymatic in uitvo Synthesis of Polylactosamine Backbones Polylactosamines consist of N-acetyllactosamine (LacNAc) chains linked to sphingoglycolipids of neolacto series and to proteins; both N- and 0-glycosidic forms of protein-bound polylactosamines are common. Polylactosamines have been known for a long time as the backbones of keratan sulfates and as undersulfated components in membranes of red blood cells and embryonic carcinoma cells. The polylactosamine backbones are expressed with and without distal decorations, which can consist of sulfate groups, single monosaccharides, e.g. a-NeuSAc, a-Fuc, a-Gal, a-GalNAc, P-GalNAc, a-GlcNAc, and sulfo-3-P-GlcA (HNK-1) and remarkably numerous oligosaccharide determinants. Fucose and sulfate decorations are expressed commonly also along the polylactosamine chain. The multitude of different capping decorations on lactosamineglycans are responsible for a large number of different saccharide-lectin interactions on mammalian cell surfaces.
652
25 Enzymatic Glycosylutions with Glycosyltvunsferuses
The polylactosamine backbones are bonded to: (i) the Man-cores of complex Nglycans, (ii) Cores 1-4 of O-glycans, and (iii) the lactose core of sphingoglycolipids. The same P3GlcNAc transferases are probably responsible for initiation and extension of some, but not all, polylactosamines. The present concept of polylactosamine biosynthesis after initialization involves: (i) the generation of primary backbones consisting of chains of LacNAc(P 1-3’)LacNAc type (LacNAc, Gal(P14)GlcNAc), (ii) generation of eventual backbone branches to yield arrays of the type LacNAc(pl-3’)[LacNAc( pl-6’)]LacNAc, and (iii) decoration reactions. 25.3.1 Enzymatic Synthesis of the Primary Chains of Blood Group i-Type The (33GlcNAc transferase responsible for extension of terminal N-acetyllactosamine residues of free oligosaccharides was reported independently by three groups, in Novikoff ascites tumor cells [31] and in human serum [27,28]. Yates and Watkins [27] succeeded in synthesizing 3.8 pmol purified [ ‘‘C]GlcNAc(~l-3)Gal(pl-4)GlcNAc from 80 pmol LacNAc and 12 pmol UDP-[ 14C]GlcNAc.They also synthesized 7.5 pmol pure [14C]GlcNAc(P1-3)Gal(P1-4)Glc from 200 pmol Gal@-4)Glc and 30 pmol UDP-[ 14C]GlcNAc. P-N-Acetylhexosaminidase treatment liberated the newly transferred [ 14C]GlcNAcresidues from both products, implying that Plinkages had been formed by the serum transferase. Methylation analysis revealed that the galactosyl groups of both products were 3-O-substituted with GlcNAc. Simple glycosides of LacNAc and lactose were also good acceptors, as was Gal(p 1-4)GlcNAc( P1-3)Gal( P 1-4)Glc. Piller and Cartron 1281 reported similar experiments. Their data show that Fuc(al-2)Gal(P1-4)GlcNAc and Gal(P1-4)[Fuc(al-3)]GlcNAc are not acceptors for the serum p3-GlcNAc-transferase, and the disaccharide Gal@-3)GlcNAc is a poor acceptor. By contrast, the polyand Gal(P1-4)lactosamines Gal(~l-4)GlcNAc(~1-3)Gal(~l-4)GlcNAc-O-Me GlcNAc(~1-3)Gal(~l-4)GlcNAc(~l-3)Gal(~1-4)GlcNAc-O-Me were as good acceptors as Gal(P 1-4)GlcNAc; the resulting products were not structurally characterized, however. Because linear polylactosamines are known as blood group i-determinants, the enzymes responsible for these reactions are called iGnT. The serum iGnT transferred GlcNAc effectively also to desialylated fetuin and desialylated a1 -acid glycoprotein and to the pentasaccharides Gal(P1-4)and Gal(Pl-4)GlcNAc(P1-2)GlcNAc(pl-2)Man(al-3)Man( P 1-4)GlcNAc Man(al-6)Man( Pl-4)GlcNAc, representing distinct branches of complex N-glycans [28] Accordingly, serum iGnT is able to transfer to terminal Gal(p1-4)GlcNAc residues of glycoprotein-linked complex N-glycans. By contrast, asialoglycophorin, which mostly contains Gal(Pl-3)GalNAc bound to serine or threonine residues, was not an acceptor. The serum iGnT converts also GalNac(P1-4)GlcNAc to GlcNAc(~1-3)GalNAc(~1-4)GlcNAc 1321. The serum iGnT extends also P6-linked branches of polylactosamines, converting the hexasaccharide LacNAc(P 13’)[LacNAc(~l-6’)]LacNAc into the octasaccharide GlcNAc(PI-3’)LacNAc(Pl3’)[GlcNAc(pl-3)’ LacNAc(p 1-6’)ILacNAc [ 331. iGnT has been purified from calf serum [34], and a human cDNA clone encoding a similar enzyme has been isolated by Sasaki et al. [35]. A transferase experiment
25.3 Enzymatic in vitvo Synthesis of Polylactosamine Backbones
653
with the crude recombinant enzyme and 500 nmol lacto-N-neo-tetraose gave 0.7 nmol [ 3H]GlcNAc(pl-3)Gal( p1-4)GlcNA( pl-3)Gal( pl-4)Glc.
25.3.2 Distal Branching of i-Type Polylactosamine Backbones
The first branching reaction of polylactosamine backbones was described by Piller et al. [36] who found an appropriate p1,6-GlcNAc transferase activity in hog gastric mucosal microsomes. This activity catalyzed the reaction shown in eq. (1).
GlcNAc(pl-3)Gal(pl-4)Glc + UDP-GlcNAc + GlcNAc(
p 1-3) [GlcNAc(pl-6)] Gal(pl-4)Glc
(1)
The product was identified by methylation, followed by methanolysis, O-acetylation and GLC-mass spectrometry of the derivatized monosaccharides. The GLC which was present data showed that 1,2,4,6-tetra-O-methyl-3-O-acetylgalactose, among the components obtained from the trisaccharide acceptor, had completely disappeared. It was replaced by a and p isomers of 1,2,4-tri-O-methy1-3,6-di-Oacetylgalactose. Compared with the acceptor, the amount of 1,3,4,6-tetra-O-methyI2-deoxy-N-methylacetamidoglucoseobtained from the tetrasaccharide product was twofold. Mass spectrometric analysis of the derivatized galactose obtained from the tetrasaccharide product confirmed that the new substituent was linked to carbon 6. The activity in hog gastric mucosal microsomes was unable to transfer to acceptors that terminated with a complete LacNAc unit at the non-reducing end. The microsomes transferred also to the glycolipid GlcNAc(p1-3)Gal( pl-4)Glcpl-Ceramide. The primary experiment of Piller et al. (loc. cit.) was repeated by Seppo et al. [37], using GlcNAc(pl-3)Gal(pl-4)GlcNAcas the acceptor. Here, the tetrasaccharide GlcNAc(~l-3)[GlcNAc(~l-6)]Gal(~l-4)GlcNAc was formed, as was shown by partial acid hydrolysis of the product and chromatographic identification of all diand trisaccharides of the hydrolysate. More recently, Helin et al. [38] incubated the doubly labeled pentasaccharide GlcNAc(pl-3)[ 3H]Gal([31-4)GlcNAc(p1-3)[ 14C]Gal(pl-4)GlcNAc with UDPGlcNAc and the gastric mucosal microsomes, obtaining the hexasaccharide GlcNAc(P 1-3)[GlcNAc(P 1-6)][3H]Gal(p 1-4)GlcNAc( p 1-3)[ 14C]Gal(p 1-4)GlcNAc. Here, product analysis was performed by partial acid hydrolysis. Paper chromatography of the hydrolysate gave [3H]-labeled, but not [ 14C]-labeled,glycans that contained the newly formed GlcNAc(p1-6)Gal bond. The diagnostic cleavage products included the disaccharide GlcNAc( pl-6)[ 3H]Gal, the trisaccharides GlcNAc(p1-6)[ 3H]Gal(pl -4)GlcNAc and GlcNAc( p1-3)[GlcNAc( pl-6)][ 3H]Gal, and the tetrasaccharide GlcNAc(p1-3)[GlcNAc(p1-6)][ 3H]Gal(P1-4)GlcNAc. In addition to the degradation data, positive-ion MALDI-TOF mass spectrometry of the original hexasaccharide product revealed a major peak of monoisotopic m/z 1177.1 that was assigned to (M + N)+ of GlcNAc4Galz (calculated m/z 1177.4). The 'H NMR spectrum of the hexasaccharide product also confirmed its structure. The spectrum revealed reporter group signals similar to those of the acceptor
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25 Enzymatic Glycosylations with Glycosyltransferases
pentasaccharide, but also contained an additional, one-proton doublet at 4.592 ppm that was assigned H-1 of the pl,6-bonded GlcNAc. In summary, the data of Helin et al. show that only the penultimate galactose unit close to the non-reducing end of the pentasaccharide served as a primary acceptor site, despite the presence of an additional, internal galactose residue. We call the branching activity of the gastric distal site specificity and the formucosal microsomes as dIGnT to emphasize its mation of blood group 1-type structures. Interestingly, incubation of the hexasaccharide GlcNAc(pl-3)[GlcNAc( P1-6)]Gal(~1-4)GlcNAc(~l-3)Gal(~l-4)GlcNAc with UDP-GlcNAc and gastric mucosal microsomes gave a small amount of the doubly branched heptasaccharide product GlcNAc(pl-3)[ GlcNAc( 81-6)]Gal( Pl-4)GlcNAc( pl-3)[GlcNAc( pl-6)]Gal(P1-4)GlcNAc [38]. It was not clear whether an analogous, slow reaction at the mid-chain galactose had occurred also at the linear pentasaccharide GlcNAc(pl3)Gal(~lL4)GlcNAc(~1-3)Gal(~l-4)GlcNAc at a rate that was below the detection limit. Alternatively, dIGnT can catalyze reactions of different rates at the midchain galactoses of different polylactosamine acceptors. Quite recently, Yeh et al. [ 191 cloned the cDNA of C2GnT-M and expressed it in functionally active form. Besides the C2GnT activity, the recombinant enzyme had significant C4GnT activity and a small amount of dIGnT activity when tested using the acceptors GlcNAc(P1-3)GalNAcul-para-nitrophenol and GlcNAc(Pl-3)respectively. These data support Gal(pl-4)GlcNAc( ~1-6)Man(u1-6)Man~l-octyl, the notion that the dIGnT-activity of hog gastric mucosal microsomes used in the experiments of Piller et al. [36], Seppo et al. [37], and Helin et al. [38] might have represented C2GnT of M-type. Starting from GlcNAc, a polylactosamine consisting of seven N-acetyllactosamine units in a highly branched array was synthesized in our laboratory, by use of a multistep process involving the dIGnT branching reaction [ 391. Besides reactions catalyzed by hog gastric mucosal microsomes at two distinct stages, the process consisted also of several reactions catalyzed by the chain extension enzymes iGnT and P4GalT I (see below). The tetradecameric product was synthesized in milligram amounts and subsequently decorated by: (i) four distal al,3-bonded Gal residues, (ii) four distal pl,3-linked GlcNAc units [39], (iii) four distal u2,3-linked Neu5Ac residues, (iv) four peridistal a3-bonded Fuc units, and (v) a combination of four distal a2,3-linked NeuSAc and four peridistal a3-bonded Fuc units [40]. The products were characterized by NMR spectroscopy and MALDI-TOF mass spectrometry, and were studied as putative inhibitors of murine sperm-to-egg adhesion and binding of lymphocytes to capillary endothelium of inflamed tissue. A glycolipid from hog gastric mucosa contains a large, branched polylactosamine backbone [41] that is strikingly similar to the tetradecameric polylactosamine backbone of Seppo et al. [39]. 25.3.3 Central Branching of i-Type Polylactosamine Backbones
Another type of polylactosamine branching was reported by Anne Leppanen et al. [42]. In their experiments, the doubly labeled linear pentasaccharide GlcNAc(pl-3)[ 3H]Gal(p 1-4)GlcNAc( p1-3)[ I4C]Gal(p1-4)GlcNAc was converted
25.3 Enzymatic in vitvo Synthesis of Polylactosamine Backbones
655
into the branched hexasaccharide GlcNAc(P1-3)[ 3H]Gal(pl-4)GlcNAc(~1-3)[GlcNAc(P1-6)][ 14C]Gal(Pl-4)GlcNAc when incubated with UDP-GlcNAc and human serum. The product was identified by digestion with endo-P-galactosidase that cleaved the hexasaccharide completely as shown by paper chromatography. All of the 3H radioactivity of the digest was found in the disaccharide fraction, cochromatographing with the GlcNAc( pl-3)Gal marker. Nearly all of the [ 14C]label in the digest was found in a tetrasaccharide, which was identified as GlcNAc(P13)[GlcNAc(Pl-6)][ ‘‘C]Gal(~l-4)GlcNAcby partial acid hydrolysis and subsequent chromatography. These data imply that the original hexasaccharide product was cleaved by endoP-galactosidase only at the galactosidic bond of the [ 3H]Gal located close to the non-reducing end. This was compatible with the postulated structure of the hexadetersaccharide product, the GlcNAc(~l-3)[GlcNAc(~1-6)]Gal(~l-4)GlcNAc minant is known to resist the action of endo-P-galactosidase. Accordingly, the serum enzyme transferred solely to the [‘4C]-labeledmid-chain Gal of the pentasaccharide acceptor. We call this activity cIGnT to emphasize the central sitespecificity of its action. Gu et al. [43] have reported similar activity in rat serum. Later experiments showed that human serum cIGnT transfers to position 6 of the internal Gal also in the tetrasaccharide Gal(~1-4)GlcNAc(~1-3)Gal(~l-4)GlcNAc [44]. The cIGnT of rat serum gave a similar product. Accordingly, this enzyme is not sensitive to the distal galactosylation status of the chain. It works with chains capped by many different groups (Leppanen et al., unpublished experiments), but it transfers effectively only to acceptors having at least one complete Gal(p 1 4)GlcNAc unit bonded at position 3 to the acceptor Gal. The structure of the pentasaccharide product generated by rat serum cIGnT was established firmly by 2D NMR experiments, for which a relatively large sample of the pure pentasaccharide had to be prepared [45]. The tetrasaccharide Gal(814)GlcNAc(~1-3)Gal(Pl-4)GlcNAc was first synthesized in amounts of a few micromoles, starting from LacNAc that was extended in a reaction catalyzed by the iGnT activity of human serum; the resulting GlcNAc( Pl-3’)LacNAc was purified and then P1,4-galactosylated by bovine milk P4GalT I. Finally, the branching reaction of the tetrasaccharide using rat serum cIGnT gave 2.7 pmol of the pure pentasaccharide product. Enzymatic generation of multiple branches to a linear polylactosamine backbone was first described by Leppanen et al. [44]. In these experiments the linear hexasaccharide LacNAc( PI -3’)LacNAc( P 1-3’)LacNAc was converted into the doubly branched octasaccharide LacNAc(Pl-3’)[GlcNAc( p1-6’)]LacNAc( PI-3’)[GlcNAc(pl-6’)ILacNAc by incubation with UDP-GlcNAc and cIGnT activity of rat serum. Analogous experiments of Salminen et al. [46] with the linear octasaccharide LacNAc(~1-3’)LacNAc(~1-3’)LacNAc(~1-3’)LacNAc gave the triply branched undecasaccharide LacNAc( ~1-3’)[GlcNAc(~l-6’)]LacNAc(~1-3’)[GlcNAc(~l-6’)]LacNAc(~l-3’)[GlcNAc(~l-6’)]LacNAc, that was converted to the tetradecasaccharide LacNAc(~1-3’)[LacNAc(~l-6’)]LacNAc(~l-3’)[LacNAc(p1-6’)]LacNAc( p 1-3’)[LacNAc( p I -6’)lLacNAc by a treatment with UDP-Gal and P4-GalT I. Branched polylactosamine backbones of this type are expressed in human erythrocytes and embryonic carcinoma cells. Recent experiments have led to identification of cIGnT in human embryonic -
656
25 Enzymatic Glycosylations with Glycosyltransferases
carcinoma cells [47], cloning of the cDNA encoding the cIGnT of these cells [48], expression and purification of a truncated, functional form of this cIGnT [49], as well as purification of a cIGnT from hog intestinal mucosa [50].The recombinant cIGnT transfers efficiently also to Gal(~l-4)GlcNAc(~l-3)Gal(~l-4)GlcNAc(~l6)Man(al-6)Manpl-octyl, a mimic of the polylactosamine-bearing branch of Nglycans [ 191. In due time, these advances might enable transfer of multiple branches to linear polylactosamines on a large scale. 25.3.4 f34-Galactosylation in Polylactosamine Backbones
The P4GalT I from bovine and human milk was recognized as the only enzyme of its kind for a while, but novel members of this family (P4GalT-I1 to P4GalT-VI) have been isolated recently (reviewed by Ujita et al. [26]). p4GalT-IV initiates polylactosamine synthesis in the 6-linked arm of Core 2 0-glycan more effectively than P4GalT-I [26], but the bovine milk P4GalT-I elongates primary polylactosamine chains efficiently in vitro [44, 511. P4GalT-I initiates the growth of p1,6-linked GlcNAc branches of polylactosamines efficiently [52], and it also elongates P1,6bonded GlcNAc(~1-3)Gal(~1-4)GlcNAc branches [53].
25.4 a3-Sialylation of N-Acetyllactosaminoglycans at the Terminal Gal Distal u3-sialylation of branched polylactosamines was catalyzed successfully by the activity (ST3Gal IV?) present in human placental microsomes [40, 521; both 3and 6-linked short polylactosamine branches reacted well. More recently, large, tetravalent sialopolylactosamines have been synthesized in low micromolar amounts (L. Penttila et al., unpublished experiments). Even long polylactosamine chains of blood group i-type are effectively sialylated by placental microsomes [54]. Several a3-sialyltransferases have been cloned, and ST3Gal I11 is commercially available in recombinant form. A particularly interesting report of Gilbert et al. [55] describe a fusion protein consisting of CMP-Neu5Ac synthetase and a2,3-sialyltransferase from Neisseria meningitidis. This polypeptide was able to catalyze the reactions shown in eqs (2) and (3).
+ Neu5Ac + CMP-Neu5Ac + PP CMP-Neu5Ac + Gal-OR -+ Neu5Ac(a2-3)Gal-OR + CMP CTP
(2) (3)
In small-scale reactions the fusion protein worked well with glycosides of LacNAc and lactose, as well as the bi-antennary N-linked glycan, and used as donors N-acetylneuraminic acid, N-propionylneuraminic acid, and N-glycolylneuraminic acid. The two tethered enzymes were recovered by a simple procedure in functionally pure form, free from contaminating enzyme activity that can hydrolyze sugar nu-
25.5 ct3-Furosylationof Lactosamine Saccharides
651
cleotides or other components of the cofactor regeneration system. Accordingly, the fusion protein could be used in a sugar nucleotide cycle in which only catalytic amounts of expensive sugar nucleotides and transferase-inhibiting nucleoside phosphates are used; the former are continuously regenerated in situ from low cost precursors by appropriate enzyme reactions [56].In this way the fusion protein was used to produce a2,3-sialyllactose on a 100-g scale starting from lactose, sialic acid, phosphoenolpyruvate, and catalytic amounts of ATP and CMP. Taken together, the data of Gilbert et al. 15.51 show that production of the enzymes required for oligosaccharide synthesis might be simplified by fusing together carefully selected enzymes that catalyze neighboring steps in the synthetic processes.
25.5 a3-Fucosylation of Lactosamine Saccharides Gal(p1-4)[ Fuc(al-3)]GlcNAc- and NeuSAc(a2-3)Gal( pl-4)[Fuc(al-3)]GlcNAc sequences, known as Lewis’- and as sialyl Lewisx-determinants, respectively, are common distal elements in lactosaminoglycans. Lewis’ (Lex) is an adhesive sugar that can bind to other epitopes of its kind in the presence of Ca2+ [57],and sialyl Lewis’ (sLex) is the prototype ligand of the E-, P- and L-selectins, participating in leukocyte-endothelium adhesion, which is a process of considerable biomedical importance [58, 591. Also a3’-galactosylated and a2’-fucosylated LacNAc units at chain termini are acceptors for a3-fucosylation, and the products, i.e. Gal(a13)Gal(pl-4)[Fuc(a 1-3)IGlcNAc and Fuc(a 1-2)Gal( p I -4)[Fuc(ul-3)]GlcNAc, respectively, are also exciting cell adhesion molecules, participating in murine sperm-to-egg adhesion [60] and adhesion of Helicobacter pylorii to human stomach [61]. In man, at least five different transferases (FucT I11 to VII) catalyze a3-fucosylation. These reactions occur at distal LacNAc termini and also at mid-chain positions of polylactosamine extensions. Enzyme-assisted in vitro synthesis of a3-fucosylated LacNAc-glycans was initially performed with naturally occurring transferases, often with enzymes from human milk. For instance, de Vries et al. [62] showed that partially purified human milk a3/4 transferases (now assumed to be a mixture of Fuc TI11 and VI) reacted with all LacNAc units of LacNAc(~1-3’)LacNAc(~l-3’)LacNAcpl-OR converting the acceptor into the triply fucosylated product Lex(pl-3’)Lex(p1-3’)Lex~lOR [Lex, Lewis’, Gal(~1-4)[Fuc(ul-3)]GlcNAc]. More recently, Niemela et al. [63] studied partial u3-fucosylation of the free hexasaccharide LacNAc(pl-3’)LacNAc( pl -3’)LacNAc with a similar enzyme sample isolated from human milk. They found that among the three acceptor sites of the unconjugated hexasaccharide, the middle LacNAc reacted most rapidly and the non-reducing LacNAc most slowly. All individual isomers of the resulting monofucosyl- and difucosylderivatives were isolated in the pure form and characterized in these experiments. The ‘H NMR spectra of the monofucosyl hexasaccharides revealed distinct differences between the isomers.
658
25 Enzymatic Glycosylations with Glycosyltransferases
Conversion of the trisaccharide NeuSAc(a2-3’)LacNAc into the tetrasaccharide NeuSAc(a2-3’)Lex, was first performed using cloned human Fuc-TI11 [64]. In a similar experiment, de Vries et al. [65]incubated 6.6 pmol NeuSAc(a2-3’)LacNAc and 8.1 pmol GDP-[ ‘‘C]fucose for 96 h at 22 “C in 6 mL 50 mM sodium cacodylate buffer, pH 6.5 in the presence of 60 pmol MnC12 with 490 mU recombinant Fuc-TV immobilized uia protein A on 0.6 mL IgG-Sepharose. The resulting mixture was applied to a Bio-Gel P-2 column and eluted with 500 mM ammonium acetate, pH 6.8. The sialyl Lewis’ tetrasaccharide, eluting as a sharp peak at a position corresponding to 0.53 x Vtot, contained 81Y0 of the total [ 14C]fucosepresent in the reby action mixture. It was identified as Neu5Ac(a2-3)Gal(~l-4)[Fuc(al-3)]GlcNAc ID ‘ H NMR spectroscopy. Using NeuSAc(a2-3’)LacNAc( pl-3’)LacNAc( PI -3’)LacNAc as the acceptor Niemela et al. [54] showed that that the full length recombinant Fuc-TVII transfers primarily at the distal, sialylated LacNAc unit, converting the acceptor into a long chain [sialyl-Lexl-glycan. By contrast, recombinant Fuc-TIV transferred fastest to the two other LacNAc residues of this acceptor. Using Fuc-TIV and Fuc-TVII in a concerted manner, the authors synthesized in vitro the triply fucosylated determinant Neu5Ac(a2-3’)Lex(~l-3’)Lex(pl-3’)Lex,that is expressed on the P-selectin ligand PSGL-1 of HL-60 cells. Even polylactosamines carrying a3-fucosyl units at specific sites along the chains can be synthesized with enzyme preparations isolated from human milk, although these enzymes per se transfer in a rather random fashion. Fucosylation performed early, before the polylactosamine chains have been fully developed, leads to glycans which contain the Lewis’ epitope close to the reducing end [66, 671. Another possibility is to generate temporary GlcNAc branches to the linear i-type chains; the ensuing a3fucosylation reaction is prevented at the branch-bearing LacNAc units, but not elsewhere along the acceptor chain [68]. a3-Fucosyltransferase preparations isolated from human milk have also been used to convert two tetravalent [sialyl(a2-3’)LacNAc]-polylactosaminesinto the corresponding tetravalent [sialyl(a2-3’)Lex]-glycans [40,521. The fucosylation transfer was restricted to the distal, sialylated LacNAc units, because all other LacNAc residues carry branches. This restriction is probably general; all human a3fucosyltransferases seem to require the presence of a free hydroxyl group at position 6’ of the acceptor LacNAc units. As first described, these syntheses yielded ca 50-100 nmol of the final products, sufficient for Stamper-Woodruff binding experiments between lymphocytes and capillary-wall endothelium on tissue slices, MALDI-TOF mass spectrometry, and one dimensional H NMR spectroscopy. The tetravalent [sialyl-Lex] glycans proved to be very potent inhibitors of L-selectin mediated adhesion of lymphocytes to capillary endothelium of rejecting organ transplants [40, 52, 691. They also inhibited L-selectin-mediated adhesion of lymphocytes to peripheral lymph node high endothelial venules [70]. As L-selectin is expressed on lymphocyte surfaces in clustered arrays on tips of microvilli, we believe that the oligovalent [sialyl Lex] glycans might bind particularly tightly to lymphocyte L-selectin by crosslinking several monovalent L-selectin molecules in the cellular clusters. More recently, the syntheses of the two tetravalent [sialyl-Lex] glycans have been scaled up 20-50 fold, to
References
659
generate material for experiments involving in vivo inhibition of leukocyte rolling in mouse cremaster muscle capillaries (L. Penttila et al., unpublished work). Multifucosylated and terminally a3-sialylated polylactosamine chains of i-type, are also effective E-selectin ligands [71, 721. Using an enzyme sample purified from human milk, Toppila et al. [53] transferred four a3-fucose units to a bi-antennary array of five LacNAc units that was a3-sialylated at both long antennal. The resulting tetrafucosyl bisialosaccharide was a very potent inhibitor of L-selectinmediated lymphocyte-endothelium adhesion, both at capillaries of rejecting heart and at peripheral lymph nodes. Accordingly, the determinant Neu5Ac(a2-3’) Lex(pl-3’)Lex is recognized better than NeuSAc(a2-3’)Lex also by L-selectin. Of particular interest was that in the experiments of Toppila et al. (loc. cit.), the tetrafucosyl bisialosaccharide inhibitor was much more potent in its action at the rejecting heart than at the lymph node. This specificity of action suggests that by use of saccharide inhibitors of cell adhesion it might be possible to inhibit lymphocyte recruitment at inflamed tissues without endangering the normal lymphocyte patrolling that requires extravasation at lymph nodes. The molecular mechanisms responsible for this organ selectivity of the saccharide inhibitor remain unknown at present; one possibility is that endothelium of inflamed tissues carries less adhesionpromoting saccharides, or less active saccharides, than the endothelium of lymph nodes. References I. 2. 3. 4.
Laine, R. A (1994) Glycobiology, 4, 759-67. Leloir, L. F. (1971) Science 172, 1299-303. Sears, P. and Wong, C.-H. (1998) Cellular and Molecular Life Sciences 54, 223-252. Van den Steen, P., Rudd., P., Dwek. R . A., and Opdenakker, G. (1998) Crit. Rev. Biochem. Mol. Biol., 33, 151-208. 5. Jentoft, N (1990) Trends Biochem. Sci. 15, 291-94. 6. Elhammer, A. and Kornfeld, S. (1986) J. Biol. Chem. 261, 5249-55. 7. Wandall H. H., Hassan H., Mirgorodskaya E., Kristensen A. K., Roepstorff P., Bennett E. P., Nielsen P. A,, Hollingsworth M. A., Burchell J., Taylor-Papadimitriou J., Clausen H. (1997) J. Biol. Chem. 272, 23503-14. 8. Hagen, F. K. and Nehrke, K. (1998) J. Biol. Chem. 273, 8268-77. 9. Hagen, K. B., Hagen, F. K., Balys, M. M., Beres, T. M., Van Wuyckhuyse, B., Tabak, L. A. (1998) J. Biol. Chem. 273, 27749-54. 10. Hansen, J. E., Lund, O., and Brunak, S. (1997) Nucleic Acids Res. 25, 278-82. 11. Schachter H., McGuire, E. J. and Roseman, S. (1971) J. Biol. Chem. 246, 5321-28. 12. Mendicino, J., Sivakami, S., Davila, M. and Chandrasekaran, E. V. (1982) J. Biol. Chem. 257, 3987-94. 13. Cheng, P.-W and Bona, S. J. (1982) J. Biol. Chem. 257, 6251-58. 14. Amado M., Almeida R., Carneiro F., Levery S. B., Holmes E. H., Nomoto M., Hollingsworth M. A,, Hassan H., Schwientek T., Nielsen P. A., Bennett E. P., Clausen H. (1998) J. Biol. Chem. 273, 12770-08. 15. Williams D. and Schachter, H. (1980) J. Biol. Chem. 255, 11247-52. 16. Williams D., Longmore G. D.,~Matta, K. L. and Schachter, H. (1980) J. Biol. Chem. 255, 11253-61. 17. Kuhns, W., Ruth, V., Paulsen, H., Matta, K. L., Baker, M. A,, Barner, M., Granovsky, M., Brockhausen, I. (1993) Glycoconjugate J. 10, 381-394. 18. Bierhuizen, M. F. A. and Fukuda, M. (1992) Proc. Natl. Acad. Sci. USA, 89, 9326-30.
660
25 Enzymatic Glycosylations with Glycosyltransferases
19. Yeh, J-C., Ong, E. and Fukuda, M. (1999) J. Biol. Chem. 274, 3215-21. 20. Maaheimo, H., Penttila L. and Renkonen, 0. (1994) FEBS Lett. 349, 55-59. 21. Li, F., Wilkins, P. P., Crawley, S., Weinstein, J., Cummings, R. D. and McEver, R. P. (1996) J. Biol. Chem. 271, 3255-64. 22. Brockhausen I., Matta, K. L., Orr, J., and Schacter H. (1985) Biochemistry, 24, 1866-74. 23. Vavasseur, F., Yang, J.-M. Dole, K., Paulsen, H. and Brockhausen, I. (1995) Glycobiology, 5, 351-57. 24. Brockhausen, I., Orr, J. and Schachter, H. (1984) Can. J. Biochem. Cell Biol. 62, 1081-90. 25. Brockhausen, I., Williams, D., Matta, K. L., Orr, J. and Schachter, H. (1983) Can. J. Biochem. Cell Biol. 61, 1322-33. 26. Ujita, M., McAuliffe, J., Schwientek, T., Almeida, R., Hindsgaul, O., Clausen, H. and Fukuda, M. (1998) J. Biol. Chem. 273, 34843-49. 27. Yates, A. D. and Watkins, W. M. (1983) Carbohydrate Res. 120, 251-268. 28. Piller, F. and Cartron, J.-P. (1983) J. Biol. Chem. 258, 12993-99. 29. Hosomi, O., Takeya, A, and Kogure, T. (1984) J. Biochem. 95, 1655-59. 30. Podolsky D. K. (1985) J. Biol. Chem. 260, 8262-71. 31. Van den Eijnden, D. H. Winterwerp, H., Smeeman, P., and Schiphorst, W. E. C. M. (1983) J. Biol. Chem. 258, 3435-37. 32. Salo, H., Niemeli, R.. Ilves, K., Aitio, 0. and Renkonen, 0 (1998) XIXth International Carbohydrate Symposium, San Diego, Abstract BP 255. 33. Vilkman, A,, Niemela, R., Penttila, L., Helin, J., Leppanen, A,, Seppo, A,, Maaheimo, H., Lusa, S., and Renkonen, 0. (1992) Carbohydrate Res. 226, 155-74. 34. Kawashima, H., Yamamoto, K., Osawa, T. and Irimura, T. (1993) J. Biol. Chem. 268, 2711826. 35. Sasaki, K., Kurata-Miura K., Ujita, M., Angata, K., Nakagawa, S., Sekine, S., Nishi, T. and Fukuda, M. (1997) Puoc. Nutl. Acud. Sci. USA 94, 14294-99 36. Piller, F., Cartron, J-P., Maranduba, A,, Veyrieres, A,, Leroy, Y., Fournet, B. (1984) J. Biol. Chem. 259, 13385-90. 37. Seppo, A. Penttila, L., Makkonen, A,, Leppanen, A,, Niemela, R., Jantti, J., Helin, J., and Renkonen, 0. (1990) Biochem. Cell Biol. 68, 44-53. 38. Helin, J., Penttila, L., Leppanen, A., Maaheimo, H., Laauri, S. Costello, C. E., and Renkonen, 0. (1997) FEBS Lett. 412, 637-642. 39. Seppo, A,, Penttila, L., Niemela, R., Maaheimo, H., Renkonen, 0. and Keane, A. (1995) Biochemistry 34, 4655-61. 40. Seppo A,, Turunen, J.-P. Penttila, L., Keane, A. Renkonen, 0. and Renkonen, R. (1996) Glycobiology, 6, 65-71. 41. Slomiany, A. and Slomiany, B. L. (1980) Biochem. Biophys. Res. Commun. 93, 770-775 42. Leppanen A., Penttila, L., Niemela, R., Helin, J., Seppo, A,, Lusa, S., and Renkonen, 0. (1991) Biochemistry, 30, 9287-96. 43. Gu, J., Nishikawa, A,, Fujii, S., Gasa, S. and Taniguchi, N. (1992) J. Biol. Chem. 267, 2994-99. 44. Leppanen, A,, Salminen, H., Zhu, Y., Maaheimo, H., Helin, J., Costello, C. E. and Renkonen, 0. (1997) Biochemistry, 36, 7026-36. 45. Maaheimo, H., Rabina, J. and Renkonen, 0. (1997) Carbohydr. Res. 297, 145-51. 46. Salminen. H., Ahokas. K., Niemela, R., Penttila, L., Maaheimo, H, Helin, J., Costello, C. E. and Renkonen, 0.(1997) FEBS Lett. 419, 220-26. 47. Leppanen, A,, Zhu, Y., Maaheimo, H., Helin J., Lehtonen, E., Renkonen, 0. (1998) J. Biol. Chem. 273, 17399-405. 48. Bierhuizen, M. F. A. Mattei, M.-G. and Fukuda. M. (1993) Genes Dev. 7, 468-78. 49. Mattila, P., Salminen, H., Hirvas, L., Niittymaki, J., Salo, H., Niemela, R., Fukuda, M., Renkonen, 0. and Renkonen, R. (1998) J. Biol. Chem. 273.27633-39. 50. Sakamoto, Y. Taguchi, T. Tano Y., Ogawa, T., Leppanen, A., Kinnunen, M., Aitio, O., Parmanne, P., Renkonen, 0. and Taniguchi, N. (1998) J. Biol. Chem. 273, 27625-32. 51. Helin, J., Maaheimo, H., Seppo, A , Keane, A., Renkonen, 0. (1995) Carbohydrate Res. 266, 191-209. 52. Renkonen, 0, Toppila, S., Penttila, L., Salminen, H., Helin, J. Maaheimo, H., Costello, C. E., Turunen, J. P. and Renkonen, R. (1997) Glycobiology 7, 453-61.
References
661
53. Toppila, S., Renkonen, R. Penttila, L., Natunen, J., Salminen, H., Helin, J., Maaheimo, H. and Renkonen 0. (1999) Eur. J. Biochem. 261, 208-215. 54. Niemela R., Natunen, J., Majuri, M. L., Maaheimo, H., Helin, J., Lowe, J. B., Renkonen, O., and Renkonen, R (1998) J. Biol. Chem. 273, 4021--26. 55. Gilbert, M., Bayer R., Cunningham, A,-M., Defrees, S., Gao, Y., Watson, D. C., Young, N. M., and Wakarchuk, W. W. (1998) Nature Biotechnology, 16, 769-72. 56. Ichikawa, Y., Shen, G.-J., and Wong. C.-H. (1991) J. Am. Chem. SOC.113, 4698-700. 57. Eggens, I., Fenderson, B. A. Toyokuni, T., Dean, B., Stroud, M.; and Hakomori, S. (1989) J. Biol. Chem. 264, 9476-84. 58. Lasky, L. A. (1995) Annu. Rev. Biochem. 64, 113-39. 59. Springer, T. A. (1995) Annu. Rev. Physiol. 57, 827-72. 60. Johnston, D. S., Wright, W., Shaper, J. H., Hokke, C. H., van den Eijnden, D. H., Joziasse, D. H. (1998) J. Biol. Chem. 273, 1888-95. 61. Boren, T., Falk, P., Roth K. A,, Larson G., and Normark, S. (1993) Science, 262, 1892-95. 62. de Vries, T., Norberg, T., Lonn, H. and van den Eijnden, D. H. (1993) Eur. J. Biochem. 216, 769-77. 63. Niemela, R. Natunen, J. Penttila, L., Salminen, H., Helin, J., Maaheimo, H. Costello, C. E. and Renkonen 0, (1999) Glycobiology, 9: 517-526. 64. Dumas, D. P., Ichikawa, Y., Wong, C. H. Lowe, J. B. and Nair, R. P. (1991) Bioorg. Med. Chem. Lett. 8, 425-28. 65. de Vries, T. van den Eijnden, D. H., Schultz, J. and O’Neill, R. (1993) FEBS Lett., 330, 24348. 66. Kashem., M. A. Wlasichuk, K. B., Gregson, J. M. and Venot, A. P. (1993) Carbohydrate Res. 250. 129-44. 67. Rabina, J., Natunen, J., Niemela, R., Salminen, H., Ilves K., Aitio, O., Maaheimo, H., Helin, J. and Renkonen, 0. (1998) Carbohydrate Res. 305, 491-99. 68. Niemela, R., Rabina, J., Leppanen, A., Maaheimo, H Costello, C. E. and Renkonen, 0. (1995) Carbohydrate Res. 279, 331-38. 69. Turunen J. P., Majuri, M. L., Seppo, A., Seppo, A,, Tiisala, S., Paavonen, T., Miyasaka, M., Lemstrom, K., Penttila, L., Renkonen, 0. and Renkonen, R. (1995) J. Exp. Med. 182, 113342. 70. Toppila, S., Lauronen, J., Mattila, P., Turunen, J. P., Penttila, L., Paavonen, T., Renkonen, 0. and Renkonen, R. (1997) Eur. J. Immunol. 27, 1360-65. 71. Patel, T. P., Goelz, S. P., Lobb, R. R. and Parekh, R. B. (1994) Biochemistry, 33, 14815-24. 72. Stroud, M. R., Handa, K., Dalyan. M. E. K., Ito, K., Levery, S. B., Hakomori, S., Reinhold, B. B., and Reinhold V. N. (1996) Biochemistry, 35, 770-78.
Carbohydrates in Chemistry and Biology Editor Beat Ernst, Gerald W. Hart, Pierre Sinay copyright OWILEY-VCH Verlag GmbH, 2000
26 Recycling of Sugar Nucleotides in Enzymatic G1ycosylation Kathryn M. Koeller and Chi-Huey Wong
26.1 Introduction The sugar nucleotide-dependent glycosyltransferases are effective catalysts for the enzymatic synthesis of oligosaccharides, glycopeptides, and glycoproteins [ 11. These enzymes catalyze the formation of glycosidic linkages in a stereo- and regiospecific manner [2].The use of glycosyltransferases therefore largely eliminates the extensive protecting group manipulations required in the chemical synthesis of carbohydrates. Although in recent years, many of the glycosyltransferases and their sugar nucleotide substrates have become commercially available, two major drawbacks remain to utilizing glycosyltransferases for synthetic purposes. First and foremost, nucleoside diphosphates (NDPs) generated as products during transferase reactions inhibit the enzyme [3]. For micro-scale reactions this problem can be solved by addition of a phosphatase to break down the NDP product [4]. For preparative scale synthesis, however, this solution does not overcome the expense of stoichiometric quantities of sugar nucleotides. To circumvent these barriers, methods for recycling sugar nucleotides in enzymatic glycosylation have been developed [5].These multi-enzyme systems require that sugar nucleotides be present in only catalytic quantities, thereby eliminating product inhibition by NDPs and reducing expense. Regeneration strategies have been employed in multi-gram syntheses of complex carbohydrates in high yields. This chapter reviews the sugar nucleotide recycling systems utilized to date for carbohydrate synthesis and highlights related advances in the field.
26.2 Glycosyltransferasesof the Leloir Pathway and their Sugar Nucleotide Substrates A considerable number of eukaryotic glycosyltransferases have been cloned to date, each with characteristic linkage specificity and substrate preference [ 1b]. Leloir
26 Recycling of Sugar Nucleotides in Enzymatic Glycosylution
664
NH2
0.
0.
0.
no a-UDP-Glucose (UDP-Glc)
0. Ho
OH
OH
a-GDP-Mannose (GDP-Man)
“&
HO
HO
’ ” ! ! z p HOoH
Hof!)UDP
a-UDP-N-Acetylgluwsamine (UDP-GlcNAc)
a-UDP-Galactose (UDP-Gal)
0
P-GDP-L-Fucose (GDP-FUC)
CAo HOHO o& HoOUDP
PCMP-N-Acetylneuraminic acid (CMP-NeuAc)
Figure 1. Sugar nucleotide substrates for glycosyltransferases of the Leloir pathway.
pathway glycosyltransferases utilize sugar nucleotides as donor substrates, and are responsible for the synthesis of most cell-surface glycoforms in mammalian systems [6]. It is remarkable that such a broad range of enzymes has converged on eight general sugar nucleotides for use as glycosyl donor substrates (Figure 1). Glucosyl- and galactosyltransferases employ substrates activated with uridine diphosphate as the anomeric leaving group (a-UDP-Glc, a-UDP-GlcNAc, a-UDPGlcUA, a-UDP-Gal, a-UDP-GalNAc), whereas fucosyl- and mannosyltransferases utilize guanosine diphosphate (P-GDP-Fuc, a-GDP-Man). Sialyltransferases are unique in that the glycosyl donor is activated by cytidine monophosphate (PCMP-NeuAc). Preparative-scale syntheses of relevant sugar nucleotides have been developed previously [ 71. Sugar nucleotide recycling systems attempt to take advantage of the efficiency of biosynthetic pathways in enzymatic synthesis. In biological systems, simple monosaccharides are the precursors to sugar nucleotides (Figure 2) [6b]. In NDP-sugar biosynthesis an initial glycosyl phosphate is formed through kinase-mediated phosphorylation of a monosaccharide. Reaction of the glycosyl phosphate with a nucleoside triphosphate (NTP) is then catalyzed by the corre-
Gal
Glc
-
26.3 Design of Regeneration Systems
665
I--
Gal-i-P
-
e l I- , ---
Glc-6-P -Glc-1-P
GlcN-6-P
Man
J l
Glc Ac-i-P
4DP-Xyl
ManNAc
- -1NeuAc
1 UDP-GalNAc
Figure 2. Biosynthetic pathways to sugar nucleotides from simple monosaccharides.
sponding NDP-sugar pyrophosphorylase (eq. (1)). This results in the production of the activated NDP-sugar. In contrast, the nucleoside monophosphate (NMP)sugar CMP-NeuAc is formed by direct condensation of NeuAc with CTP, catalyzed by the enzyme CMP-NeuAc synthetase (eq. (2)). Sugar-1-phosphate NeuAc
+ CTP
+ NTP
+ NDP-sugar
+ CMP-NeuAc
+ PPi
+ PPi
(1)
(2)
26.3 Design of Regeneration Systems The similarities between glycosyltransferase reactions that employ NDP-sugars as substrates enable the construction of a nearly universal NDP-sugar recycling scheme (Scheme 1). In theory, the NDP that is released by the transferase can be
666
26 Recycling of Sugar Nucleotides in Enzymatic Glycosylation
NDP-sugar
pyrophosphorylase NDP-s4iar
NDP
NTpA Kinase
I!
Sugar-1-P
A;
Scheme 1. A general theoretical recycling system for NDP-sugars.
converted into the corresponding NDP-sugar in a two-step enzymatic process. Such a method would involve kinase-mediated phosphorylation of the NDP to convert it to an NTP. Condensation of the NTP with a glycosyl phosphate would then afford the activated sugar nucleotide. The regeneration of CMP-NeuAc is slightly more complicated, in that the product released by the transferase is an NMP rather than an NDP. Thus, an additional phosphorylation step and, likewise, an additional kinase are required before condensation of NeuAc with CTP can occur.
26.4 Practical Regeneration Systems In practice, the availability of the biosynthetic enzymes and substrates is often a limiting factor in the design of a sugar nucleotide recycling system. This generally makes the overall regeneration strategy more complex and lengthier than the twoenzyme theoretical scheme. The following sections describe recycling systems for sugar nucleotides that have found application in carbohydrate synthesis. 26.4.1 UDP-Galactose
In a system where UDP-Gal regeneration is the goal, the inclusion of a one-step conversion of UTP to UDP-Gal using the enzyme UDP-Gal pyrophosphorylase would be ideal. Unfortunately, this enzyme is not commercially available, nor is it easy to prepare from biological sources [8]. Luckily, an alternative biosynthetic pathway to UDP-Gal exists. In nature, UDP-Glc is a central intermediate for the biosynthesis of UDP-Gal and UDP-GlcUA, as well as other sugar nucleotides. The enzyme UDP-Glc pyrophosphorylase (UDPGP; EC 2.7.7.9) required for the conversion of UTP to UDP-Glc can be purchased. The UDP-Gal 4-epimerase
26.4 Practical Regeneration Systems
OH Ho
& O &:H
.
661
NHAcOH
A
/
NHAc
HO
HO
PPase
-
HoHO &$,.,OH
opo; OH
2 pi Scheme 2. Recycling of UDP-Gal employing UDP-Gal4-epimerase.
(UDPGE; EC 5.1.3.2) needed for the subsequent conversion of UDP-Glc to UDPGal is also readily available. A system employing these concepts was the first sugar nucleotide recycling method described in the literature. Wong et al. used this scheme to synthesize multigram quantities of N-acetyllactosamine (LacNAc) from N-acetylglucosamine (Scheme 2) [9]. This method has several salient features worth discussion that are not altogether obvious. For example, in the UDPGE-catalyzed reaction the equilibrium lies in the direction of the undesired product UDP-Glc. Continuous removal of UDP-Gal by pl,4-galactosyltransferase(P1,4-GalT; EC 2.4.1.22) drives the reaction in the direction of UDP-Gal production, however. The UDPGP substrate glucose-1-phosphate (Glc-1-P) is provided by the action of phosphoglucomutase (PGM; EC 5.4.2.2) on Glc-6-P, which is more stable and more readily available than Glc-1-P. Occasionally hexokinase (EC 2.7.1.1) can be employed to provide Glc-6-P from glucose. Finally, pyrophosphatase (PPase; EC 3.6.1.1) functions to remove the inorganic pyrophosphate product generated in the UDPGP reaction, thereby driving it forward. Other groups have used routes based on this initial concept for the synthesis of oligosaccharides [lo]. A similar recycling system was reported by Auge et al. for
668
26 Recycling of Sugar Nucleotides in Enzymatic Glycosylution
OH
HO
OR UDP-Gal
UDP
f
Scheme 3. Recycling of UDP-Gal employing sucrose synthetase for the one-pot synthesis of the a-gal epitope.
complex branched penta- and heptasaccharide synthesis [ 1I]. Thiem and Wiemann have also applied this strategy to the recycling of UDP-2-deoxy-Gal for the synthesis of 2’-deoxy-LacNAc [ 121. The UDP-Gal epimerase system was also one of the early methods to employ pyruvate kinase (PK; EC 2.7.1.40) for the conversion of UDP to UTP, with the simultaneous formation of pyruvate from phospho(eno1)pyruvate (PEP). Previous kinase systems employed were either more expensive (nucleoside diphosphate kinase(EC 2.7.4.6)/ATP) or were thermodynamically less favorable (acetate kinase(EC 2.7.2.l)/acetyl phosphate). Another potentially useful kinase system has recently been described. Noguchi and Shiba have utilized polyphosphate kinase (PPK) for the formation of UTP from UDP in a UDP-Gal recycling system [13]. Previously, PPK was shown to catalyze the reversible transfer of phosphate from ATP to ADP. Recently, it has been discovered that PPK will also accept other NDP and NTP substrates. For application to recycling systems, poly(phosphate) is more affordable than PEP as a phosphoryl donor. Synthetically, replacement of PK with PPK in the UDPGE-based recycling system efficiently provided LacNAc from GlcNAc on a multi-gram scale. The epimerase-based recycling system for UDP-Gal has been modified to contain the enzyme sucrose synthetase (EC 2.4.2.13) (Scheme 3) [14]. This enzyme catalyzes the formation of sucrose (Glc-al,2-Fru) from fructose with simultaneous conversion of UDP-Glc to UDP. The readily reversible nature of the synthetase reaction enabled the authors to employ it in the production of UDP-Glc. As previously, removal of UDP-Glc by UDPGE provided the desired forward driving force in this enzymatic cascade. Initially, this strategy was utilized for the production of LacNAc from GlcNAc. Hokke et al. then expanded this methodology to synthesis of the a-gal trisaccharide epitope, employing both a1,3-GalT and p1,4GalT in a one-pot reaction [15]. The success of this strategy relied on the fact that only the p1,4-GalT accepts the non-reducing terminal GlcNAc residue as a
26.4 Practical Regeneration Systems
HP
O
q HoOUDP
669
EzL IUTP
DVq #
“ I
I
Scheme 4. Recycling of UDP-Gal employing Gal-I-P uridyltransferase for the synthesis of LacNAc.
substrate. Subsequently, the Wang group also made use of a one-pot, two-GalT system for the synthesis of the a-gal pentasaccharide epitope [16]. In an alternative strategy for the regeneration of UDP-Gal, Wong et al. employed Gal- 1-P-uridyltransferase (Gal-1-P UT; EC 2.7.7.12) for the conversion of Gal-1-P to UDP-Gal (Scheme 4) [17]. This conversion is coupled to the formation of Glc-1-P from UDP-Glc by use of UDPGP. An attractive feature of this method is the utilization of galactose as starting material. This economical approach is possible through use of galactokinase (EC 2.7.1.6), an ATP-dependent enzyme that produces the galactosyl phosphate. LacNAc and several LacNAc derivatives have been synthesized using this recycling scheme. The Gal-1-P UT regeneration system has also been used in syntheses of glycopeptides and homogeneous glycoproteins. This UDP-Gal recycling system was combined with a subtilisin-based peptide synthesis to afford several disaccharidelinked glycopeptides [ 181. Furthermore, in a study of glycoprotein remodeling, the Gal-1-P UT method was employed to append galactose on to an N-linked GlcNAc on the surface of RNase B (Scheme 5) [19]. Further elaboration to the sialyl LewisX (sLe”) tetrasaccharide was then performed with u2,3-sialyltransferase (a2,3-SiaT) and ul,3-fucosyltransferase (al,3-FucT). Thus, recycling systems have shown their utility in simple saccharide synthesis and in the construction of complex glycopeptides and glycoproteins. 26.4.2 Other UDP-Sugars Although UDP-Glc has great utility as an intermediate in the regeneration of other sugar nucleotides, it also occasionally functions as the desired glycosyl donor.
610
26 Recycling of Sugar Nucleotides in Enzymatic Glycosylation
I -0 0
I5
26.4 Pructicul Regeneration Systems
67 1
Haynie and Whitesides have developed a regeneration scheme for UDP-Glc in the syntheses of sucrose and trehalose [20]. As previously, the sucrose synthetase conversion of UDP to UDP-Glc is coupled to the transfer of Glc from sucrose. The authors obtained the synthetase in partially purified form from wheat germ. UDPGlc is, furthermore, an intermediate in the biosynthesis of UDP-GlcUA, a donor substrate for P-glucuronosyltransferases (EC 2.4.1.17) [21]. For the recycling of UDP-GlcUA, the enzyme UDP-Glc dehydrogenase (EC 1.1.1.22) is required for the oxidation of the UDP-Glc 6-hydroxyl to the 6-carboxylic acid. This conversion is coupled with the reduction of 2 NAD+ to 2 NADH. Through this recycling system it was shown that GlcUA could be transferred to various phenolic acceptors. Instead of individually immobilized enzymes, Gygax et al. utilized a crude liver homogenate containing all the enzymes necessary for the regeneration scheme. Regenerations of UDP-GlcNAc and UDP-GlcUA have been successfully combined in the synthesis of a hyaluronic acid polymer of -1500 sugar residues (Scheme 6) [22]. In the presence of Glc-1-P and GlcNAc-1-P, both UDP-GlcUA and UDP-GlcNAc can be recycled. The presence of UDP-Glc dehydrogenase and lactate dehydrogenase are also required for the oxidation of UDP-Glc to UDPGlcUA. For this study, the hyaluronic acid synthase and most other enzymes were commercially available, although the UDP--GlcNAc pyrophosphorylase was obtained by overexpression in E. coli. A recycling system for UDP-GlcNAc has been reported for use with P1,6GlcNAc-transferase (EC 2.4.1.148) [23]. In this study, the authors attempted to use crude yeast cell extracts as the source of UDP-GlcNAc pyrophosphorylase (EC 2.7.7.23). Because of problems with contaminating phosphatases, however, this method was unsuccessful. Stepwise synthesis with whole yeast cells eventually provided the desired trisaccharide product. 26.4.3 CMP-NeuAc
The regeneration system for CMP-NeuAc is more complicated than that for NDPsugars (Scheme 7) [24]. An additional phosphorylation step must be incorporated, because CMP, a nucleoside monophosphate, is released after reaction with the sialyltransferase. For recycling purposes, nucleoside monophosphate kinase (NM K; EC 2.7.4.4) or myokinase (MK; EC 2.7.4.3) is added for the conversion of CMP to CDP. In this reaction, the phosphoryl donor is ATP. Subsequently, both CDP and ADP must be re-phosphorylated to CTP and ATP, respectively. Thus, for regeneration of CMP-NeuAc, an additional kinase and two equivalents of PEP are required. The condensation of NeuAc with CTP is catalyzed by CMP-NeuAc synthetase (EC 2.7.7.43). This system was used for the large-scale synthesis of 6’sialyl-LacNAc(6’-SLN) from LacNAc catalyzed by a2,6-SiaT (EC 2.7.7.43) in 97% yield. A recent modification of this recycling system was in the expression of an a2,3SiaT/CMP-NeuAc synthetase fusion protein [25]. This construct was more soluble than a2,3-SiaT alone, which is a poorly soluble transmembrane protein. The fusion
E7
B-co,-
PPi
-
r
J
l
UDP-GlcNAc
V
UDP-GlCUA
NADH
UTP
E6 = Lactate Dehydrogenase E7 = Inorganic Pyrophosphatase
Scheme 6 . Recycling of UDP-GlcUA and UDP-GlcNAc for the synthesis of a hyaluronic acid polymer.
HO& HO
Pi
NAD
x
= UDP-GlcNAcPyrophosphorylase
E5 = Pyruvate Kinuse
E4
E3 = UDP-Glc Pyrophosphorylase
El = Hyaluronic Acid Synthase E2 = UDP-Gk Dehydrogenase
0
R
m
ru
613
26.4 Practical Regeneration Systems
OH
H O W ; o 2 H
2Pi
€5
PPi
NeuAc
AcHN HO OH
Y CTP C
H
o OH w o
&
o
&
AcHN HO OH
OH
Ho
CMP
Scheme 7. Recycling of CMP-NeuAc for the synthesis of 3’-sialyl-LacNAc. El = a2,3-sialyltransferase; E2 = nucleoside monophosphate kinase; E.i = pyruvate kinase; Eq = CMP-NeuAc synthetase; Es = pyrophosphatase.
protein functioned efficiently at pH 7.5, even though the optimum pH for a2,3-SiaT (pH 6.0) and CMP-NeuAc synthetase (pH 8.5) differ significantly. This heterodimer was utilized in a 100-gram synthesis of 3/-sialyllactose. In another study, the CMP-NeuAc recycling system was coupled to UDP-Gal regeneration for a one-pot synthesis of 6’-SLN (Scheme 8) [26]. In the strategy reported by Ichikawa et al., NeuAc is generated from the reaction of ManNAc with pyruvate, catalyzed by NeuAc aldolase (EC 4.1.3.3). In turn, the pyruvate generated by the UDP-Gal and CMP-NeuAc recycling systems serves as a substrate for NeuAc aldolase. Remarkably, the multi-enzyme systems operated efficiently in tandem, without problems of product inhibition. Thus only the monosaccharide substrates GlcNAc, ManNAc, and Glc-1-P were required for the formation of the 6/-SLN trisaccharide. In principle, this one-pot multi-system strategy could be extended to the production of other complex oligosaccharides, such as the branched tetrasaccharide sLeX. The monosaccharide sialic acid remains relatively costly for preparative-scale synthesis. Although it has been demonstrated that NeuAc can be generated in situ by reaction of ManNAc with pyruvate, catalyzed by NeuAc aldolase [27], ManNAc is also relatively expensive and difficult to prepare. Therefore, a method for the generation of NeuAc from the inexpensive monosaccharide GlcNAc in a twoenzyme system might also prove useful in regeneration schemes. It has been shown that GlcNAc can be converted to ManNAc chemically [28], or enzymatically by the
OH NHAc
614
26 Recycling of Sugar Nucleotides in Enzymatic Glycosylution
PPi
6
noHO
on
NHAc
Scheme 8. Recycling of UDP-Gal and CMP-NeuAc for the one-pot synthesis of 6’-sialylLacNAc.
action of GlcNAc-2-epimerase [29]. Recently, an overexpression system for the epimerase was developed for industrial-scale synthesis [ 301. The resulting ManNAc product provided by the epimerase could then be condensed with pyruvate to produce NeuAc. CMP-NeuAc regeneration has also been coupled to p-galactosidase (EC 3.2.1.23) activity (Scheme 9). [31] The biological role of glycosidases is the hydro-
t
OH
ManNAc
CMP-NeuAc
Scheme 9. Combination of P-galactosidase and a2,3-SiaT for the synthesis of 3'-sialyl-LacNAc.
,OH
AcHN
Y
PEP + NeuAc
NHAc
676
26 Recycling of Sugar Nucleotides in Enzymutic Glycosylution
lysis of glycosidic linkages, but these enzymes can be made to catalyze the reverse reaction under appropriate conditions. As such, removal of product by a transferase can shift the equilibrium to the bond-forming direction. p-Galactosidases can utilize either the lactose disaccharide or a p-nitrophenyl galactoside as the galactosyl donor substrate for synthetic purposes [32]. In this respect, 0-galactosidase has been shown to be an effective catalyst in the synthesis of LacNAc and various derivatives thereof. In one system, LacNAc then served as a substrate for a2,6-SiaT in the production of 6’-SLN. Gambert and Thiem have, moreover, used this strategy in the synthesis of the sialylated Thomsen-Friedenreich antigen [33]. Compared with glycosyltransferases the use of glycosidases is advantageous in terms of stability, cost, and availability. As a drawback, glycosidases are also more promiscuous with regard to linkage specificity. Another system that couples glycohydrolase activity to glycosyltransferase activity has been reported. Ito and Paulson have developed a CMP-NeuAc recycling scheme that operates in conjunction with a trans-sialidase activity from Trypanosoma cruzi (Scheme 10) [34].This trans-sialidase catalyzes the reversible transfer of NeuAc from NeuAc(a2,3)Gal-OR to virtually any p-linked galactoside substrate. The CMP-NeuAc recycling system is employed to circumvent the expense of the sialylated pentasaccharide donor substrate. Synthetically, NeuAc is first transferred under catalysis by a2,3-SiaT. This pentato Gal(~l,3)-GlcNAc(~l,3)Gal(~1,4)Glc saccharide product is then a substrate for the trans-sialidase. Several novel sialylated molecules were produced by this technique.
26.4.4 GDP-Sugars The regeneration of GDP-Man has been employed in the synthesis of mannosebased oligosaccharides (Scheme 11) [35]. GDP-Man can be produced from Man-lP and GTP by the action of GDP-Man pyrophosphorylase (GDPMP; EC 2.7.7.22) in a manner analogous to previously described for NDP-sugars. The system described employed an a l ,2-mannosyltransferase (al,2-ManT) that was overexpressed in E. coli. The a1,ZManT accepted mannose, mannobiose, and 0-mannosyl glycopeptides as substrates. In the same fashion, GTP can be converted to GDP-Man as an intermediate in the recycling of GDP-Fuc (Scheme 12) [36]. This conversion can be accomplished by utilizing GDPMP from dried yeast cells and ‘GDP-Fuc-generating enzymes’ partially purified from the bacterium Klebsiella pneumonia. This system must be coupled to an alcohol dehydrogenase (EC 1.1.1.2), which catalyzes the oxidation of 2-propanol to acetone, along with the reduction of NADP+ to NADPH. Alternatively, Fuc-1-P can be biosynthesized from fucose by the action of fucokinase (EC 2.7.1.52) from porcine liver in the presence of ATP (Scheme 13). Reaction of Fuc-1-P with GTP catalyzed by GDP-Fuc pyrophosphorylase (EC 2.7.7.30) then affords GDP-Fuc [37]. Both of these methods have been utilized in the synthesis of sLeXfrom 3’-SLN.
26.4 Practical Regeneration Systems
QY
a
0
611
678
26 Recycling of Sugar Nucleotides in Enzymatic Glycosylation
zE
HO
HO HO
1
9
HO HO
NHCbz
f
0
OH
/
nu
GDP
HO
GTP
HO HO
OPO3'
Scheme 11. Recycling of GDP-Man for the synthesis of mannosyl glycopeptides. OH
HO OH
Sialyl Lex a1 ,&FucT
pyrophosphorylase PPiGDP-Fuc
ActlN
GW-Fuc
GDp-Man
* SynmeSIllng
OH
NHAc
NADPH
p:
dehydroge-
NADP
A
Scheme 12. Recycling of GDP-Fuc through GDP-Man for the synthesis of sLe"
PPase
2P
26.4 Practical Regeneration S y s t e m
679
680
26 Recycling of Sugar Nucleotides in Enzymatic Glycosylation
3-phosphoglycerate phosphatase
I
phosphogfucose isomerase
phosphate 4-=
Scheme 14. Multi-enzyme synthesis of D-fructose from starch.
In situ recycling methods for GDP-Fuc are not yet optimized for preparativescale synthesis. This is mainly because of difficulty obtaining adequate quantities of the key biosynthetic enzymes. GDP-Man pyrophosphorylase, ‘GDP-synthesizing enzymes’, fucokinase, and GDP-Fuc pyrophosphorylase are currently not available commercially.
26.4.5 Other Carbohydrate-Based Regeneration Systems
Other recycling systems that do not involve the synthesis of sugar nucleotides are also relevant in carbohydrate synthesis. For example, the production of fructose from starch has been accomplished in a five-enzyme system (Scheme 14) [38]. Conversion of starch to Glc-1-P is catalyzed by phosphorylase a. Isomerization to Glc6-P, and further conversion to Fru-6-P is then achieved by phosphoglucomutase and phosphoglucose isomerase, respectively. A trans-aldolase then converts Fru-6-P to fructose, with the simultaneous conversion of glycerol to 3-phosphoglycerate. The action of 3-phosphoglycerate phosphatase then irreversibly hydrolyses the phosphate, providing the forward driving force, and completing the cycle by recycling inorganic phosphate. Sulfation of carbohydrates is a topic of growing interest. The first recycling system used for the sulfation of carbohydrates was reported by Lin et al. The regeneration of 3’-phosphoadenosine 5’-phosphosulfate (PAPS), the universal biological sulfuryl donor, was accomplished by a multi-enzyme cascade (Scheme 15) [39]. In conjunction with a Nod factor sulfotransferase, N,N’-diacetylchitobiose was sulfated at the reducing terminal GlcNAc 6-hydroxyl. This sulfation system was also used to generate 6-sulfo-LacNAc, a key intermediate in the preparation of 6-sulfo-sLe' . A newly developed sulfation system accomplishes a similar goal, while also reducing the number of coupling enzymes necessary for the regeneration of PAPS (Scheme 16) [40]. In this protocol, PAPS is recycled from PAP through catalysis
J
0
n
- '\ -
n
.
HO
.\I OH
.
JCOi
0
ope,*-
E1+mco,
ATP
ir,
Scheme 15. Recycling of PAPS for the synthesis of sulfated carbohydrates.
M A O Y
O
ecoi\/*Dp
6- (PAPS)
~~
GDP-FUC al,3-FucT
~
CMP-NeuAc a2.3-SiaT
E3: Pyrophosphatase
E2: Myokinase
El: Pyruvate Kinase
SLex 6-6ulfate
Q
P
hJ
682
26 Recycling of Sugar Nudeotides in Enzymatic Glycosylation
HO
HO
0
OH
PAPS
0
OH
PAP
I 0
Scheme 16. Two-enzyme system for the recycling of PAPS.
by a single enzyme, aryl sulfotransferase. Because the only cofactor in this scheme is p-nitrophenol sulfate, the regeneration system has been greatly simplified, and is much more cost-effective. Chitotriose was found to be the best acceptor for the sulfotransferase. Notably, although LacNAc was a poor substrate, LacNAc(bl,3)LacNAc was a good acceptor in this system.
26.5 Conclusion Methods for the regeneration of sugar nucleotides in enzymatic glycosylation have proven extremely useful for synthetic purposes. Enzymatic synthesis can be both efficient and cost-effective in an appropriate system. Although recycling schemes for sugar nucleotides such as UDP-Gal and CMP-NeuAc are well-stocked by a commercial arsenal, others have not been as readily developed. For example, GDP-Fuc and UDP-GlcNAc regenerating schemes have yet to prove effective on a preparative-scale, because of lack of availability of the relevant biosynthetic enzymes. This field is, however, actively maturing with the continuous development of novel en-
References
683
zymes for use in recycling schemes. Complex substrates, such as glycopeptides, glycoproteins, and sulfated oligosaccharides, are currently being pursued to further the utility of recycling systems in carbohydrate synthesis.
References I. a) Y. Ichikawa in Glycopeptides und Related Compounds (Eds. D. G. Large, C. D. Warren), Marcel Dekker, New York, 1997, p. 79; b) P. Sears, C.-H. Wong, Cell Mol. Life Sci. 1998. 54, 223; c) C.-H. Wong, R. L. Halcomb, Y. Ichikawa, T. Kajimoto, Angew. Chem. Znt. Ed. Engl. 1995, 34, 412 and 521. 2. A. T. Beyer, J. E. Sadler, J. I. Rearick, J. C. Paulson, R. L. Hill, Adv. Enzymol. 1981, 52, 24. 3. B. W. Weston, R. P. Nair, R. D. Larsen, J. B. Lowe, J. Biol. Chem. 1992, 267, 4152. 4. C. Unverzagt, H. Kunz, J. C. Paulson, J. Am. Chen7. Soc. 1990, 112, 9308. 5. Y. Ichikawa, R. Wang, C.-H. Wong, Methods Enzymol. 1994, 247, 107. 6. a) L. F. Leloir, Science 1971, 172, 1299; b) R. Kornfeld, S. Kornfeld, Ann. Rev. Biochem. 1985, 54, 63 1. 7. C.-H. Wong, G. M. Whitesides, Enzymes in Synthetic Organic Chemistry, Pergamon, Oxford, 1994. D. 252. 8. S. E.'Heidlas, W. J. Lees, G. M. Whitesides, J. Org. Chem. 1992, 57, 152. 9. C.-H. Wong, S. L. Haynie, G. M. Whitesides, J. Org. Chem. 1982, 47, 5418. 10. a) M. M. Palcic, 0. P. Srivastava, 0. Hindsgaul, Curbohydr. Res. 1987, 159, 315; b) C. Auge, S. David, C. Mathieu, C. Gautheron, Tetrahedron Lett. 1984, 25, 1467. 11. C. Auge, C. Mathieu, C. Merienne, Curbohydr. Res. 1986, 151, 147. 12. J. Thiem, T. Wiemann, Angew. Chenz. Int. Ed. Engl. 1991, 30, 1163. 13. T. Noguchi, T. Shiba, Biosci. Biotechnol. Biochem. 1998, 62, 1594. 14. L. Elling, M. Grothus, M.-R. Kula, Glycohiology 1993, 3, 349. 15. C. H. Hokke, A. Zervosen, L. Elling, D. H. Joziasse, D. H. van den Eijnden, Glycoconjugate J. 1996, 13, 687. 16. J. Fang, J. Li, X. Chen, Y. Zhang, J. Wang, Z. Guo, W. Zhang, L. Yu, K. Brew, P. G. Wang, J. Am. Chem. Soc. 1998, 120, 6635. 17. C.-H. Wong, R. Wang, Y. Ichikawa, J. Org. Chem. 1992, 57, 4343. 18. C.-H. Wong, M. Schuster, P. Wang, P. Sears, J. Am. Chem. Soc. 1993, 115, 5893. 19. K. Witte, P. Sears, R. Martin, C.-H. Wong, J. Am. Chem. Soc. 1997, 119, 2114. 20. S. L. Haynie, G. M. Whitesides, Appl. Biochem. Biotech. 1990,23, 155. 21. D. Gygax, P. Spies, T. Winkler, U. Pfaar, Tetrahedron 1991, 47, 5119. 22. C. DeLuca, M. Lansing, 1. Martini, F. Crescenzi, G.-J. Shen, M. O'Regan, C.-H. Wong, J. Am. Chem. Soc. 1995, 117, 5869. 23. G. C. Look, Y. Ichikawa, G.-J. Shen, P.-W. Cheng, C.-H. Wong, J. Org. Chem. 1993, 58, 4326. 24. Y. Ichikawa, G.-J. Shen, C.-H. Wong, J. Am. Chem. SOC.1991,113, 4698. 25. M. Gilbert, R. Bayer, A.-M. Cunningham, S. DeFrees, Y. Gao, D. C. Watson, N. M. Young, W. W. Wakarchuk, Nat. Biotechnol. 1998, 16, 769. 26. a) Y. Ichikawa, J. L.-C. Liu, G.-J. Shen, C.-H. Wong, J. Am. Chem. Soc. 1991, 113, 6300; b) P. Stangier, W. Treder, J. Thiem, GlycoconjuguteJ. 1993, 10, 26. 27. a) C. Auge, S. David, C. Gautheron, Tetruhedron 1984,25, 4663; b) M.-J. Kim, W. J. Hennan, H. M. Sweers, C.-H. Wong, J. Am. Chem. SOC.1992,114, 10138. 28. E. S. Simon, M. D. Bednarski, G. M. Whitesides, J. Am. Chem. Soc. 1988, 110, 7159. 29. U. Kragl, D. Gygax, 0. Ghisalba, C. Wandrey, Angew. Chem. Int. Ed. Engl. 1991, 30, 82. 30. I. Maru, J. Ohnishi, Y . Ohta, Y. Tsukada, Carbohydr. Res. 1998, 306, 575. 31. G. F. Herrmann, Y. Ichikawa, C. Wandrey, F. C. A. Gaeta, J. C. Paulson, C.-H. Wong, Tetrahedron Lett. 1993, 34, 3091. 32. S. Takayama, M. Shimazaki, L. Qiao, C.-H. Wong, Bioorg. Med. Chem. Lett. 1996, 6 , 1123. 33. U. Gambert, J. Thiem, Eur. J. Org. Chem. 1999, 1, 107.
684
26 Recycling of Suyur Nucleotides in Enzymatic Glycosylation
34. Y. Ito, J. C. Paulson, J. Am. Chem. SOC.1993, 115, 7862. 35. P. Wang, G.-J. Shen, Y.-F. Wang, Y. Ichikawa, C.-H. Wong, J. Ory. Chem. 1993, 58, 3985. 36. Y. Ichikawa, Y.-C. Lin, D. P. Dumas, G.-J. Shen, E. Garcia-Junceda, M. A. Williams, R. Bayer, C. Ketcham, L. E. Walker, J. C. Paulson, C.-H. Wong, J. Am. Chem. SOC.1992, 114, 9283. 37. See also R. Stiller, J. Thiem, Liebigs Ann. Chew. 1992, 5, 467. 1992, 114, 6980. 38. A. Moradian, S. A. Benner, J. An?. Chem. SIC. 39. C.-H. Lin, G.-J. Shen, E. Garcia-Junceda, C.-H. Wong, J. Am. Chem. Soc. 1995, 117, 8031. 40. M. D. Burkart, M. Izumi, C.-H. Wong, Anyew. Clzrm. Int. Ed. Enyl. 1999, 38, 2147.
Carbohydrates in Chemistry and Biology Editor Beat Ernst, Gerald W. Hart, Pierre Sinay copyright OWILEY-VCH Verlag GmbH, 2000
27 Enzymatic Glycosylations with Non-Natural Donors and Acceptors Xiangping Qian, Keiko Sujino, and Monica M. Palcic
27.1 Introduction The demonstration that oligosaccharides can serve as recognition markers in diverse biological events [ I , 21 has stimulated interest in the synthesis of oligosaccharides and their analogs [3-71. The availability of such molecules can provide further insights into their biological functions and might lead to the discovery of novel carbohydrate-based therapeutics [ 81. Despite many advances in the chemical synthesis of oligosaccharides, it still remains a challenge, especially for the preparation of analogs where additional steps must be employed in the synthetic route to achieve a modification at a specified position [4]. As well, a replication system for amplifying minute amounts of carbohydrates is not currently available, nor is instrumentation commercially available for the solid-phase synthesis of oligosaccharides. Oligosaccharides are biosynthesized in vivo by glycosyltransferases that sequentially transfer a single pyranosyl residue from a sugar nucleotide donor to a growing carbohydrate chain [9- 121. Enzymatic synthesis using glycosyltransferase has several advantages over the chemical synthesis of oligosaccharides-high regio- and stereoselectivity, no requirement for chemical protection and deprotection, and very mild reaction conditions [ 13-1 51. Although there are more than 100 different mammalian glycosyltransferases, each biosynthesizing a unique glycosidic linkage, only nine main nucleotide donors are used as building blocks by these glycosyltransferases to construct the diverse and complex oligosaccharides found in mammals. Recognition of the donor substrates by glycosyltransferases is primarily based on the nucleotide portion. Nonnatural oligosaccharide analogs can be prepared by using glycosyltransferases to transfer a modified monosaccharide from donors. Studies probing the specificity of glycosyltransferases using acceptor analogs where the hydroxyl group is replaced with H, OCH3, NH2 or other substituents, have helped to establish structural requirements for acceptor recognition [16]. It has been found that only a few of the
686
27 Enzymatic Glycosylations with Non-Natural Donors and Acceptors
hydroxyl groups of the acceptor, termed ‘key polar groups’ [ 17- 191, are necessary for binding to glycosyltransferases. Chemical modifications can therefore be introduced at positions other than those bearing key polar groups to produce nonnatural acceptor precursors for enzymatic synthesis. With advances in molecular biology and biotechnology more than 30 glycosyltransferases have been cloned and many glycosyltransferases are now readily available in quantities sufficient for use in in vitro synthesis [20]. The increasing availability of glycosyltransferases along with their flexibility in the recognition of both donor and acceptor substrates makes enzymatic glycosylation with non-natural donors and/or non-natural acceptors an increasingly practical alternative for the preparation of non-natural oligosaccharides [ 16, 2 1-24]. The term ‘non-natural’, in the current context, is used to indicate that the acceptor or donor is chemically modified and different from the natural ones that are used by glycosyltransferases. Biosynthetically glycosyltransferases act on very large and complex glycoproteins and glycolipids, therefore small molecule acceptors might be viewed as non-natural. In this contribution, however, we do not consider a change of aglycone or adding/ removing a sugar unit(s) at the reducing end as creating a non-natural acceptor.
27.2 Enzymatic Glycosylations 27.2.1 Galactosylations
p1,4-Galactosyltransferase ~1,4-Galactosyltransferase(Pl,4-GalT, E.C. 2.4.1.22/38/90) has been commercially available for many years in unit quantities. It is the most widely studied glycosyltransferase with regard to substrate specificity and use in preparative synthesis. It catalyzes the transfer of Gal with inversion of configuration from UDP-Gal to OH4 of terminal P-linked GlcNAc to form N-acetyllactosamine. In the presence of alactalbumin, the enzyme prefers to use glucose as an acceptor to produce lactose. Non-natural donors ~l,4-Galactosyltransferasehas been shown to tolerate modifications on any OH group of the Gal residue of UDP-Gal donors. It transfers 2-deoxy-Gal at a rate similar to Gal [25-281. 3-Deoxy [29], 4-deoxy [30], 6-deoxy [31, 321, and 6-deoxy-6fluoro Gal [31, 321 are also transferred. The enzyme can also utilize UDP-Ara [30, 331, UDP-GalNAc [34], UDP-GalNH2 [27], UDP-Glc [30, 35, 361, and UDPGlcNH2 [34] as donors (Scheme 1). Interestingly, UDP-5’-thio-Gal, with the ring oxygen of Gal replaced by a sulfur atom, was active as a donor substrate for 01,4-GalT [37]. UDP-5’-thio-GalNAc was also found to be a substrate in the presence of lactalbumin [38].
27.2 Enzymatic Glycosylations
687
HO
H
I
H, NHz, NHAc
NHAc
I Non-Natural Donors I OMe, 0-Fucose, 0-Sialic acid methyl ester, H, F, SH
r-
A
H, 0x0, 0-Ally1
HO
OR
HO
NHAc
1
N33 NHAcyl
OH
HO
NHAc
OH I-Deoxy-nojirimycin
aHoaO 4 HO&
AcHN OH (-) Conduritol B
Glucal
a
" 1
" 1
H0-L
OH
AcHN
AcHN
OH
OH
-.
nu,
OH
OH
OH
I Non-Natural Acceptors Scheme 1. Non-natural donors and acceptors for P1,4-GalT.
Non-natural acceptors
As shown in Scheme 1, the acceptor specificity of B174-GalTis extremely relaxed because numerous modified GlcNAc analogs are active as acceptors. Basically, Pl,4-GalT tolerates modifications everywhere on the sugar ring, including the ring oxygen, as long as the 4-OH remains available for glycosylation.
688
27 Enzymatic Glycosylations with Non-Natural Donors and Acceptors
The 2-NHAc group can be replaced with azido [27, 391, N-propanoyl [40, 411, N-butanoyl [40], allylcarbamate [42], and many amide derivatives [43] including charged groups, highly bulky heterocycles, and glycuronamides. 2-Ethylamino-2deoxy, 2-N-methylacetamido-2-deoxy, and 2-O-acetyl-P-~-glucosidewere, however, inactive as acceptors and inhibitors for P1,4-GalT [44]. Analogs with the 3-OH group deoxygenated [44, 451, alkylated with a methyl or ally1 group, or oxidized to the ketone are active as acceptors although the relative rates of transfer are much lower than to N-acetylglucosamine [45]. The 6-OH group of GlcNAc can be methylated [41, 451, fucosylated [41], deoxygenated [44], or substituted with F or SH [44]. Although addition of a-linked sialic acid to 6-OH of GlcNAc is not tolerated [41], when the carboxylic acid of NeuAc is derivatized to the methyl ester, the resulting compound proved to be a weak acceptor for p1,4-GalT. Although the relative rate of transfer is only 4% that for GlcNAc, this is sufficient for preparative synthesis and generation of product [411. The great tolerance of pl,4-GalT for acceptor modifications is further exemplified by its transfer of Gal from UDP-Gal to 5’-thio-Glc and l-deoxynojirimycin, which have the ring oxygen modified, and to glucal, which has a flattened ring conformation [45]. The enzyme can even resolve racemic (+) conduritol B to give a single galactosylated product of (-)-conduritol B [46]. More interestingly, the enzyme transfers galactose to the p-anomeric position of 3-acetamido-3-deoxy-~glucose acceptors resulting in the formation of an unusual pl-1 (trehalose type) linkage [47]. This ‘frame-shifted’ galactosylation [48] was also observed with Nacetylgentosamine [49], N-acetyl-5’-thiogentosamine[ 501, and xylose [ 5 11 acceptors. A large variety of immobilized acceptors has also been employed in preparative reactions with Pl,4-GalT [52-571. al,3-Galactosyltransferase
a1,3-Galactosyltransferase (al,3-GalT, E.C. 2.4.1.151) catalyzes the transfer of Gal with retention of configuration from UDP-Gal to 3-OH of the Gal residue in Gal(P1-4)GlcNAc-R to form Gal(al-3)Gal(Pl-4)GlcNAc epitopes [58-611, the major xenoactive antigens responsible for hyperacute rejection in xenotransplantation [62].An enzyme for preparative synthesis has been isolated from porcine and bovine tissues and recombinant porcine al,3-galactosyltransferase is now commercially available in unit quantities. Non-natural donors Unlike pl,4-GalT, al,3-galactosyltransferase has a rather restricted specificity for the nucleotide donor substrate as revealed by studies with recombinant murine al,3-GalT [63]. Although UDP-Glc, UDP-GalNAc, UDP-GlcNAc, and UDPglucuronic acid are not substrates for the enzyme [63], UDP-2-deoxy-Gal is a better substrate than UDP-Gal. The relative rates of transfer of 3-deoxy, 4-deoxy, or 6deoxy donors are 0.2, 0.6 and 2% that of UDP-Gal [64].
27.2 Enzymatic Glycosylutions
689
I Non-Natural DonorsI
H, OCHzR
OH, N3! Succinimido, NHCHO, NHAcyl
1
6 -
HO
OH
OR
H, OCH2R
N HAc
v
H -
Non-Natural Acceptors Scheme 2. Non-natural donors and acceptors for ctl,3-GalT.
Non-natural acceptors
Besides using Gal(p1-4)GlcNAc as an acceptor, the enzyme can transfer Gal to Gal(plL3)GlcNAcand Gal(al-4)Glc [65]. Substitutions of 2-NHAc of the GlcNAc residue with azido and succinimido groups are tolerated [63]. The N-acetyl group can be replaced with many acyl groups of various sizes, hydrophilicities, or lipophilicities [66], although replacement of the 2-NHAc with an amino group abolishes activity [63]. Deoxygenation of the 3-OH of the GlcNAc residue is tolerated whereas any substitution or derivatization at this position is not. As shown in Scheme 2, analogs with modifications (deoxygenation and 0-alkylation) on 6-OH of GlcNAc, or 2-OH, and 6-OH of the terminal Gal residue are substrates [67]. Modification on 4-OH of Gal is not tolerated by the enzyme, suggesting that this group is a key polar group essential for binding to a1,3-GalT [67]. The 4-deoxy analog of Gal(p1-3)GlcNAcp-OR was found to be as active as Gal(p1-3) GlcNAcp-OR [67].
OR
690
27 Enzymatic Glycosylations with Non-Nutural Donors and Acceptors
27.2.2 Fucosylations The fucosylated oligosaccharides on cell surfaces are involved in numerous intercellular recognition events [ 11. al,3/4-Fucosyltransferasescatalyze the transfer of a fucose residue with retention of configuration from GDP-fucose to a variety of acceptors to form the blood-group related antigenic determinants Lea and LeX[68]. al,3/4-Fucosyltransferasesare a multigene family of enzymes with different substrate specificities toward Gal(p1-4)GlcNAc, Gal(pl-3)GlcNAc, and NeuAc(a23)Gal(p1-4)GlcNAc acceptors, inhibitor sensitivity, pH optima, and tissue distributions [12]. The most common source for isolation of a preparatively useful fucosyltransferase is human milk [ 101. There are two different human milk fucosyltransferases, al,3/4-fucosyltransferase and al,3-fucosyltransferase.Five different human al,3/4-fucosyltransferaseshave been cloned, FucT 111 to FucT VII, and both FucT V and VI are commercially available. Human Milk a1,3/4-Fucosyltransferase Non-natural donors
GDP-3-deoxyfucose, GDP-arabinose and GDP-L-galactose are active as donor substrates for human milk a1,3/4-FucT (E.C. 2.4.1.65) when Gal(plL3)GlcNAcpO(CH2)gCOOMe is used as an acceptor [69]. GDP-3-deoxy-Fuc and GDP-L-Gal were shown to be preparatively useful, using Gal( pl-4)GlcNAcp-O(CH2)&OOMe as an acceptor for human milk a1,3/4-FucT [70]. The enzyme was also shown to tolerate the addition of a propyl group at 6-OH of L-Gal in GDP-L-Gal. Large substituents, e.g. tethered blood group B trisaccharide, can be introduced at C-6 of the Fuc residue in GDP-Fuc [71].Essentially, any group can be attached to the C-6 position of FUC,as was demonstrated by the transfer of modified fucose residues containing biotin and blood group A trisaccharide biolabels (Scheme 3 ) [72]. Non-natural acceptors
Human milk a1,3/4-FucT uses both Gal(p1-3)GlcNAc (type I) and Gal(p14)GlcNAc (type 11) acceptors. Chemical mapping studies with a series of monodeoxygenated and modified acceptor substrates showed that modifications are tolerated at every hydroxyl group in the sugar rings except 6-OH of the Gal and 3- or 4-OH of the GlcNAc residue to which fucose is transferred [73].The 2-NHAc group of the GlcNAc residue in NeuAc(a2-3)Gal(~l-4)GlcNAc~-O(CH~)~COOMe or NeuAc(a2-3)Gal(~1-3)GlcNAc~-O(CH~)~COOMe can be replaced with azido, amino, or propionamido groups [74].Thio-linked N-acetyllactosamine, in which the inter-glycosidic oxygen is replaced by sulfur, is also a good acceptor for the enzyme [75]. Ether- and imino-linked octyl N-acetyl-5a’-carba-~-lactosamides were also found to be acceptors for human milk al,3/4-FucT [76]. Surprisingly, human milk al,3/4-FucT can even tolerate the introduction of a large methyl group directly at the site of fucosylation; it transfers fucose to the hindered tertiary alcohol acceptor 3-C-methyl-N-acetyllactosamine[ 771. This unexpected finding demonstrated that glycosyltransferases can overcome inherent steric limitations and can be used to
27.2 Enzymatic Glycosylations
CHPOH, H &OH
691
0-GDP
I I
CH20CH2CH2CH3
0 aGal( 1-13),
aFuc(l+2)
0
II
L N , C 6 - F u c PGal-0 H
,
I Non-Natural Donors I
NHAc OR
OR
NH
S
HO
H AcHN O W
i
@0 O
o
%
OH
NHAc
HO
I Non-Natural Acceptors1 Scheme 3. Non-natural donors and acceptors for human milk a1,3/4-FucT.
OR
692
27 Enzymutic Glycosylations with Non-Natural Donors and Acceptors
produce oligosaccharide analogs that cannot easily be prepared by chemical methods. FucT I11 and VI FucT I11 transfers fucose on to the 4-OH group of GlcNAc residues of Gal(b1-3)GlcNAc or NeuAc(a2-3)Gal( PI-3)GlcNAc to form Lea or sialyl Lea respectively, whereas FucT VI uses Gal(PI-4)GlcNAc or NeuAc(a2-3)Gal(~l-4)GlcNActo form LeXor sialyl Le" products. Despite their close sequence homologies, they behave differently in their recognition of non-natural donors [78]. FucT 111 transfers LGal, arabinose, L-Glc, 2-amino-2-deoxyfucose, and 2-fluoro-2-deoxyfucosewhereas FucT VI cannot tolerate modifications on the 2-OH group of the fucose. FucT VI still transfers L-Gal and arabinose, however. Both enzymes tolerate diverse replacements of the N-acetyl group of the GlcNAc unit, as seen with Pl,4-GalT [78SO]. FucT VI has been shown to tolerate replacement of the GlcNAc unit with glucal and even with cyclohexane diol[24]. The utility of FucT I11 and FucT VI has further been expanded to enable construction of libraries of sialyl Lea or sialyl Le" derivatives using both non-natural donors and non-natural acceptors (Schemes 4 and 5) [Sl-831. FucT I11 and FucT VI have been probed with non-natural donors where the purine base was modified. Both enzymes can tolerate exchange of the guanine by other purine bases such as adenine [84]. These non-natural donors proved to be preparatively efficient in the enzymatic synthesis of Lea or Le", suggesting that costly sugar nucleotide donors can be replaced with inexpensive ones to reduce the cost of enzymatic synthesis. FucT V Similar to human milk a1,3/4-FucT, FucT V requires the 6-OH group of Gal and OH-3 or OH-4 of GlcNAc for substrate binding. Gal(P1-4)Glucal [39], Gal(P1-4) (-5-S)-Glc [39], 3-sulfo-Gal(Pl-4)GlcNAc [85] and Gal(~l-4)-6-sulfo-GlcNAc[85] are acceptors for FucT V. 27.2.3 Sialylations
Cell-surface sialic acid residues play important roles in diverse biological processes [86]. Sialic acids are usually found in terminal positions linked through an a-glycosidic linkage. The stereoselective synthesis of a-sialosides remains a challenge because the glycosides have the thermodynamically unfavorable equatorial orientation and the anomeric carbon is a very hindered quaternary center [4]. Enzymatic sialylation is, therefore, an attractive alternative means of preparation of a-sialosides in an efficient and stereocontrolled manner. a2,3-Sialyltransferase and a2,6-Sialyltransferase
a2,3-Sialyltransferase and a2,6-sialyltransferase from rat liver (a2,3-SialT, E.C. 2.4.99.6; a2,6-SialT, E.C. 2.4.99.1) have been cloned and expressed and are now
27.2 Enzymatic Glycosylations
693
{f: N
L-Glc
I
I CHPOH,H
OH OH
1 Non-Natural Donors I
HOWi&\EJ HO
AcHN
OH
HO
NHAc
NHAcyl
OR
I
I Non-Natural Acceptors) Scheme 4. Non-natural donors and acceptors for FucT 111.
commercially available in quantities sufficient for use in preparative synthesis. a2,3SialT from rat liver transfers a sialic acid unit from CMP-sialic acid to OH-3 of the terminal Gal residue in Gal(P1-4)GlcNAc or Gal(pl-3)GlcNAc sequences whereas rat liver a2,6-SialT transfers the sialic acid to OH-6 of the terminal Gal residue in Gal@-4)GlcNAc [87]. Non-natural donors
The finding that CMP-sialic acid synthetase can accept many sialic acid analogs has facilitated the synthesis of CMP-sialic acid analogs for studies of the donorspecificity of these enzymes [88-901. Both a2,3-SialT and a2,6-SialT tolerate substitutions at C-9 of CMP-sialic acid (Schemes 6 and 7) [89, 91, 921. The 9-OH group can be replaced with fluoro, azido, amino, acetamido, hexanoylamido, benzamido, and even fluorescent labels. 9-0-Acetyl sialic acid can also be transferred. Donors in which the 5-NHAc group of sialic acid is replaced with OH, NHC(O)CHzOH, or NHCbz (Cbz = benzyloxycarbonyl) are also accepted by a2,3-SialT [93]. CMP-4-deoxysialic acid was found to be a donor substrate for a2,6-SialT [94].
694
27 Enzymatic Glycosylations with Non-Natural Donors and Acceptors
OH OH
I Non-Natural Donors)
NHAcyl
Ho
A
C
H HO
N
~
HO
O ,H
OH
I Non-Natural Acceptors] Scheme 5. Non-natural donors and acceptors for FucT VI.
Non-natural acceptors
Chemical mapping studies have indicated that OH-6 of Gal and 2-NHAc are required for binding to rat liver a2,6-SialT whereas rat liver a2,3-SialT requires an intact 3,4,6-triol system on the Gal residue [95].Analogs of Gal(pl-4)GlcNAc(pl2)Mana-O-Oct, where the 3- or 4-OH group of the Gal residue is deoxygenated or substituted with a fluoro atom, were found to be active as substrates for a2,6-SialT (Scheme 7) [96]. The 4/’-O-methyl derivative of the trisaccharide is an acceptor for a2,3-SialT [96]. A wide range of substitutions on the N-acetyl group of both type I or type I1 acceptors were accepted by a2,3-SialT [97, 981. a2,3-SialT also transfers sialic acid to lactal, lactose, and 2-0-pivaloyllactose [39].
27.2 Enzymatic Glycosylutions F, N3, NH2, NHAcyl, NH-FIuows, OAC
695
0
cop
AcHN
HO
I
OH
Ho OH, NHCOCHzOH, NHCbz
I Non-Natural DonorsI N3, NH2, NHCOCH2CH3, NHAcyl, OH, OCOC(CH3)3 H, Br H H, OCH3
HO
OR
HO
I H
N3, NH2, NHAcyl
OMe
-@
I
H
,OH
OOct
1 Non-Natural Acceptor3 Scheme 6. Non-natural donors and acceptors for a2,3-SialT
696
27 Enzymatic Glycosylutions with Non-Natural Donors and Acceptors F, N3, NH2, NHAcyl, NH-FIuo~~s, OAC
0
AcHN
I Non-Natural Donors I
I
ooci
I Non-Natural Acceptors) Scheme 7. Non-natural donors and acceptors for a2,6-SialT.
27.2.4 N-Acetylglucosaminylation The branching pattern of complex N-glycans is controlled by a series of N-acetylglucosaminyltransferases numbered GnT I-VI [ 12, 991. These enzymes transfer an N-acetylglucosamine residue from UDP-GlcNAc to different sites on the tri-mannose core structure Man(a1-6 [Man(al-3)]ManfJ as shown in Scheme 8. Biosynthetically, N-acetylglucosaminyltransferases catalyze the transfer of a GlcNAc residue from UDP-GlcNAc to oligosaccharide acceptors having the trimannose core structure attached to GlcNAc(81-4)GlcNAcp-Asn. The finding that GlcNAc(~1-4)GlcNAc~-Asn can be replaced with a hydrophobic group made it feasible to study the donor and acceptor specificities of these enzymes using smaller oligosaccharide acceptors which are readily prepared [ 100, 1011. N-AcetylglucosaminyltransferaseI, 11, and I11
The C-3, C-4, and C-6 deoxy analogs of UDP-GlcNAc were found to act as donor substrates for GnT I (E.C. 2.4.1.101) from human milk using Man(a1-6 [Man(al-
27.2 Enzymutic Glycosylutions
GnT VI
Hm
HO HO
GnT 111
GnT IV -HO
697
HoJ
H
AH-
O
=
o
~
e
~
&
HO Asn-R
\OH
NHAc
GnTl
Scheme 8. Glycosylation sites for GnT ILVI.
OMe OR
H
HO
m
OOct
HNHAc 0-UDP
Non-Natural Donors
HO
IGnTl
Non-Natural Acceptors
Scheme 9. Non-natural donors and acceptors for GnT I
3)]Manp-O(CH2)sCOOMeas the acceptor [ 1021. The tetrasaccharide products of the above reactions have been evaluated with GnT I1 [102]. Deoxygenation at C-3 of GlcNAc unit was not tolerated by GnT I1 and removal of the 4-OH or 6-OH of GlcNAc caused only modest decreases in activity [ 1021. As shown in Scheme 9, GnT I can use non-natural acceptors that are deoxygenated or 0-alkylated on the aMan(1-6) residue [103-1051. Alkylations at OH-4 of clMan1-3, the mannose residue to which transfer occurs, are tolerated. Analogs where OH-3, 4, or 6 of the aManl-6 residue was deoxygenated or methylated were found to be substrates of GnT I1 (Scheme 10) [103].
698
27 Enzymatic Glycosylations with Non-Natural Donors und Acceptors
H,OMe H,OMe
-
0 NHAc
H,OMe
I
-
HO
ooct H,OMe
-
H
#" HoHO
Scheme 10. Non-natural acceptors for GnT I1 and 111.
Deoxygenation at C-4 [ 1061, C-6 [ 1071 or methylation at C-4 [99, 1031 of aMan13 are also tolerated by GnT XI. A dideoxygenated pentasaccharide, which was chemo-enzymatically synthesized by use of GnT I1 [ 1061, and a di-0-methylated pentasaccharide were active as substrates for GnT 111 (E.C. 2.4.1.144) producing the expected hexasaccharide analogs [108, 1091. N-AcetylglucosaminyltransferaseV
There has been particular interest in N-acetylglucosaminyltransferase V (GnT V, E.C. 2.4.1.155) because the activity of this enzyme has been shown to correlate with the metastatic potential of tumor cell lines [ 110-1 141. Whereas a heptasaccharide is the smallest physiological substrate, trisaccharides GlcNAc(~l-2)Man(alL6)ManP-OR[115, 1161 and GlcNAc(P1-2)Man(al-6)GlcP-OR [ 1171 were found to be excellent acceptors for GnT V. The 0-GlcNAc residue can also be replaced by a P-Glc residue [ 1181. Studies using analogs where OH-3, 4, or 6 of the P-GlcNAc residue of GlcNAc(~l-2)Man(al-6)Glc~-OR were modified indicated that an intact 3,4,6-triol system is required for recognition by GnT V [ 119, 1201. Modifications on the a-Man residue and P-Glc residues are tolerated by this enzyme (Scheme 11) [117, 121-1231. Kinetic studies using restricted trisaccharide analogs revealed that GnT V preferentially recognizes the gg rotamer [124]. Analogs where the oxygen atoms in the glycosidic linkages were replaced with sulfur are also good acceptors for GnT V producing the corresponding tetrasaccharides [ 1251.
699
27.2 Enzymatic Glycosylutions
\ o
8'
c
3
d
X 8
I 0
Z
c
o x I
-0
8
0
s
700
27 Enzymatic Glycosylations with Non-Natural Donors and Acceptors
27.3 Summary Glycosyltransferases are remarkably flexible in their donor and acceptor specificities. The use of glycosyltransferases greatly simplifies the synthetic procedure for the preparation of non-natural oligosaccharides starting from non-natural donors and/or non-natural acceptors. With progress in molecular biology, even more glycosyltransferases will become available in sufficient quantities for use in chemical synthesis. As the structures of glycosyltransferases become well defined, they can be tailored using site-directed mutagenesis to have enhanced stabilities, even broader specificities and high affinities towards non-natural substrates while maintaining regio- and stereospecificity. Large-scale synthesis of oligosaccharides using a CMPfusion has been reported [ 1261. CombinNeuSAc synthetase/a2,3-~ialyltransferase ing two or more enzymes into a fusion protein will simplify the production and purification of enzymes, reduce costs and facilitate synthesis. As improved and alternate methods for the regeneration [ 1I] and large-scale production [ 1271 of sugar nucleotide donors are developed, enzymatic glycosylation will become increasingly routine for the construction of large quantities of many non-natural oligosaccharides. Acknowledgments Research support from the Natural Sciences and Engineering Research Council of Canada (to M.M.P.) and the Alberta Research Council for a graduate scholarship in Carbohydrate Chemistry (to X.Q.) are gratefully acknowledged.
References 1. 2. 3. 4.
Varki, A. Glycobiology 1993, 3, 97. Dwek, R. A. Chem. Rev. 1996, 96, 683. Toshima, K.; Tatsuta, K. Chem. Rev. 1993, 93, 1603. Khan, S. H.; Hindsgaul, 0. In Moleculur Clycobiology; Fukuda, M., Hindsgaul, 0. Eds.; Oxford: New York, 1994; pp 206. 5. Barresi, F.; Hindsgaul, 0. J. Carbohydr. Chem. 1995, 14, 1043. 6. Boons, G.-J. Tetrahedron 1996, 52, 1095. 7. Schmidt, R. R. In Glycosciences;Gabius, H.-J., Gabius, S. Eds.; Chapman & Hall: Weinheim, 1997; pp 31-53. 8. McAuliffe, J.; Hindsgaul, 0. Chem. Ind. 1997, 170. 9. Wong, C.-H.; Whitesides, G. M. Enzymes in Synthetic Organic Chemistry; Elsevier: Oxford, 1994; pp. 252-311. 10. Palcic, M. M. Methods Enzjwzol. 1994, 230, 300. 11. Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T. Anyew. Chem., Int. Ed. Engl. 1995, 34, 521. 12. Brockhausen, I.; Schachter, H. In Glycosciences;Gabius, H.-J., Gabius, S. Eds.; Chapman & Hall: Weinheim, 1997; pp 79-113. 13. Gijsen, H. J. M.; Qiao, L.; Fitz, W.; Wong, C.-H. Chem. Rev. 1996, 96, 443. 14. Takayama, S.; McGarvey, G. J.; Wong, C.-H. Chem. Soc. Rev. 1997,26,407.
References
701
15. Ichikawa, Y. In Glycopeptides and Related Compounds: Synthesis, Analysis and Application, Large, D. G., Warren, C. D. Eds.; Dekker: New York, 1997; pp 79-205. 16. Palcic, M. M.; Hindsgaul, 0. Trends Glycosci. Glycotechnol. 1996, 8, 37. 17. Lemieux, R. U. In Proc. VIIIth Int. Symp. Med. Chem.; Swedish Pharmaceutical Press: Stockholm, 1985; pp. 329-351. 18. Lemieux, R. U. Chem. SOC.Rev. 1989, 18, 347. 19. Lemieux, R. U. Acc. Chem. Res. 1996,29, 373. 20. Sears, P.; Wong, C.-H. Cell. Mol. Life Sci. 1998, 54, 223. 21. Matta, K. L. In Modern Methods in Carbohydrate Synthesis; Khan, S . H.; O’Neil, R. A. Eds.; Harwood Academic: Amsterdam, 1995; pp 437-466. 22. Crawley, S. C.; Palcic, M. M. In Modern Methods in Carbohydrate Synthesis; Khan, S. H.; O’Neil, R. A. Eds.; Hanvood Academic: Amsterdam, 1995; pp 492-517. 23. Elhalabi, J. M.; Rice, K. G. Curr. Med. Chem. 1999, 6, 93. 24. Ohrlein. R. Top. Curr. Chem. 1999, 200, 227. 25. Thiem, J.; Wiemann, T. Angew. Chem., Intl. Ed. Engl. 1991, 30, 1163. 26. Thiem, J.; Wiemann, T. Synthesis 1992, 141. 27. Wong, C.-H.; Wang, R.; Ichikawa, Y. J. Org. Chem. 1992, 57, 4343. 28. Srivastava, G.; Hindsgaul, 0.;Palcic, M. M. Carbohydr. Res. 1993, 245, 137. 29. Hindsgaul, 0.;Kaur, K. J.; Gokhale, U. B.; Srivastava, G.; Alton, G.; and Palcic, M. M. ACS Symp. Ser. 1991, 466, 38. 30. Berliner, L. J.; Robinson, R. D. Biochemistry 1982, 21, 6340. 31. Kodama, H.; Kajihara, Y.; Endo, T.; Hashimoto, H. Tetrahedron Lett. 1993, 34, 6419. 32. Kajihara, Y., Endo, T., Ogasawara, H., Kodama, H.; Hashimoto, H. Carbohydr. Rex 1995, 269, 273. 33. Arlt, M.; Hindsgaul, 0. J. Org. Chem. 1995, 60, 14. 34. Palcic, M. M.; Hindsgaul, 0. Glycobiology 1991, I , 205-209. 35. Do, K.-Y.; Do, S.-I.; Cummings, R. D. J. Biol. Chem. 1995, 270, 18447. 36. Andree, P. J.; Berliner, L. J. Biochim. Biophys. Acta 1978, 544, 489. 37. Yuasa, H.; Hindsgaul, 0.;Palcic, M. M. J. Am. Chem. Soc. 1992, 114, 5891. 38. Tsuruta, 0.;Shinohara, G.; Yuasa, H.; Hashimoto, H. Bioorg. Med. Chem. Lett. 1997, 7,2523. 39. Ichikawa, Y.; Lin, Y.-C.; Dumas, D. P.; Shen, G.-J.; Garcia-Junceda, E.; Williams, M. A,; Bayer, R.; Ketcham, K.; Walker, L. E.; Paulson, J. C.; Wong, C.-H. J. Am. Chem. Soc. 1992, 114, 9283. 40. Berliner, L. J.; Davies, M. E.; Ebner, K . E.; Beyer, T. A.; Bell, J. E. Mol. Cell. Biochem. 1984. 62, 37. 41. Palcic, M. M.; Srivastava, 0. P.; Hindsgaul, 0. Carbohydr. Res. 1987, 159, 315. 42. Ohrlein, R.; Ernst, B.; Berger, E. G. Carbohydr. Res. 1992,236, 335. 43. Baisch, G.; Ohrlein, R.; Ernst, B. Bioory. Med. Chem. Lett. 1996, 6, 749. 44. Kajihara, Y.; Kodama, H.; Endo, T.; Hashimoto, H. Carbohydr. Res. 1998, 306, 361. 45. Wong, C.-H.; Ichikawa, Y.; Krach, T.; Gautheron-Le Narvor, C.; Dumas, D. P.; Look, G. C. J. Am. Chem. SOC.1991, 113, 8137. 46. Yu, L.; Cabrera, R.; Ramirez, J.; Malinovskii, V. A,; Brew, K.; Wang, P. G. Tetruhedron Lett. 1995, 36, 2897. 47. Nishida, Y.; Wiemann, T.; Sinnwell, V.; Thiem, J. J. Am. Chem. Soc. 1993, 115, 2536. 48. Gambert, U.; Thiem, J. Top. Curr. Chem. 1997, 186, 21. 49. Nishida, Y.; Wiemann, T.; Thiem, J. Tetrahedron Lett. 1992, 33, 8043. 50. Nishida, Y.; Wiemann, T.; Thiem, J. Tetrahedron Lett. 1993, 34, 2905. 51. Wiemann, T.; Nishida, Y.; Sinnwell, V.; Thiem, J. J. Org. Chem. 1994, 59, 6744. 52. Nishimura, S.-I.; Lee, K. B.; Matsuoka, K.; Lee, Y. C. Biochem. Biophys. Res. Commun. 1994, 199, 249. 53. Matsuoka, K.; Nishimura, S.-I. Macromolecules 1995, 28, 2961. 54. Wiemann, T.; Taubken, N.; Zehavi, U.; Thiem, J. Carbohydr. Res. 1994,257, C1. 55. Kopper, S. Carbohydr. Res. 1994,265, 161. 56. Schuster, M.; Wang, P.; Paulson, J. C.; Wong, C.-H. J. Am. Chem. Soc. 1994, 116, 1135. 57. Meldal, M.; Auzanneau, F.-I.; Hindsgaul, 0.;Palcic, M. M. J. Chem. Soc., Chem Commun. 1994, 1849.
102
27 Enzymatic Glycosylations with Non-Natural Donors and Acceptors
58. Joziasse, D. H.; Shaper, N. L.; Salyer, L. S.; van den Eijnden, D. H.; van der Spoel, A. C.; Shaper, J. H. Eur. J. Biochem. 1990, 191, 75. 59. Fang, J.; Li, J.; Chen, X.; Zhang, Y.; Wang, J.; Guo Z.; Zhang, W.; Yu, L.; Brew, K.; Wang, P. G. J. Am. Chem. Soc. 1998,120,6635. 60. Hokke, C. H.; Zervosen, A.: Elling, L.; Joziasse, D. H.; van den Eijinden, D. H. Glycoconjugate Journal 1996, 13, 687. 61. Galili, U. Immunol. Today 1993, 14, 480. 62. Butler, D. Nature 1998, 391, 320. 63. Stults, C. L. M.; Macher, B. A.; Bhatti, R.; Srivastava, 0. P.; Hindsgaul, 0. Glycobiology 1999, 9, 661. 64. Sujino, K.; Uchiyama, T.; Hindsgaul, 0.;Seto, N. 0. L.: Wakarchuk, W. W.; Palcic, M. M. J. Am. Chem. SOC.2000, 122, 1261. 65. Blanken, W. M.; Van den Eijnden, D. H. J. Biol. Chem. 1985,260, 12927. 66. Baisch, G.; Ohrlein, R.; Kolbinger, F.; Streiff, M. Bioorg. Med. Chem. Lett. 1998, 8, 1575. 67. Sujino, K.; Malet, C.; Hindsgaul, 0.;Palcic, M. M. Carbohydr. Rex 1998, 305, 483. 68. Erika, S. Trends Glycosci. Glycotechnol. 1996, 8, 391. 69. Gokhale, U. B.; Hindsgaul, 0.;Palcic, M. M. Can. J. Chem. 1990, 68, 1063. 70. Stangier, K.; Palcic, M. M.; Bundle, D. R.; Hindsgaul, 0. Thiem, J. Curbohydr. Res. 1998, 305, 511. 71. Srivastava, G.; Kaur, K. J.; Hindsgaul, 0.;Palcic, M. M. J. Biol. Chem. 1992, 267, 22356. 72. Hallgren, C.; Hindsgaul, 0. J. Curbohydr. Chem. 1995, 14, 453-464. 73. Gosselin, S.; Palcic, M. M. Bioorg. Med. Chem. 1996, 4, 2023. 74. Nikrad, P. V.; Kashem, M. A,; Wlasichuk, K. B.; Alton, G.; Venot, A. P. Carbohydr. Res. 1993,250, 145. 75. Ding, Y . ;Hindsgaul, 0.; Li, H.; Palcic, M. M. Bioorg. Med. Chem. Lett. 1998, 8, 3199. 76. Ogawa, S.; Matsunaga, N.; Li, H.; Palcic, M. M. Eur. J. Org. Chem. 1999, 631. 77. Qian, X.; Hindsgaul, 0.;Li, H.; Palcic, M. M. J. Am. Chem. Soc. 1998, 120, 2184. 78. Baisch, G.; Ohrlein, R.; Katopodis, A,; Streiff, M.; Kolbinger, F. Bioorg. Med. Chem. Lett. 1997, 7, 2441. 79. Baisch, G.; Ohrlein, R.; Katopodis, A.; Ernst, B. Bioorg. Med. Chem. Lett. 1996, 6, 759. 80. Baisch, G.; Ohrlein, R.; Streiff, M. Bioorg. Med. Chem. Lett. 1998, 8, 161. 81. Baisch, G.; Ohrlein, R.; Katopodis, A. Bioorg. Med. Chem. Lett. 1997, 7, 2431. 82. Baisch, G.; Ohrlein, R.; Streiff, M.; Kolbinger, F. Bioorg. Med. Chem. Lett. 1998, 8, 755. 83. Baisch, G.; Ohrlein, R. Bioorg. Med. Chem. 1998, 6, 1673. 84. Baisch, G.; Ohrlein, R.; Katopodis, A. Bioorg. Med. Chem. Lett. 1996, 6, 2953. 85. Chandrasekaran, E. V.; Jain, R. K.; Larsen, R. D.; Wlasichuk. K.; DiCioccio, R. A.; Matta, K. L. Biochemistry 1996, 35, 8925. 86. Reuter, W.; Stache, R.; Stehling, P., Baum, 0. In Glycosciences; Gabius, H.-J., Gabius, S. Eds.; Chapman & Hall: Weinheim, 1997; pp 245-259. 87. Harduin-Lepers, A,; Recchi, M.-A.; Delannoy, P.; Glycobiology 1995, 5 , 741. 88. Higa, H. H.; Paulson, J. C. J. Biol. Chem. 1985, 260, 8838. 89. Gross, H. J.; Bunsch, A.; Paulson, J. C.; Brossmer, R. Eur. J. Biochem. 1987, 168, 595. 90. Brossmer, R.; Gross, H. J. Methods Enzymol. 1994, 247, 153. 91. Gross, H. J.; Rose, U.; Krause, J. M.; Paulson, J. C.; Schmid, K.; Feeney, R. E.; Brossmer, R. Biochemistry 1989, 28, 7386. 92. Gross, H. J.; Brossmer, R. Eur. J. Biochem. 1988, 177, 583. 93. Chappell, M. D.; Halcomb, R. L.; J. Am. Chem. Soc. 1997, 119, 3393. 94. Gross, H. J.; Brossmer, R. Glycoconjugate J. 1987, 4 , 145-156. 95. Wlasichuk, K. B.; Kashem, M. A,; Nikrad, P. V.; Bird, P.; Jiang, C.; Venot, A. P. J. Biol. Chem. 1993,268, 13971. 96. Van Dorst, J. A. L. M.; Tikkanen, J. M.; Krezdorn, C. H.; Streiff, M. B.; Berger, E. G.; Van Kuik, J. A.;,Kamerling, J. P.; Vliegenthart, J. F. G. Eur. J. Biochem. 1996,242, 674. 97. Baisch, G.; Ohrlein, R.; Streiff, M.; Ernst, B. Bioorg. Med. Chem. Lett. 1996, 6, 755. 98. Baisch, G.; Ohrlein, R.; Streiff, M. Bioorg. Med. Chem. Lett. 1998, 8, 157. 99. Taniguchi, N.; Ihara, Y. Glycoconjugate J. 1995, 12, 733. 100. Vella, G. J.; Paulsen, H.; Schachter, H. Can. J. Biochem. Cell. Biol. 1984, 62, 409.
References
703
101. Palcic, M. M.; Heerze, L. D.; Pierce, M.; Hindsgaul, 0. Clycoconjugate J. 1988, 5, 49. 102. Srivastava, G.; Alton, G.; Hindsgaul, 0. Carbohydr. Res. 1990, 207, 259. 103. Reck, F.; Springer, M.; Paulsen, H.; Brockhausen, I.; Sarkar, M.; Schachter, H. Carbohydr. Res. 1994, 259, 93. 104. Moller, G.; Reck, F.; Paulsen, H.; Kaur, K. J.; Sakar, M.; Schachter, H.; Brockhausen, I. Glycoconjugate J. 1992, 9, 180. 105. Reck, F.; Springer M.; Meinjohanns, E.; Paulsen, H.; Brockhausen, I.; Schachter, H. Clycoconjugate J. 1995, 12, 747. 106. Kaur, K. J.; Hindsgaul, 0. Carbohydr. Res. 1992, 226, 219. 107. Reck, F.; Meinjohanns, E.; Springer M.; Wilkens, R.; Van Drost J. A. L. M.; Paulsen, H.; Moller, G.; Brockhausen, I.; Schachter, H. Glycoconjugate J. 1994, 11, 210. 108. Alton, G.; Kanie, Y.; Hindsgaul, 0. Carhohydr. Rex 1993,238, 339. 109. Khan, S. H.; Compston, C. A,; Palcic, M. M.; Hindsgaul, 0. Carbohydr. Res. 1994,262, 283. 110. Yamashita, K.; Tachibana, Y.; Ohkura, T.; Kobata, A. J. Biol. Chem. 1985, 260, 3963. 1 1 1. Arango, J.; Pierce, M. J. Cell. Biochem. 1988, 37, 225. 112. Dennis, J. W.; Kosh, E.; Bryce, D.-M.; Breitman, M. L. Oncogene 1989, 4, 853. 113. Dennis, J. W.; Laferte, S.; Waghorne, C.; Breitman, M. L.; Kerbel, R. S. Science 1987, 236, 582. 114. Dennis, J. W. Cancer Surveys 1988, 7, 573. 115. Tahir, S. H.; Hindsgaul, 0. Can. J. Chem. 1986, 64, 1771. 116. Hindsgaul, 0.;Tahir, S. H.; Srivastava, 0. P.; Pierce, M. Carbohydr. Res. 1988, 173, 263. 117. Srivastava, 0. P., Hindsgaul, 0.;Shoreibah, M.; Pierce, M. Carbohydr. Res. 1988, 179, 137. 118. Kanie, 0.;Palcic, M. M.; Hindsgaul, 0. RZKEN Rev. 1995, 853. 119. Kanie, 0.;Crawley, S. C.; Palcic, M. M.; Hindsgaul, 0. Carbohydr. Rex 1993, 243, 139. 120. Kanie, 0.;Crawley, S. C.; Palcic, M. M.; Hindsgaul, 0. Bioorg. Med. Chem. 1994, 2, 1231. 121. Linker, T.; Crawley, S. C.; Hindsgaul, 0. Carbohydr. Res. 1993,245, 323. 122. Khan, S. H.; Duus, J. D.; Crawley, S. C.; Palcic, M. M.; Hindsgaul, 0. Tetrahedron Asym. 1994, 5, 2415. 123. Ogawa, S.; Furuya, T.; Tsunoda, H.; Hindsgaul, 0.;Stangier, K.; Palcic, M. M. Carhohydr. Res. 1995,271, 197. 124. Lindh, I.; Hindsgaul, 0. J. Am. Chern. Soc. 1991, 113, 216. 125. Lu, P.-P.; Hindsgaul, 0.;Li, H.; Palcic, M. M. Cun. J. Chem. 1997, 75, 790. 126. Gilbert, M.; Bayer, R.; Cunningham, A.-M.; DeFrees, S.; Gao, Y.; Watson, D. C.; Young, N. M.; Wakarchuk, W. W. Nature Biotech. 1998, 16, 769. 127. Koizumi, S.; Endo, T.; Tabata, K.; Ozaki, A. Nature Biotech. 1998, 16, 847.
Carbohydrates in Chemistry and Biology Editor Beat Ernst, Gerald W. Hart, Pierre Sinay copyright OWILEY-VCH Verlag GmbH, 2000
28 Solid-Phase Synthesis with Glycosyltransferases Claudine A @ , Christine Le Nurvou, and And& Lubineau
28.1 Introduction In recent years chemical solid-phase synthesis has gained growing interest in parallel to the development of the concept of combinatorial chemistry, a promising approach to the discovery of new biologically active compounds. In peptide and oligonucleotide chemistry this technology has been established for a long time, but not in oligosaccharide chemistry. Pioneering studies in this field suffered from the lack of effective glycosyl donors and high stereoselective coupling methods [ 1-31, Recent advances in this area, and development of highly reactive sugar donors and protective group chemistry have stimulated a renewed interest in solid-phase synthesis of oligosaccharides [4-61 and glycopeptides [7, 81.
28.2 General Aspects Solid-phase synthesis has several advantages over traditional solution-based methods. Firstly, large excesses of reagents can be used so that the coupling steps can be repeated to drive reactions to completion. Secondly, products are isolated at each step by simple filtration, an obvious simplification of the work-up procedures, which might enable future automation. Development of polymer-supported synthesis of oligosaccharides using purely chemical glycosylation methods, a chemo-enzymatic approach based on the use of glycosyltransferases has also started to develop. Glycosyltransferases have become widely used in the past ten years as efficient tools for glycosylation; as opposed to glycosidases, another class of enzymes of interest in oligosaccharide solution phase synthesis, these enzymes catalyze sugar unit transfer from a sugar-nucleotide donor on to an unprotected sugar acceptor, with complete regio and stereoselectivity.
106
28 Solid-Phase Synthesis with Glycosyltransferases
Thus these also seem appropriate reagents for use in solid-phase synthesis. These enzymes have, moreover, become increasingly available as a result of genetic engineering, and soluble forms of glycosyltransferases, originally membrane-bound proteins, have been generated. Solid-phase enzymatic synthesis raises extra problems, however, such as accessibility of the interior of the solid matrix to the enzyme or, for chemo-enzymatic synthesis, the biocompatibility of the support with both aqueous and organic solvents. Indeed this approach requires that the resins swell in aqueous buffers which, for example, precludes the use of the hydrophobic Merrifield type resin. Roughly, the amounts of swelling in different solvents give information on the overall polar or hydrophobic character of the support [9]. But even the microporous polyoxyethylene-polystyrene, sold under the trade name of Tentagel, which has good swelling properties in polar solvents, does not allow macromolecules such as enzymes of 20-50 kDa and more, to penetrate into the interior of the beads. A recent study on chymotrypsin-catalyzed proteolysis of peptide substrates bound to Tentage1 beads has shown that only the surface of the beads, i.e. 15% of the total functional sites at most, are available to the enzyme [lo]. The general scheme for solid-phase synthesis using glycosyltransferases is depicted in Scheme 1. Solid-phase synthesis requires a covalent linker group to attach the small molecule, a monosaccharide or an amino acid in the case of a glycopeptide, on to the polymeric resin. Such a linker must be stable to the reaction consupport
monosaccharide cleavable linker
SugarGlycosyltransferase 1
Y c
Sugarnucleotide 2
Glycosyltransferase 2
Oligosaccharide release from matrix
Scheme 1 . General schemc for the solid-phase synthesis of oligosaccharides using glycosyltransferases.
28.3 Enzymatic Synthesis on Insoluble Supports
701
ditions used during the synthesis but it needs to be cleaved selectively at the end of the synthesis, releasing the oligosaccharide or the glycopeptide from the resin into solution; light-sensitive or enzyme-susceptible linkers are particularly suitable for this purpose. A spacer-arm providing distance between the support and the acceptor substrate to eliminate steric problems can be optionally intercalated between the matrix and the linker. Sugar-chain elongation is the result of the sequential action of glycosyltransferases in the presence of the corresponding sugar-nucleotide donor. We shall describe enzymatic glycosylations that have been achieved on both insoluble and soluble supports, because, irrespective of the nature of the support, polymer-supported synthesis is based on the same concept, and good results with soluble supports have been reported in the literature.
28.3 Enzymatic Synthesis on Insoluble Supports 28.3.1 Enzymatic Synthesis of Oligosaccharides Use of an Amino-Functionalized Water-Compatible Polyacrylamide Gel Pioneering work in the solid-phase enzymatic synthesis of oligosaccharides was accomplished by Zehavi and co-workers. The authors used an amino-functionalized water-compatible polyacrylamide gel; it is well-known that polyacrylamide gels, the preferred polymer gels for electrophoresis, allow proteins to diffuse inside. Zehavi et al. first attached one glucose unit to a photo-removable linker, methyl 4-hydroxymethyl-3-nitrobenzoate. In the second step the unprotected glycoside 1 was linked to aminoethyl-substituted polyacrylamide gel beads via an amide linkage (Scheme 2). The glucopolymer 2 although obtained in a low yield, served as an acceptor in p( 1-4)galactosyltransferase-catalyzed reaction, in the presence of a-lactalbumin, with I4C radiolabeled UDP-Gal. The lactose disaccharide 4 was subsequently released from the lactosyl polymer 3 by irradiation at 320 nm, in quantitative yield, but nevertheless the overall yield of lactose, estimated from radiolabelling was disappointingly very low (< YO), probably because of inaccessibility of the substrate to the enzyme [ 111. Such light-sensitive polymers with maltose and maltotriose side chains were also prepared for use as acceptors in the glycogen synthase reaction, but only slightly better transfers (7%) were observed [12, 131. To improve the accessibility of saccharide acceptors in glycosylation reactions, polymers with longer spacers were prepared [14]. Thus a glucopolymer 5 with an aminooctyl spacer between the polyacrylamide support and the photolabile linker became an effective support for enzymatic glycosidation catalyzed by p( 1-4)galactosyltransferase. The reaction was conducted on 0.5 mmol scale with in situ generation of UDP-Gal from UDP-Glc by adding UDP-Glc epimerase. Lactose 4 released from lactopolymer 6 by photolysis was isolated in an overall yield of 51% (Scheme 2) [15].
708
28 Solid-Phase Synthesis with Glycosyltransferases
HO
OH
HO
OH
OH
3,n=2
6,n=8
I
iii
4
Scheme 2. Synthesis of lactose uia enzymatic glycosylation using a photolabile solid support. Reagents: i) NH2-(CH2),-NH-CO-P, water-DMF, EDCD, 10% for 2, 37% for 5; ii) a-lactalbumin, GT, UDP-Gal (3 eq.), cacodylate buffer pH 7, 3 mM MnC12, for 3; a-lactalbumin, GT, UDP-Glc (1.1 eq), UDPGE, cacodylate buffer pH 7, 0.3 mM MnC12, 0.02% NaN3, for 6; iii) hv, 1% for 4 from 2. 51% for 4 from 5.
Use of a Sepharose Matrix
In 1980 Barker and Nunez [16] reported the synthesis of Gal~l-4GlcNAcp-hexanolamine by use of a solid-phase system in which the GlcNAcp-hexanolamine glycoside was covalently linked to CNBr-activated Sepharose 4B, with 6 to 10 pmol ligand mL-' packed wet gel; it is noteworthy that the same coupling method has been widely applied by David et al. to enzyme immobilization [17]. With partially purified p( 1-4)galactosyltransferase from bovine milk, and a twofold excess of UDP-Gal, the Sepharose-bound N-acetylglucosamine 7 was converted into 8 in
28.3 Enzymutic Synthesis on Insoluble Supports
709
iii HO
NHAc
HO
bH
'OH
9 Scheme 3. Enzymatic synthesis of LacNAcD-hexanolamine on Sepharose. Reagents: i) GT, UDPGal (2 eq.), cacodylate buffer pH 7, 5 mM MnC12, 0.1'%1mercaptoethanol, 18 h, 35"C, 85%; ii) 2 M NaOH, 40°C, 3 h, 80%.
85% yield; then the disaccharide glycoside 9 could be cleaved from the matrix, in 80% yield, by alkaline treatment (Scheme 3). Very recently, in a related approach, Norberg et al. presented the solid-phase enzymatic fucosylation of a disaccharide acceptor linked to Sepharose via a disulfide linkage and a linker arm of 12 atoms with a loading of 5 to 10 pmol disaccharide ligand mL-' wet gel [ 181. The sepharose-bound disaccharide 10 incubated with human milk a( 1-3/4)fucosyltransferase and GDP-Fuc in fivefold excess was converted into the Sepharose-bound trisaccharide 11 in 68% glycosylation yield (Scheme 4). The Lewisa trisaccharide derivative 12 could be released from the matrix, together with the starting disaccharide 13, by treatment with 2-mercaptoethanol, in 91% yield. Because the difficulties encountered in this enzymatic fucosylation step could not be improved, even after extensive washing of the gel and recycling with fresh enzyme and sugar-nucleotide, it was assumed that low reactivity was a result of steric factors. The same matrix was, therefore, further substituted with longer linkers of the PEG-type. Sepharose gels with linker lengths of 35, 47, 59 and 71 atoms between the matrix and the N-acetylglucosamine acceptor were built and subjected to enzymatic galactosylation with /3( 1-4)galactosyltransferase and excess UDP-Gal [19]. Aliquots were removed and treated with DTT; the 'H NMR spectrum of the
7 10
28 Solid-Phase Synthesis with Glycosyltransferases
10
ii
Scheme 4. Solid-phase enzymatic synthesis of a Lewisa trisaccharide by use of a disaccharide acceptor bound to Sepharose. Reagents: i) cacodylate buffer pH 6.8, 5 mM MnC12, 0.05% NaN3, FT, GDP-Fuc ( 5 eq.) 68%; ii) 2-mercaptoethanol, 1 h, 60"C, 91%.
filtrate enabled estimation of the yield. The longest linker turned out to give the best yield (98%). The GlcNAc Sepharose with the longest linker was then successfully converted into the sialyl Lewis' tetrasaccharide 16, by successive incubation of the gel 14 with p( 1-4)galactosyltransferase, recombinant a(2-3)sialyltransferase and human milk a(1-3/4)fucosyltransferase, in the presence of the corresponding sugar-nucleotides, used in three to fourfold excess, and alkaline phosphatase,
28.3 Enzymatic Synthesis on Insoluble Supports
71 1
14
15, R =
15
I1
16, R = H
Scheme 5. Solid-phase enzymatic synthesis of a Lewis" tetrasaccharide using a linker of 71 atoms between the monosaccharide acceptor and the Sepharose matrix. Reagents: i) 1. cacodylate buffer pH 7.5, 5 mM MnC12, x-lactalbumin, GT, UDP-Gal (3 eq.), CIAP, 3 7 T , 5 days; 2. cacodylate buffer pH 7.35, 0.1% triton X-100, ST3, CMP-NeuAc (2.5 eq.), 3 mM MnCll, 0.02% NaN3, 35 "C, 5 days; 3. cacodylate buffer pH 6.5, 5 mM MnC12, FT, GDP-Fuc (4 eq.), 37"C, 5 days; ii) 1. DTT, 0.1 M phosphate buffer; 2. BioGel P-2, 57% from 14.
followed by final treatment of the gel 15 with dithiothreitol (57% overall yield, Scheme 5 ) . Use of Controlled-Pore Glass
Wong and coworkers promoted the use of aminopropyl silica for solid-phase enzymatic synthesis [20]. This rigid, non-swelling support turned out to be compatible with biomolecules, but it required a spacer for rendering the substrate more accessible to the enzyme. The authors prepared a disaccharide target carrying at the reducing end a C-6 spacer-arm with a carboxylic function that was condensed via its cesium salt to commercially available N-iodoacetylaminopropyl controlled-pore glass, affording the solid-supported disaccharide acceptor 17 (Scheme 6). This ac-
712
28 Solid-Phase Synthesis with Glycosyltransferuses
17
OH AcHN ~
Ji
OH
~
o
H
HO
~ OH
o
+
o
OH
~ OH
o
~
H o 0
18
J ii
HO
OH
OH
OH
19 Scheme 6. Solid-phase enzymatic synthesis of NeuAca2-3Gal/ll-4GlcNAc~1-3Gal on controlledpore glass. Reagents: i) 1. HEPES buffer pH 7.2, 5 m M MnC12, GT, UDP-Gal (3 eq.), 0.5% DTT, 48 h; 2. cacodylate buffer pH 7.5, CMP-NeuAc (2 eq.), CIAP, 48 h, 9.y.; ii) NHz-NHz, room temp., 24 h.
ceptor could be enzymatically galactosylated with /I1( -4)galactosyltransferase and UDP-Gal, and then sialylated upon treatment with CMP-NeuAc, a(2-3)sialyltransferase and alkaline phosphatase, commonly used to hydrolyze the inhibitor CMP, released in the reaction. Both glycosylation steps seemed quantitative, because after cleavage of the ester linkage on the conjugate 18 by treatment with hydrazine, the tetrasaccharide hydrazide 19 was the only observed product.
28.3.2 Enzymatic Synthesis of Glycopeptides Use of Controlled-Pore Glass
The same functionalized silica support was used for glycosyltransferase-catalyzed sugar-chain elongation on a glycopeptide [21]. A N-linked glycodipeptide was attached to the support via a spacer made of six glycine units and an a-chymotrypsinsensitive phenylalanine ester bond, giving the silica-supported N-Boc-Asn (G1cNAcp)-Gly-Phe 20 with a loading of ca 0.2 mmol glycopeptide gg’ dry silica (Scheme 7). This supported glycopeptide was first galactosylated with p( 1-4)
~
28.3 Enzymatic Synthesis on Insoluble Supports
O 'H
I1 3
Ji
Scheme 7. Solid-phase synthesis of a sialyl LewisXglycopeptide on aminopropyl silica. Reagents: i) 1. HEPES buffer pH 7, 10 mM MnC12, GT, UDP-Gal (1.5 eq.), 55%; 2. HEPES buffer pH 7, 5 mM MnC12, ST3, CMP-NeuAc (1.5 eq.), 65%; ii) 1. a-chymotrypsin; 2. ultrafiltration; 3. FT, GDP-Fuc (2.5 eq.), HEPES buffer pH 7, 95%).
7 14
28 Solid-Phase Synthesis with Glycosyltransferases
galactosyltransferase and UDP-Gal in 55% yield, and subsequently sialylated in the presence of CMP-NeuAc and a(2-3)sialyltransferase in 65% yield affording the silica-bound sialylated glycopeptide 21. Yields were evaluated by cleavage of the glycopeptide from an aliquot of the functionalized silica with a-chymotrypsin. Because galactosylation and sialylation reactions were incomplete, the sialylated triwas released from peptide Boc-Asn-(NeuAca2-3Gal~l-4GlcNAc~)-Gly-Phe-OH the solid support, together with partially glycosylated products, by cleavage with achymotrypsin. Then fucosylation in a traditional solution-phase reaction with a(13/4)fucosyltransferase and GDP-Fuc, afforded a mixture of the sialyl Lewis' tripeptide 22 (35%), the fucosylated tripeptide 23 (20%) and unreacted glycopeptide 24 (45%). The solid-phase synthesis of an 0-glycopeptide by a chemo-enzymatic approach was reported by Wong and Seitz [22]. By use of the same support, aminopropylCPG, the starting amino acid, alanine, was attached through the flexible, acid- and base-stable Hycron anchor, first introduced by Kunz [23], which can be cleaved as an ally1 ester with palladium(0) under mild conditions. The glycooctapeptide 26 incorporating the preformed glycosyl amino acid, Thr(GlcNAcp), was first assembled according to classical solid-phase peptide synthesis in an overall yield of 20% starting from 25 (Scheme 8). According to the authors, CPG turned out not to be the
Ac-Lys-Pro-Pro-Asn-Tr-Tr-Ser-Ala-HY CRON-NHGlcNAc
GalPl -4GlcNAc
+
b
CPG
GalP 1-4GlcNAc
Ac-Lys-Pro-Pro-Asn-Thr-Thr-Ser-Ala-OH
B
NeuAccr2-3Gal~l-4GlcNAc
29
Scheme 8. Chemo-enzymatic synthesis of a 0-sialyl-LacNAc octapeptide. Reagents: i) HEPES buffer pH 7, 5 mM MnC12, UDP-Gal (2 eq.), GT, CIAP, 3 7 T , 3 days; ii) HEPES buffer pH 7, ST3, CIAP, CMP-NeuAc (1.5 eq.), 3 7 T , 4 days, 76%.
28.4 Enzymatic Synthesis of Oligosaccharides and Glycoconjugates
7 15
optimum support for chemical peptide synthesis, but the most suitable for enzymatic reactions [24]. The loading of the support used in this preparation was also very low (60 pmol g-'). The glycopeptide 26 was then submitted to enzymatic galactosylation in the presence of p( l -4)galactosyltransferase and a twofold excess of UDP-Gal under the usual conditions. Unexpectedly, during incubation at neutral pH and 37"C, hydrolysis of the ally1 ester linkage was observed in 53% yield, affording the O-LacNAc octapeptide 27. The remaining supported product could be released from the silica beads by treatment with palladium(0) and morpholine. Altogether the O-LacNAc peptide 27 was synthesized in 15% yield on the basis of the loading of the initial support 25, giving a 75% yield for the galactosylation step. Enzymatic sialylation was subsequently performed on the supported glycopeptide 28 but again, peptide cleavage from the support occurred during incubation and the major part (76%) of the sialylated glycopeptide 29 was finally recovered from the filtrate. Use of Polyethylene Glycol Polyacrylamide (PEGA)
An alternative to the use of silica gel described above is a PEGA resin originally developed by Meldal for peptide solid-phase synthesis [25]. PEGA is a poly(ethy1ene glycol)-polyacrylamide copolymer with high swelling capacity both in organic media and in water, and has free amino groups for functionalization. The acid-labile Rink's linker (4-(a-amino-2',4'-dimethoxybenzyl)phenoxyaceticacid) was first attached to the PEGA resin and this supported linker was then reacted with FmocGly-O-pentafluorophenyl ester and, after removal of the Fmoc group, the resinbound amino acid was coupled to protected glycopeptide Asn(G1cNAcp) by conventional peptide chemistry, to give, after deprotection, the resin-bound glycopeptide 29 with a loading capacity of 0.16 mmol gg' resin (Scheme 9). Compound 29 was then galactosylated in the presence of p( 1-4)galactosyltransferase and a large excess of UDP-Gal, in quantitative yield [26]. It is worth mentioning that pre-incubation of the resin with enzyme and buffer for three days at 4 "C before adding UDP-Gal was beneficial, affording conversion of 29 into 30 in a much shorter time. The final lactosamine glycopeptide 31 was recovered by cleavage of 30 with 95% aqueous trifluoroacetic acid. Diverse PEGA resins with different amounts of cross-linking and loading capacity could be prepared and studied for their compatibility with biomolecules; this study led to the conclusion that a more cross-linked resin gave a lower yield in enzymatic galactosylation [27].
28.4 Enzymatic Synthesis of Oligosaccharides and Glycoconjugates on Soluble Supports 28.4.1 Enzymatic Synthesis of Oligosaccharides Use of Water-Soluble Amino-Substituted Poly(viny1 alcohol)
In view of the low yields observed for enzymatic transfer of galactose on insoluble polyacrylamide polymers [ 111, Zehavi et al. developed the use of a water-soluble
7 16
28 Solid-Phase Synthesis with Glycosyltransferuses
,OJ . ti
'N H
..J H
0
PEG$
0 Me0
Po
" H
HO
29
OH OH
OMe Me
,4 H
0
PEGL
HO
30
OMe
N
HO
OH
OH
H
31
Scheme 9. Chemo-enzymatic synthesis of a N-glycopeptide on a polyethylene glycol polyacrylamide (PEGA) resin. Reagents: i) cacodylate buffer pH 7.4, 5 mM MnC12, UDP-Gal (3 eq.), GT, 23 "C, 48 h, 95%; ii) 95% aqueous TFA, 23 "C, 2 h.
polymer that afforded improved accessibility of substrate to enzyme. This linear polymer consisted of poly(viny1 alcohol) substituted with amino groups. The lightsensitive gluco polymer 32 carrying 0.15 mmol D-glucose g-', was glycosylated as previously described using 14C-labeledUDP-Gal and p( 1-4)galactosyltransferase, affording, after purification by ultrafiltration, the lactopolymer 33 in 34% yield (Scheme 10). Upon UV irradiation of 33, radiolabeled lactose 4 was subsequently recovered in 88% yield [28].
28.4 Enzymatic Synthesis of Oligosaccharides and Glycoconjugates
'OH
7 17
bH
32
HO
-!&&:=-$7-+fi 'OH
O 'H 4
Scheme 10. Enzymatic synthesis of lactose by use of a photolabile water-soluble amino-substituted poly(viny1 alcohol). Reagents: i) cacodylate buffer pH 7, 3 mM MnC12, UDP-Gal (1 eq.), GT, alactalbumin, 0.1% mercaptoethanol, 37 "C, 18 h, 34%; ii) 1. hv, 20 h, 88%);2. ultrafiltration.
Use of Water-Soluble Glycopolymer Synthesized by Polymerization
Nishimura et al. introduced the use of water-soluble glycopolymers. They synthesized the glycopolymer 34 by radical copolymerization of sugar monomer having an n-pentenyl group as a polymerizable aglycone, with acrylamide in an aqueous medium in the presence of ammonium persulfate and N,N,N',N'-tetramethylethylenediamine as initiators, according to Scheme 11 [29, 301. Polymers of more than 40 kDa were obtained with a composition varying from 1 : 5 to 1 :23 for the carbohydrate monomer-to-acrylamide ratio, depending on copolymerization conditions. Galactosylation of the GlcNAc polymer 34, performed by means of UDP-Gal and /I( 1 -4)galactosyltransferase, led to the LacNAc polymer 35 in quantitative yield, the n-pentenyl group providing the acceptor sugar with an adequately flexible spacer-arm. As opposed to insoluble polymer, such a water-soluble polymer enabled direct reaction monitoring by ' H NMR measurement in D20 [31]. As an extension of this work, the authors applied the same approach to the preparation of free oligosaccharides, by introducing suitable, removable linker arms between the saccharide and the polymer backbone. Thus, radical copolymerization with acrylamide of monomers 36 and 37 having spacer-arms of different lengths, under the previous conditions, yielded water-soluble GlcNAc acceptor polymers 38 and 39, both having a linker selectively cleavable by hydrogenolysis [32, 331. Enzy-
7 18
28 Solid-Phase Synthesis with Glycosyltransferases NHAc
NHAc 1
OH
x:y=1:8
%
0
Scheme 11. Enzymatic galactosylation of a water-soluble GlcNAc-bearing polyacrylamide. Reagents: i) CH2=CH-CO-NH2, TEMED, APS, H20, room temp., 2 h; ii) 1. HEPES buffer pH 6, 10 mM MnC12, UDP-Gal (1.5 eq.), GT, 37"C, 24 h; 2. gel filtration on Sephadex (3-50, q.y.
matic sugar chain-elongation was performed with p( 1-4)galactosyltransferase and UDP-Gal. Quantitative galactosylation could be achieved with polymer 39 bearing the long spacer-arm, whereas only 30% galactosylation was observed with polymer 38 bearing the short spacer-arm. N-acetyllactosamine 40 was finally released in high yield from polymer 41 by hydrogenolysis (Scheme 12). Other types of GlcNAc polymer, 43 and 44, containing an L-phenylalanine residue in the spacer-arm moiety as an a-chymotrypsin-sensitive structure, were also prepared in a similar manner [33, 341. Galactosylation with /3( 1-4)galactosyltransferase and subsequent sialylation with rat liver a(2-6)sialyltransferase in the presence of the sugar-nucleotides were conducted as shown in Scheme 13. Sugar transfer reactions were achieved with high efficiency using only a small excess of UDP-Gal and CMP-NeuAc, affording, after filtration and lyophilization, the polymer 45 bearing sialyl a(2-6)N-acetyllactosamine branches, in almost quantitative yield. Finally by hydrolytic action of a-chymotrypsin, sialyl a(2-6)N-acetyllactosamine derivative 46 was recovered in 72% overall yield from 44.It is noteworthy that whereas quantitative galactosylation was also observed with polymer 43, it was not possible to release lactosamine from the polymer by the action of achymotrypsin, because of the lack of the flexible spacer-arm between phenylalanine and the polymer backbone [33].
28.4.2 Enzymatic Synthesis of Glycolipids on Water-Soluble PolyacrylamidePoly(N-acryloxysuccinimide) (PAN) The water-soluble polyacrylamide-poly(N-acryloxysuccinimide) (PAN) first described by Whitesides for enzyme immobilization [35],was used by Zehavi et al. in
28.4 Enzymatic Synthesis of Oligosaccharides and Glycoconjugates
36 R = NHCOCH=CH2 37 R = NHCO(CH~)SNHCOCH=CH~
7 19
38 R = S 39 R = (CH2)sNHCO-S
HO OH
42 R = S 41 R = (CH2)sNHCO-S
HO HO OH
OH
40
Scheme 12. Enzymatic synthesis of N-acetyllactosamine using a water-soluble glycopolymer with a linker cleavable by hydrogenolysis. Reagents: i) CHz=CH-CO-NH2, TEMED, APS, DMSOH20, room temp., 24 h, 61% for 37, 83% for 38; ii) 1. HEPES buffer pH 6, 10 mM MnC12, UDPGal (1 eq.), GT, 37 "C, 24 h; 2. gel filtration on Sephadex G-50, q.y. for 40, 30% for 41; iii) H2, Pd/ C, H20-MeOH, 95%.
enzymatic supported synthesis of glycolipids. The hydrazide function in compound 47 was reacted with PAN yielding the glucopolymer 48 which served as an acceptor in the p( 1-4)galactosyltransferase-catalyzed reaction in the presence of a-lactalbumin and 14C labeled UDP-Gal [36]. The lactosyl sphingosine polymer 49 was obtained in 36% yield, after purification by extensive dialysis; further photolysis of the 2-nitrobenzylurethane group followed by acylation with stearoyl chloride, provided lactosylceramide 50 in 54% yield (Scheme 14). Alternatively the polymer 51 carrying 0.24 meq lactosyl/3(l-1)sphingosine g-', chemically prepared from PAN, was sialylated in 55% yield by use of recombinant rat liver a(2-3)sialyltransferase, 14C labeled CMP-NeuAc, and calf intestinal alkaline phosphatase [37]. Again photolysis of the 2-nitrobenzylurethane group in the sialylated polymer 52 followed by acylation with stearoyl chloride afforded the GM3 ganglioside, NeuAccc(2-3)Galp( 1-4)Glcp( l-1)Cer 53.
720
28 Solid-Phase Synthesis with Glycosyltransferases
NHAc
OH
43 n=O 44 n = 6
x:y= 1 :4 Ii
AcHN HO
AcHN
HO
N H 2O 46
Scheme 13. Enzymatic synthesis of a sialylated trisaccharide derivative, by stepwise sugar-chain elongation, using a water-soluble chymotrypsin-sensitive glycopolymer. Reagents: i) 1. HEPES buffer pH 6, UDP-Gal (1.5 eq.), GT, 37"C, 24 h; 2. cacodylate buffer pH 7.4, ST6, CIAP, CMPNeuAc (1.5 eq.), 37 "C, 48 h, gel filtration on Sephadex G-25, 9.y.; ii) 1. a-chymotrypsin, Tris HCl buffer pH 7.8, 4 0 T , 24 h; 2. gel filtration on Sephadex G-15, 87%.
In conclusion, compared with solution-phase synthesis, solid-phase enzymatic synthesis, generally, needs higher concentrations of enzyme, longer reaction times, and a large excess of donors. Although there is still room for improvement in terms of yields and scale-up of syntheses, in solid-phase synthesis purification procedures are straightforward; so it is expected that this approach will open the route to automated oligosaccharide synthesis in the near future.
28.4 Enzymatic Synthesis of Oligosucchurides
72 1
OH
OH 49 R = NHNHCO-P 51 R = NH(CH*)*NHCO-P
OH OH
OAR
p AcHN +o& oHO” & :
o HO
OH
OH
y
(
H
N
C
H
d 12CH3
y (CH2) 16CH3
53 0 Scheme 14. Enzymatic synthesis of lactosyl ceramide and GM3 using a water-soluble polyacrylamide gel. Reagents: i) 1. PAN, EtjN; 2. Sephadex (2-25; ii) cacodylate buffer pH 7, 3 mM MnC12, UDP-Gal (0.3 eq.), GT, a-lactalbumin, 0.1% mercaptoethanol, 37 “C,24 h, 36%; iii) 1. hv, THF-water, room temp., 9.5 h, 2. 50% CH3COONa-THF, CH3(CH2)16COCl, 54% for 50; iv) cacodylate buffer pH 6.5, ST3, CIAP, CMP-NeuAc (2 eq.), 55%).
722
28 Solid-Phase Synthesis with Glycosyltransferases
References 1. J. M. Frechet, C. Schuerch, Carbohydr. Res., 1972,22, 399-412. 2. G. Excoffier, D. Gagnaire, J.-P. Utille, M. Vignon, Tetrahedron, 1975, 31, 549-553. 3. R. Eby, C. Schuerch, Carbohydr. Res., 1975,39, 151-155. 4. S. J. Danishefsky, K. F. McClure, J. T. Randolph, R. B. Ruggeri, Science, 1993, 260, 13071309. 5. R. Liang, L. Yan, J. Loebach, M. Ge, Y. Uozumi, K. Sekanina, N. Horan, J. Gildersleeve, C. Thompson, A. Smith, K. Biswas, W. C. Still, D. Kahne, Science, 1996,274, 1520-1522. 6. J. Rademann, R. R. Schmidt, Tetrahedron Lett., 1996,23, 3989-3990. 7. Z.-W. Guo, Y. Nakahara, Y. Nakahara, T. Ogawa, Angew. Chem. Int. Ed. Engl., 1997, 36, 146441466, 8. W. Kosch, J. Marz, H. Kunz, Reactive Polymers, 1994, 22, 181-187. 9. M. Meldal, Methods Enzymol., 1997, 289, 83-104. 10. J. Vagner, G. Barany, K. S. Lam, V. Krchnak, N. F. Sepetov, J. A. Ostrem, P. Strop, M. Lebl, Proc. Null. Acad. Sci, USA, 1996, 93, 8194-8199. 11. U. Zehavi, S. Sadeh, M. Herchman, Carbohydr. Rex, 1983, 124, 23-34. 12. U. Zehavi, M. Herchman, Carbohydr. Res., 1986,151, 371-378. 13. U. Zehavi, M. Herchman, S. Kopper, Carbohydr. Rex, 1992, 228, 255-263. 14. S. Kopper, U. Zehavi, Reactive Polymers, 1994,22, 171-180 15. S. Kopper, Carbohydr. Rex, 1994,265, 161-166. 16. H. A. Nunez, R. Barker, Biochemistry, 1980, 19, 489-495. 17. S. David, C. AugC, C. Gautheron, Ado. in Carbohydr. Chem. Biochem., 1991, 49, 175-237. 18. 0. Blixt, T. Norberg, J. Carbohydr. Chem., 1997, 16, 143-154. 19. 0. Blixt, T. Norberg, J. Org. Chem., 1998,63, 2705-2710. 20. R. L. Halcomb, H. M. Huang, C.-H. Wong, J. Am. Chem. Soc., 1994, 116, 11315-11322. 21. M. Schuster, P. Wang, J. C. Paulson, C.-H. Wong, J. Am. Chem. SOC.,1994, 116, 1135-1136. 22. 0. Seitz, C.-H. Wong, J. Am. Chem. SOC.,1997,119, 8766-8776. 23. 0. Seitz, H. Kunz, J. Org. Chem., 1997, 62, 813-826. 24. U. Slomczynska, F. Albericio, F. Cardenas, E. Giralt, Biomed. Biochim. Acta, 1991, 50, 67-73. 25. M. Meldal, Tetrahedron Lett., 1992,33, 3077-3080. 26. M. Meldal, F.-I. Auzanneau, 0. Hindsgaul, M. Palcic, J. Chem. SOC.Chem. Commun., 1994, 1849-1 850. 27. F.-I. Auzanneau, M. Meldal, K. Bock, J. Peptide Sci., 1995, I , 31-34. 28. U. Zehavi, M. Herchman, Carbohydr. Res., 1984, 128, 160-164. 29. S.-I. Nishimura, K. Matsuoka, K. Kuritd, Macromolecules, 1990, 23, 41 82-4184. 30. K. Matsuoka, S.-I. Nishimura, Macromolecules, 1995, 28, 2961-2968. 31. S.-I. Nishimura, K. Matsuoka, Y. C. Lee, Tetrahedron Lett., 1994, 35, 5657-5660. 32. %-I. Nishimura, K. B. Lee, K. Matsuoka, Y. C. Lee, Biochem. Biophys. Rex Conimun., 1994, 199, 249--254. 33. K. Yamada, E. Fujita, S.-I. Nishimura, Carbohydr. Res., 1997, 305, 443-461. 34. K. Yamada, S.-I. Nishimura, Tetrahedron Lett., 1995, 36, 9493-9496. 35. A. Pollak, A. Blumenfeld, M. Wax, R. L. Baughn, G. M. Whitesides, J. Am. Chem. Soc., 1980, 102, 6324-6336. 36. U. Zehavi, M. Herchman, R. R. Schmidt, T. Bar, GZycoconjugateJ., 1990, 7, 229-234. 37. U. Zehavi, A. Tuchinsky, Glyconconjugate J., 1998, 15, 657-662.
Carbohydrates in Chemistry and Biology Editor Beat Ernst, Gerald W. Hart, Pierre Sinay copyright OWILEY-VCH Verlag GmbH, 2000
29 Glycosidase-Catalysed Oligosaccharide Synthesis David J. Vocadlo and Stephen G. Withers
29.1 Introduction The use of glycosidases for the synthesis of oligosaccharides has a long history, dating back over 85 years [ 11. The most difficult problem with their use as synthetic catalysts relates to their normal role being in hydrolysis. Given the 55 M concentration of water, hydrolysis is generally the preferred process in aqueous solution. A second problem can be that of control of the regiochemistry of bond formation. Several approaches have been followed in attempts to solve these problems, and these are discussed briefly within this article. More detailed accounts can be found in some useful recent reviews [2-41 and in some earlier overviews [5-7]. In this chapter we have chosen to present a brief review including some background on the enzymes themselves, the basic approaches currently used to solve the hydrolysis and regiochemistry problems, and some future perspective. The major content of this chapter is, however, a tabulation of a large number of examples of the use of glycosidases in the synthesis of specific linkages. Hopefully this will prove a useful resource for those interested in the application of such technology.
29.2 Background on Glycosidases Glycosidic bonds occur within a fantastically broad range of contexts in natural systems, as described in Volume 2 of this series. Given the large number of glycosidic bonds and the frequent need for relatively specific cleavage thereof it is no surprise that very large numbers of glycosidases exist to carry out this function. Amino acid sequences of well over 1000 different glycoside hydrolases are now available, primarily deduced from their gene sequences, and these have been arranged into some 70 different families on the basis of sequence similarities [8]. At the time this article is being written an excellent web site is available that gives up-
724
29 Glycosiduse-Cutulysed Oligosucchuride Synthesis
dated listings of these family members, plus some other information on the identities of important amino acid residues (http://afmb.cnrs-mrs.fr/-pedro/CAZY/ db.html). The site also provides links to the original papers and three-dimensional structures of enzymes, where available. This site therefore provides an excellent starting point in the search for a specific enzyme activity because each family contains enzymes of closely related activity and the full spectrum of specificities is represented.
29.3 Basic Mechanisms Hydrolysis occurs with one of two possible stereochemical outcomes, inversion or retention of anomeric configuration. All enzymes within a family hydrolyze their substrate with the same stereochemical outcome. These different outcomes demand different mechanisms, proposals for which were made by Koshland in 1953 [9]. Subsequent mechanistic and structural studies have largely substantiated these proposals and have identified the key active site residues as almost always a pair of carboxylic acids [ 10- 131. Glycosidases hydrolysing with inversion of anomeric configuration employ a mechanism involving direct displacement of the leaving group in an acid- or base-catalyzed process proceeding via an oxocarbenium ionlike transition state as shown in Figure 1. The two carboxylic acids are appropriately spaced (10-1 1 A)to perform their role as general acidic or basic catalysts. The vast majority of glycosidases that hydrolyze with net retention of anomeric configuration also have an active site containing a pair of carboxylic acids, but now only approximately 5 A apart. This smaller separation of the two groups reflects their different roles in the two-step double-displacement mechanism. One residue functions as a general acid catalyst, protonating the glycosidic oxygen, whereas the other functions as a nucleophile, attacking at the anomeric center to form a covalent glycosyl-enzyme intermediate as shown in Figure 2. In a second step, water attacks this intermediate in a general base-catalyzed process to yield the product of retained anomeric configuration. Both steps again proceed via oxocarbenium ion-like transition states (not shown). A variation on this mechanism is seen for some N-acetylhexosaminidases (at this stage apparently restricted to those from family 20 and chitinases from family 18). Such enzymes do not seem to contain a carboxylic acid that functions as a nucleophile. Rather, the Nacetyl group functions in that role, the reaction proceeding through an oxazoline intermediate as shown in Figure 3.
29.4 Synthesis by the ‘Thermodynamic’ Approach One approach that has been employed to favor synthesis over hydrolysis involves running the reaction in the presence of very high concentrations (typically molar) of
29.4 Synthesis by the ‘Thermodynamic’ Approach 0
3
14
0
k
P
0
3:
0
I
a:
tt
I
I
I
0
I
I
I 0
0
d oI $ z
t
0
Y
P
725
726
29 Glycosidase-Catalysed Oligosaccharide Synthesis
29.4 Synthesis by the ‘Thermodynamic’ Approach
727
OH
A. phoenicis a-Mannosidase
HO
HO OH
HO ‘OH
22% Yield Figure 4. Enzymatic synthesis of a-D-Man-(1 -2)-~-Man using a-mannosidase from Aspergillus phoenicis.
sugar under so-called ‘thermodynamic’ conditions such that the condensation reaction becomes favorable. Organic solvents can help in displacing this equilibrium, as long as the enzyme is compatible with, and the sugars soluble in, this medium. Similarly, high salt concentrations can be used to reduce the activity of the water and reduce product oligosaccharide solubility, thereby displacing the equilibrium [ 141. The advantage of this approach is its simplicity, hence its cost-effectiveness. Yields are, however, typically poor, rarely exceeding 15%, and control of regiochemistry is essentially non-existent. The approach is therefore of limited use for the coupling of two sugars because it frequently produces intractable mixtures. There are, however, occasional exceptions involving the coupling of two units of the same sugar; useful (20%) yields of a disaccharide have occasionally been obtained [15, 161, Figure 4. This approach can, however, provide an attractive route to the synthesis of glycosides of simple hydrophilic (ideally liquid) alcohols; reactions are performed in the presence of a very high concentration of the alcohol as shown in the example in Figure 5.
HO
A. oryzae P-Galactosidase *
HO
OH
HO-(CH,),-OH
”&
(cH2I6oH
I
HO
OH
48% Yield
Figure 5. Enzymatic synthesis of 6-hydroxyhexyl P-D-galactoside using P-galactosidase from Aspergillus oryzae.
728
29 Glycosidase-Catalysed Oligosaccharide Synthesis
Crout has recently shown that this approach can provide good (up to 61%) yields if the reaction is performed at an elevated temperature (50 "C) and time is spent in determining the optimum water activity to be used for the enzyme under study [ 171. This can be achieved either by directly adjusting the alcohol/water ratio or by varying the concentration of a co-solvent. This approach can therefore provide useful syntheses of simple glycosides as starting materials for chemical or enzymatic synthesis.
29.5 The Kinetic Approach The alternative approach, which can be employed only with retaining glycosidases, involves intercepting the glycosyl-enzyme intermediate that is formed at the active site with an acceptor moiety which reacts in place of water as shown in Figure 6. This approach relies upon the presence of an aglycone binding site that will bind a sugar in the correct orientation for one of the hydroxyl groups to react with the glycosyl-enzyme. Interactions between this second sugar and the protein will serve to stabilize the transition state for glycosyl transfer, reducing the activation barrier relative to that for hydrolysis in exactly the same way that such interactions serve to stabilize the transition state for disaccharide hydrolysis. The process therefore involves reaction of an activated donor sugar with a suitable acceptor sugar in the presence of the appropriate retaining glycosidase as shown in the example in Figure 7. The success of this approach depends upon two factors. Firstly, the transglycosylation process must occur in preference to hydrolysis of the glycosyl-enzyme. This can be controlled to some extent through the concentration of the acceptor sugar used, because occupancy of the acceptor site requires sugar concentrations higher than the dissociation constant for that site. The second important point is that the product disaccharide must function as a significantly poorer glycosyl donor than the activated donor substrate employed. This is best controlled by use of the most reactive (highest kCat/Km)donor possible, because the ratio of the rates of consumption of donor and product depends upon the relative kcat/Kmvalues for the two species and on the concentration of each species present at that time according to the expression:
Nitrophenyl glycosides and glycosyl fluorides are particularly attractive donors in this regard, not only because of their generally high k,,,/K,,, values, but also because the departed aglycone is itself a very poor acceptor and thus will not compete as an acceptor. It is also very easily separated from the products. Although some disaccharides, e.9. lactose, can serve as very inexpensive donors, complications can arise if the glucose liberated itself acts as an acceptor. A more unusual donor that has been used is the sialyl lactoside of a water-soluble polymer,
729
29.5 The Kinetic Approach
9 v)
VJ \
8
0 10
cd
~
t
I
0
4: s x 0
0 x
v)
c-
0
d8 +
d
3: 0
3: 0
730
29 Glycosiduse-Cutulysed Oligosacchuride Synthesis
which was used in conjunction with a leech ceramide glycanase for the transfer of sialyl lactose to ceramide, thereby yielding ganglioside GM3 in high (61%) yield [ 181. Sugar oxazolines have been used as donors for N-acetylhexosaminidases that react through the oxazoline mechanism. For example, incubation of Bacillus sp. chitinase with the oxazoline derivative of chitobiose resulted in rapid production of chito-oligosaccharides as shown in Figure 8 [ 191. If these factors are controlled well, respectable yields in the range of 10-60% can be obtained. This, however, generally requires exquisite timing of product harvesting or the use of some trick to ‘pull’ the equilibrium of the reaction in the synthetic direction. Because no universal approach exists, these ‘tricks’ these must be developed separately for each enzyme. One way of achieving this end again involves the use of an organic co-solvent to reduce the activity of the water, thereby minimizing the hydrolytic process. For example, when p-nitrophenyl p-galactoside was used as donor and 3-0-methyl glucose as acceptor in reactions catalyzed by the Kluyveromycesfragilis P-galactosidase a 14% yield of the 6-linked disaccharide product was obtained if reaction was performed in 67%)triethyl phosphate whereas only hydrolysis was observed when the reaction was conducted in aqueous buffer [20]. Another approach has been to remove one product continuously in some way. In a very few fortuitous cases this has occurred via crystallization of the product in situ; this is obviously not a general approach. Removal of disaccharide products by preferential absorption on to activated charcoal chromatography columns has also been reported [21]. A more generally useful approach has been to use a second ‘coupling’ enzyme, which will react with the product of interest only and, ideally, convert it directly to the desired product. This approach is particularly useful if the second enzyme is a glycosyl transferase, directly yielding the trisaccharide desired as shown below in Wong’s synthesis of sialyl LacNAc (Figure 9) [22]. Other examples include Paulson’s synthesis of sialyl 2,3-Gal P-OR [23] and Thiem’s preparation of the sialyl T-antigen [24]. Control of the regiochemistry of bond formation is more difficult to achieve. Probably the best method at present is to screen the available glycosidases, hopefully guided by the literature, for the enzyme that gives the product desired. We hope our compilation of enzymes will be useful in this process. A logical approach to such screening, if the product is available, is to screen the enzymes for their ability to cleave the product of interest. Although the principle of microscopic reversibility would indicate that an enzyme capable of hydrolysing the desired product can form the product of interest, a positive result would not necessarily prove that it would form the desired product preferentially. A somewhat improved approach, which does not require access to the product, is described in Section 29.6. All such screening approaches, however, are currently limited by the availability of enzymes. A second approach that has met with some success has involved manipulation of solvent conditions to change the regiochemistry of bond formation. For example, in studies on the Bacillus circulans pgalactosidase using p-nitrophenyl galactoside as donor and p-nitrophenyl GlcNAc as acceptor the Gal+- 1,4-GlcNAc-pNP product was obtained as the exclusive product (21% yield) when reaction was performed in the presence of 50% acetoni-
29.5 The Kinetic Approach
3: 0
8$ 03: 3:
d
0
X 0
z
0
731
732
29 Glycosidase-Catalysed Oliyosaccharide Synthesis
trile. When reaction was performed at lower (20%) acetonitrile concentrations, however, Gal-p-1,6-GlcNAc-pNP was instead obtained in 25.5% yield [25]. Finally, there have been several examples of success in manipulating regiochemical outcomes by changing the anomeric stereochemistry of the acceptor sugar. Thus, for example, Nilsson has shown that when methyl a-galactoside is used as the acceptor for the transfer of Gal from p-nitrophenyl a-galactoside by the coffee bean a-galactosidase, the predominant product (27%) is the 1-3 linked isomer whereas when methyl p-galactoside is the acceptor the 1-6 linked product is formed preferentially (18% compared with 9% of the 1-3 isomer). Similarly with E. coli pgalactosidase, using p-nitrophenyl p-galactoside as donor, the 1-6 isomer was dominant (14% yield) for transfer to methyl a-galactoside whereas when transfer was to methyl p-galactoside the 1-3 linked product predominated over the 1--6 (22% compared with 3%) [26]. In a further example Crout demonstrated a major difference in product distribution when the N-acetylgalactosaminidase from Aspergillus ovyzae transfers GalNAc to methyl aGalNAc or to methyl PGalNAc. Transfer to methyl a-galactoside resulted in 81% yield of the 1-4 linked product and only 9% of the 1-6 isomer whereas transfer to methyl P-galactoside resulted in almost equal amounts of the two isomers, but at lower yields (20Y0of the 1-4 isomer and 23% of the 1-6 isomer) ~71. In evaluating the synthetic potential of glycosidases using the ‘kinetic’ method it is important to recognize that the yields and even the regiochemical outcomes will vary significantlywith enzyme concentration and the reaction time employed. These differences in regiochemical outcome can arise because the transglycosylation products obtained are, themselves, substrates for the enzyme and can, therefore, be processed further. The net consequence will therefore tend to be a progression from an initial ‘kinetically controlled’ product mix towards a ‘thermodynamically controlled’ transglycosylation product mixture and ultimately to hydrolysis products. Consequently it is frequently observed that the product ratios obtained do not reflect expectations on the basis of cleavage specificities because the product that forms fastest will probably also be rearranged fastest. Different regiochemical outcomes arising from variation of solvent composition or acceptor structure could, therefore, be a consequence of the different relative rates of these processes rather than specific effects on enzyme structure or binding mode.
29.6 Recent Developments and New Directions A completely new approach to the problem of controlling the hydrolytic reaction has been developed in the Withers group through the use of glycosynthases, specific mutants of retaining glycosidases that are used in conjunction with glycosyl fluoride donors of the opposite anomeric configuration to that hydrolyzed by the wild-type enzyme [28]. These mutants are those in which the amino acid functioning as the catalytic nucleophile has been replaced by a residue with a non-nucleophilic sidechain, alanine being the substitution of choice at the time of writing. Such mutants
29.6 Recent Developments and New Directions
733
are completely hydrolytically inactive towards disaccharide substrates because they are unable to form the reactive glycosyl-enzyme intermediate. If, however, the mutant is presented with a glycosyl fluoride substrate of the same anomeric configuration as the intermediate that would ordinarily have formed, it is accepted by the enzyme as a surrogate for this intermediate and is transferred to a suitable acceptor as shown in Figure 10. Because the products so formed are completely hydrolytically stable, excellent yields (up to 92% isolated yields to date) can be obtained. This approach was published first with the P-glucosidase from Agrobacterium sp. [28]. A second example has recently appeared [29]and several others are being developed at the time of writing. A more direct large-scale screen for enzymes of interest has very recently been developed (Blanchard & Withers, unpublished). This involves first inactivating the enzyme under investigation via the trapping of a 2-deoxy-2-fluoroglycosyl-enzyme intermediate by treatment of the enzyme with the appropriate 2,4-dinitrophenyl 2deoxy-2-fluoro glycoside. Once freed of excess inactivator aliquots of this inactive, yet catalytically competent, intermediate species are incubated in the presence of a series of potential acceptors for a defined time period during which transglycosylation, releasing free enzyme, can occur, Figure 11. Each aliquot is then assayed with a standard substrate, positive results indicating that the ligand in question is capable of acting as an acceptor. The approach has been adapted to a 96-well format, enabling the facile screening of large numbers of acceptors, although the approach does not provide any insights into the regiochemistry of bond formation. Most published work to date has been on the use of exo-glycosidases, which usually cleave a single sugar residue at a time from the non-reducing terminus. Approaches involving the transfer of large blocks of sugars will, however, require the use of endo-glycosidases. Some very impressive work in this area has been pioneered by Takagawa and coworkers, who demonstrated that an endo-N-acetyl glucosaminidase from Arthrobacter protophormiae (endoA) has transglycosylation activity and could be used to synthesize neoglycoproteins. They used (Man)6(GlcNAc)2 as donor and transferred to GlcNAc(Asn)peptide acceptors, thereby producing (Man)6(GlcNAc)Z(Asn)-peptide products. They also showed that the approach could be used with partially deglycosylated ribonuclease B as acceptor thereby producing a modified glyco form, as shown below (Figure 12) [30-341. This approach was further developed by Y.-C. Lee for the production of (Man)g(GlcNAc)z-peptide analogs by use of pre-synthesized GlcNAc-peptide analogs as acceptors [35]. Lee also showed that organic solvents such as acetone could significantly improve (up to 89%) yields of products in such block transfer reactions [33, 361. This approach has enormous potential in the remodeling of glycoproteins. The role of oligosaccharides in biological processes has become a field of intensive research and accordingly facile methods for their preparation are highly desirable. The following tables should hopefully provide a useful resource for the researcher interested in synthesizing such oligosaccharides via an enzymatic or chemoenzymatic route. Table 1 lists linkages formed by exo-glycosidases, Table 2 includes all products formed by endo-glycosidases, and Table 3 includes alkyl glycosides synthesized by exo-glycosidases. References included in each table were selected primarily on the basis of interest and adequate product characterization.
HO CH3
F
Figure 11. Reactivation of a 2-fluoro-a-glucosyl-enzyme intermediate by transglycosylation to an acceptor sugar.
Figure 10. Glucosynthase-catalyzed synthesis of a cellobioside.
HO
0
-0A
%
s
F
v3
ru
P
W
4
29.6 Recent Developments and New Directions
0
(GlcNAc)2(Man),
Ribonuclease
EndoA
a
___)
GlcNAc
Ribonuclease
735
0
(GlcNAc)2(Man)6
End?
Ribonuclease
Figure 12. Remodeling of RNase B glycoforms through the use of endo A.
In each table the linkages formed are listed alphabetically with a-linkages listed first followed by P-linkages. In the linkage column, where the configuration of the anomeric carbon in the second saccharide unit is indicated, the acceptor molecule is either an oligosaccharide or a glycoside. Glycoside acceptors are differentiated by the inclusion of the hetero-atom involved in the glycosidic linkage after the second saccharide unit. Yields listed are calculated based on donor, acceptor, or in a few cases, transfer ratios. Identical products synthesized by different groups are listed as separate entries. When the same principal investigator has referred to the synthesis in different publications there is only one entry listing each reference.
12
11
10
9
8
I
6
5
4
3
2
1
Enzyme Source
Trehdlase ~ - ~ - 2 d G1-])-alc( Lobosphaera sp. D-2dGIC Trehalase u - D - ~ ~ Gl-I)-UIc( Lohospkaera sp. D-G~ ~ - ~ - 2 d G1-3)-Dlc( Glucoamylase Aspergillus niger 2dGlc a-~-2dGlc( 1-3)-~- a-Glucosidase Aspergillus niger 2dGlc Glucoamylase ~ - ~ - 2 d G1l-4)-Dc( Aspergillus niger 2dGlc u-D-2dGIc(1-4)-D- a-Glucosidase Asperg illus n ig er 2dGlc a-D-2dGk(I - 6 ) - ~ - Glucoamylase 2dGlc Aspergillus niger ~ - ~ - 2 d G1-6)-Dk( a-Glucosidase Aspergillus niger 2dGlc a-D-Gal(l-l)-a-D- a-Galactosidase Gal Candida guilliermondii H-404 P-Galactosidase a-D-Gal(1-1)-DEscherichia coli Fruj P-Galactosidase a-D-Gal(l-I)-DAsperg illus Fwf oryzae u-D-Gal(1-2)-U-D- a-Galactosidase Coffeebean Gal-0
Entry Linkage
16
~ - ~ - 2 d G Il -cI)-a-D-2dGk ( u-D-2dGk(1-l)-a-~-Glc ~ - ~ - 2 d G 1l -3)-D-2dGk c( a-D-ZdGIc(1-3)-~-2dGlc 1 -4)-~-2dGlc u-~-2dGlc( u-D-~~GIc( 1-4)-~-2dGk
2dGlc Glc 2dGlc 2dGlc 2dGlc 2dGlc
2dGlc 2dGlc 2dGlc 2dGlc 2dGlc 2dGlc
NA
1-6)-~-2dGlc ~~-~-2dG lc(
a-D-Gal(I-l)-a-D-Gal
U - D - G d ( 1-I)-D-Fruf
a-D-Gal(1-1)-u-Fruf
a-u-Gal( 1 -2)-a-~-Gal-OpNP
2dGlc Lactose
Fruj
Fruf
apNPGal
2dGlc Lactose
Gal Gdl
upNPGal
2
52
34
NA
NA
~ - ~ - 2 d G Il -6)-D-2dGk c(
2dGlc
2dGlc
NA
NA
NA
NA
6
Yield ("/')
Product
Accept01
Donor
~
Table 1. Enzymatic synthesis of oligosaccharides using em-glycosidases.
26
40
40
39
38
38
38
38
38
38
31
31
Reference
26
25
24
23
22
21
20
19
18
17
16
15
14
13
a-Galactosidase Coffee bean a-Galactosidase Coffee bean a-Galactosidase Coffee bean a-Galactosidase Coffee bean a-Galactosidase Coffee bean a-Galactosidase Cundidu guilliermondii H-404 a-D-Gal(1-3)-a-D- a-Galactosidase Coffee bean (2,6-OAll)-Gal0 a-u-Gal( 1-3)-a-~- a-Galactosidase (2-OAlI)-Gal-O Coffee bean a-u-Gal( 1-3)-a-u- a-Galactosidase (2-0Bn)-Gal-0 Coffee bean a-D-Gal(1 -3)-a-u- a-Galactosidase Gal-0 Coffee bean a-D-Gdl(1-3)a-D- a-Galactosidase GalNAc-0 Coffee bean a-D-Gal(1-3)-a-~- P-Galactosidase Bacillus GdlNAc-O circulans a-~-Gal( 1-3)-a-u- p-Galactosidase GalNAc-0 Bacillus circuluns a-~-Gal( 1-3)-a-D- a-Galactosidase Gal-0 Coffee bean
a-u-Gal( I -2)-a-DGal-0 a-D-Gal( 1-2)-a-DGlc a-D-Gal(I-z)-a-DGlc a-D-Gal(1-2)-a-DGlc a-~-Gal( 1-2)-a-~Glc a-D-Gal(1 -2)-DGal
a-D-GalNAc-OEt a-D-GalNAc-OBn
apNPGal PpNPGal
apNPGal
PpNPGal
a-u-Gal-OMe
a-u-GalNAc-OMe
a-~-Gal( 1-3)-a-~-GalNAcOMe
a-D-Gal-OAll
Raffinose
46 26
53 27
46
a-D-Gal(1-3)-a-~-GalNAcOEt a-D-Gal(1-3)-a-~-GalNAcOBn
a-u-(2-OBn)-Gal-OMe
apNPGal
62
a-u-Gal( 1-3)-a-~-(2-OAll)Gal-OMe a-~-Gal( 1-3)-a-u-(2-OBn)Gal-OMe a-D-Gal(1-3)-a-u-Gal-oAll
a-u-(2-OAll)-Gal-OMe
apNPGal
45
43
a-D-Gal(1-3)-a-D-(2,6-OAll)- 14 Gal-OMe
apNPGal
21
39
NA
a-D-Gal(1-2)-~-Gal
Lactose
Lactose
44
42
5
a-u-Gal( 1-2)-CIs
CIS
Melibiose
4.9
41
1.8
a-~-Gal( I-2)-y-CD
Y-CD
Melibiose
43
41
2.3
a-u-Gal(1-2)-P-CD
p-CD
Melibiose
12
41
2.2
a - ~ - C a l1(-2)-a-CD
a-CD
Melibiose
26
6
a-u-Gal(l-2)-a-~-Gal-OoNP
apNPGal
apNPGal
ii.
F'
0
(0
?
-
b
35
&
a 2 a
:
g
b m
C
3\
Fu
u,
31
36
35
34
33
32
31
30
29
28
21
Enzyme Source
a-D-Gal(1-3)-a-~- a-Galactosidase Coffee bean Gal-0 a-~-Gal( 1-3)-a-~- a-Galactosidase Coffee bean Gal-0 a-D-GaI(1-3)-a-~- a-Galactosidase Coffee bean Gal-0 a-D-GaI(1-3)-a-D- a-Galactosidase Coffee bean Glc a-D-Gal(1-3)-D-D- a-Galactosidase Coffee bean (2,6-OAll)-Gal0 a-D-Gal(1-3)-P-D- a-Galactosidase (6-OAIl)-Gal-O Coffee bean a-D-Gal(I -3)-P-D- a-Galactosidase Asperg illus Gal oryzae a-D-Gal(1-3)-8-D- a-Galactosidase Gal Coffee bean a-D-Gal(I -3)-P-D- a-Galactosidase Gal Coffee bean a-D-Gal(1-3)-P-D- P-Galactosidase Asperg illus Gal oryzae a-D-Gal(1-3)-p-D- P-Galactosidase Asperg illus Gal oryzae
Entry Linkage
Table 1 (continued)
P-~-(2,6-OAll)-Gal-OMe
P-~-(6-0All)-Gal-OMe P-D-Gal(1-4)-p-~-GlcNAcSEt
P-D-Gal(1-4)-P-~-GlcNTeoc- a-D-Gal(I-~)-P-D-G~I( I-4)-P- 20 49 D-GlcNTeoc-SEt SEt P-Lactose-SEt a-D-GaI(1-3)-P-~-Gal( 1 4 - P - NA 50 D-GIC-SEt ~-D-G~I(~-~)-P-D-G~I(~-~)-P15 0-Lactose-SEt 50 D-Glc-SEt P-Lactose-SBu
apNPGal apNPGal
apNPGal
PpNPGal
upNPGal
apNPGal
a-D-Gal(1-3)-P-~-Gal( 1-4)-PD-Glc-SBU
a-D-Gal(1-3)-P-~-(6-OAll)Gal-OMe a-~-Gal(l-3)-P-~-Gal( 14-PD-G~NAC-SE t
a-D-Gal(1-3)-P-~-(2,6-OAll)Gal-OMe
18
16
3
3
5
apNPGal
a-D-Gal(I -3)-CI8
CIS
50
48
43
43
42
41
Melibiose
apNPGal
a-D-GaI(1-3)-a-~-Gal-OpNP NA
26
26
Reference
apNPGal
1
Yield ("%)
apNPGal
Product
a-D-Gal(1 -3)-a-~-Gal-OoNP
apNPGal
Acceptor
aoNPGal
apNPGal
Donor
50
49
48
41
46
45
44
43
42
41
40
39
38
P-Galactosidase Asperg illus oryzae a-D-Gal(1-3)-P-D- a-Galactosidase Coffee bean Gal-0 a-~-Gal( 1-3)-8-~- a-Galactosidase Coffee bean Gal-0 a-Galactosidase a-D-Gal(1-3-DCundida guillierGal rnondii H-404 a-D-Gal(1-4)-P-D- a-Galactosidase Morttierella Glc vinacea a-~-Gal( 1-4)-p-D- a-Galactosidase Absidiu reflexa Glc a-D-Gal(1-4)-P-D- a-Galactosidase Morttierella Glc vinacea a-D-Gal(1-4)-P-D- a-Galactosidase Absidia refexa Glc a-D-Gal(1-4)-P-~- P-Galactosidase Bacillus GlcNAc-S circulans P-Galactosidase a-D-Gal(1-4)-DEscherichia coli Fruf a-D-Gal(1-4)-~- 0-Galactosidase Asperg illus Fruf oryzue P-Galactosidase a-D-Gal(1-5)-DEscherichia coli Fruf P-Galactosidase a-D-Gal(1-5)-DAsperg illus FWf oryzae
a-D-Gal(1 - 3 ) - P - D Gal
52
13-P-~-Glc-19-a-~-Ga1( 1-6)- 11 !3-D-Glc-O-steviol 13-u-~-Gal(l-6)-a-~-Gal(l- 3.3 6)-P-D-Gk-19-P-D-GIC-Osteviol 13-a-~-Gal(l-6)-P-~-Glc-1911 P-D-Glc-0-steviol a-D-Gal(1-4)-P-~-GlcNAc- 50 SEt
Rubusoside
Rubusoside
Raffinosel Melibiose Raffinosel Melibiose Raffinosel Melibiose
10
10
6.6 9.2
a-D-Gal(1-4)-~-Fruf a-D-Gal(1-4)-~-Fruf
a-D-Gal(l-S)-D-Fruf a-D-Gal(1-5)-~-Fruf
0-D-GlcNAc-SEt
Fruf Fruf
Fruf Fruf
Gal Gal
Gal Gal
Rubusoside
Raffinosel Melibiose apNPGal
40
40
40
40
48
52
52
52
13-a-~-Gal( 1-6)-P-~-Glc-19- 13 P-D-Glc-0-steviol
Lactose
Lactose
Rubusoside
39
NA
a-D-Gal(1-3)-~-Gal
P-D-Gal-OMe
26
apNPGal
9
0-D-Gal-OMe
apNPGal
a-D-Gal(1-3)-fi-~-Gal-OMe
P-Lactose-SPh
PpNPGal
~
62
61
60
59
58
51
56
55
54
53
52
51
~~~
~
~
~~
~
Enzyme Source
a-D-Gal(1-6)-a-~- a-Galactosidase Glc Coffee bean a-D-Gal(1-6)-a-~- a-Galactosidase Gal-0 Coffee bean a-D-Gal(1-6)-a-~- a-Galactosidase Coffee bean Glc a-D-Gal(1-6)-a-~- a-Galactosidase Coffee bean Glc a-D-Gal(I -6)-a-~- a-Galactosidase Glc Coffee bean a-D-Gal(1-6)-a-~- a-Galactosidase Glc Coffee bean a-D-GaI(1-6)-a-~- a-Galactosidase Glc Coffee bean a-~-Gal( 1-6)-a-~- a-Galactosidase Morttierella Glc vinacea a-D-Gal(1-6)-p-~- a-Galactosidase (2-OAlI)-Gal-O Coffee bean a-D-Gal(1-6)-p-~- a-Galactosidase (2-0Bn)-Gal-0 Coffee bean a-D-Gal(1-6)-P-~- a-Galactosidase Asperg illus Gal oryzae a-D-Gal(1-6)-P-~- a-Galactosidase Coffee bean Gal
Entry Linkage
~~
Table 1 (continued)
24
a-D-GaI(ld)-y-CD a-~-Gal( 1-6)-a-CD a-D-Gal(1-6)-a-CD a-D-Gal(1-6)-P-CD a-D-Gal(1-6)-y-CD a-D-Gal(1-6)-a-~-GIc( 1-2)-0-
7-CD a-CD a-CD 0-CD y-CD Sucrose
Melibiose Melibiose Melibiose Melibiose Melibiose
@-D-Gal( 1-4)-b-~-GlcNAcSEt
p-D-Gal(1-4)-P-~-GlcNTeoc- a-D-GaI(1-6)-P-~-Gal( 14-0SEt D-GlcNTeoc-SEt
apNPGal
a-D-Gal(1-6)-P-~-(2-OAll)Gal-OMe a - ~ - G a l1-6)-P-~-(2-OBn)( Gal-OMe a-D-Gal(1-6)-P-~-Gal( 1-4)-PD-GlcNAc-SEt
apNPGal
apNPGal
apNPGal
Gal
D-FrUf
NA
a-D-Gal(1-6)-a-~-Gal-OMe
a-D-Gal-OMe
apNPGal
3.5
16
14
12
18
22
26
38
2
49
48
43
43
55
41
41
41
54
53
26
53
(?h)
NA
a-D-Gal(I-6)-P-CD
Melibiose
~
Reference
0-CD
~~~~
Yield
Product
~~
Acceptor
~~
Donor
74
73
72
71
70
69
68
67
66
65
64
63
a-D-Gal(1-6)-P-D- a-Galactosidase Aspergillus niger Gal a-~-Gal( 1-6)-P-D- a-Galactosidase Coffee bean Gal a-D-Gal(1-6)-P-~- P-Galactosidase -Gal Asperg illus oryzae a-D-Gal(1-~)-P-D- 0-Galactosidase Gal Asperg illus oryzae a-D-Gal(1-6)-p-~- P-Gdlactosidase Asperg illus Gal oryzue a-D-Gal(1-6)-P-D- a-Galactosidase Gal-0 Coffee bean a-D-Gal(1-~)-O-D- a-Galactosidase Gal-0 Coffee bean a-D-Gal(1-6)-P-D- P-Galactosidase Escherichia coli Glc a-D-Gal(1-6)-~- b-Gdlactosidase Escherichia coli Fmf a-~-Gal( I-~)-D- 8-Galactosidase Asperg illus Fmf oryzae a-D-Gal(I-~)-D- a-Galactosidase Candidu yuillierGal mondii H-404 a-D-GalNAc(1a-N-Acetyl3)-a-~-Gal-0 galactosaminidase Chamelea gullinu 5.2
NA
w-D-Gal(1 -6)-~-Fruf
a - ~ - G a l1-6)-~-Gal (
P-D-Gal-OMe Sucrose Fruj Fruf
apNPGal Gal Gal
Gal
a-D-Gal-OMe
40
4.4
a-D-Gal(1-6)-a-~-Glc( 1-2)-PD-Fruf a - ~ - G a l1-6)-~-Fruf (
P-u-Gal-OMe
apNPGal
aoNPGalNAc
40
a-D-GaI(1-6)-P-~-Gal-OMe NA
P-Lactose-SPh
PpNPGal
Lactose
51
a-~-Gal(l-6)-P-~-Gal-OMe 18
P-Lactose-SBu
PpNPGal
Lactose
26
a-~-Gal(l-h)-fi-~-Gal(I-4)-P17 D-Glc-SPh
P-Lactose-SEt
PpNPGal
D-Glc-SBU
11
39
55
50
b
z
3
4
n a
$
2
,$
c:
2
i
co r: co
;tr
o\
9
Eu
~-D-G~I(~-~)-P-D-G~I(~-~)-P13 50
50
50
P-Lactose-SEt
50
apNPGal
a-D-Gal(l-6)-P-~-Gal( I-4)-P- 20 D-GIC-SEt a-D-Gal(1-6)-P-~-Gal( l-4)-@- NA D-GIC-SEt a-~-Gal( 1-6)-P-~-Gal( 14-P9 D-Gk-SEt
P-Lactose-SEt
clpNPGal
142
29 Glycosidase-Catalysed Oligosaccharide Synthesis
W
d
z
0 W
0
r-
2
W
N
0
5 4
-3 v 3
&
5 b
&
&
m
0
V
5
5 0,
m
r-
w r -
r - r -
W
r-
rch
g
z
63
64
11
14
a-D-GIc(1-4)-P-D-Glc-OpNP
w-D-GIc(1-4)-~-Fruf
PpNPGlc
PpN PGlc
Fruf
PpNPGlc
PpNPGlc
Glc
96
95
94
93
92
66 66
6.4 3.5 4.5
2 6
u-D-GIc(1-4)-~-Glc a-D-Glc(1-4)-~-Glc ~-D-GIC( 1-5)-~-Fruf
a-D-GlC(1-6)-a-D-GIC(1-4)-DGlc u-D-GIc(1-6)-a-~-Gk( 1 -6)-DGlc
Glc Glc Fruf
Glc Glc
Glc Glc Glc
Glc Glc
59
66
65
60
2.2
u-~-Glc-( 1-4)-~-Glc
Glc
Glc
SP.
Glucoamylase Rhizopus sp. Glucoamylase Rhizopus oryzue Glucoamylase Rhizopus oryzae a-Glucosidase Sacchuromyces SP. U-D-GlC( 1-6)-U-D- Glucoamylase Rhizopus oryzue Glc u-D-G~c( 1-6)-a-~- Glucoamylase Rhizopus oryzue Glc
91
a-D-GIC-(1-4)-DGlc u-D-GIc(1-4)-~Glc u-D-GIc(1-4)-~Glc u-D-GIc(1-5)-DFruf
60
1.8
Glc
a-~-Glc-( 1-4)-D-Gk
Glc
a-Glucosidase Succhuromy ces
u-D-G~c-( 1-4)-DGlc
SP.
59
62
a-D-Glc(1-4)-a-D-Glc(l-l)-a2 D-GIC a-D-Glc(l-4)-fi-~-Glc-OpNP 1 1
Trehalose
Glc
SP.
~-D-CIC( 1-4)-U-D- Glucoamylase Rhizopus nivrus Glc 1-4)-P-D- P-Galactosidase u-D-G~c( Asperg illus Glc-0 oryzae (Impurity?) u-D-G~c( 1-~)-P-D- P-Galactosidase Glc-0 Asperg illus oryzue (Impurity?) a-Glucosidase Succharomyces
62
Trehalose
U - D - G ~ ( ~ - ~ ) - ~ -l-l)-uD - G ~ C (2.1 D-Glc
Glc
u-D-GIc(1-4)-a-~- a-Glucosidase Succhuromyces Glc
90
89
88
87
86
85
t
s
107
106
105
104
I03
102
U-D-Gk-(I-6)-DGlc U-D-GIC(I-6)-DGlc a-D-GIc(1-6)-DGlc
SP.
Glucoamylase Rhizopus sp. Glucoamylase Rhizopus orvzae Glucoamy lase Rhiz0pu.r oryzae Glc Glc G Ic
Glc Glc Glc
a-D-Glc(1-6)-~-Glc
60
26
26
24
24
a-D-Glc-(1-6)-~-Glc
Glc
Fruf'
Glc
Glc
Glc
Glc
Glc
1 -6)-~-a- Glucoamylase u-~-Glc( Rhizopus oryzae Glc a-D-Glc(I-6)-D-a- Glucoamylase RhizopuJ oryzae Glc a-D-Gk(1 -6)-Da-Glucosidase Saccharonzy ces Fmf SP. u-~-Glc-( I-~)-D- a-Glucosidase Sacchuromycex Glc
101
66
65
60
65
65
61
61
59
Stevioside
Maltose
a-D-GIc(1-6)-P-D- P-amylase Aspergillus sp. Glc Glc
Stevioside
Maltose
62
1- 1)-a- 13 a-D-Glc(1-6)-a-~-Gk( D-GlC 13-P-D-GIC(I-2)-P-D-GIC-I9- NA a-D-Gk(1-6)-b-~-Glc-Osteviol NA 13-a-D-Gk(I -6)-P-D-Gk(12)-P-D-Gk-19-P-D-GIC-0steviol a-D-GIc(l-6)-a-u-Glc(1-4)-~- 3.5 Glc a-D-Glc(1-6)-a-~-Glc( 1-6)-~- 6 Clc a-~-Glc( I-6)-~-Fruf 5
Trehalose
Glc
SP.
a - ~ - G k1-6)-a-~( Glucoamylase Rhizopus niveus Glc a-D-GlC(1-6)-p-O- P-amylase Aspergillus sp. Glc
62
6.4
a-D-Glc(1-6)-a-~-Glc( 1-I)-aD-GlC
Trehalose
Reference
Yield ("/.)
Product
Acceptor
Glc
Donor
a-D-Gk(1-6)-a-o- a-Glucosidase Saccharomy ces Glc
Enzyme Source
100
99
98
97
Entry Linkage
Table 1 (continued)
a-D-Man(1-2)-aD-Man-0 a-D-Man( 1-2)-aD-Man-0 a-D-Man( 1-2)-DMan a-D-Man( 1 - 2 ) - ~ Man a-D-Man( 1 - 2 ) - ~ Man a-D-Man(1-2)-DMan
a-D-Man(1-2)-DMan a-D-Man( 1 4 - D Man a-D-Man(1-3)aMan a-D-Man( 1- 3 ) - ~ Man a-D-Man( 1 - 3 ) - ~ Man a-D-Man( 1 - 4 ) - ~ Man a-D-Man( ]-4)-uMan
110
116
122
121
120
119
118
111
115
114
113
112
111
109
a-D-Man( 1 -1)-aD-Man a-D-Man( 1 -2)-aD-Man
108
a-Mannosidase Jack bean a-Mannosidase Aspergillus phoenicis a-Mannosidase Jack bean a-Mannosidase Jack bean a-Mannosidase Jack bean a-Mannosidase Jack bean a-Mannosidase Jack bean a-Mannosidase Aspergillus phoenicis a-Mannosidase Aspergillus niger a-Mannosidase Jack bean a-Mannosidase Jack bean a-Mannosidase Jack bean a-Mannosidase Aspergillus niger a-Mannosiddse Jack bean a-Mannosidase Jack bean 26 26 68
15 68
a-D-Man( 1-2)-a-~-Man-OMe 18 8 16 1.2 NA 22
2
3.6 1.3
0.2 4.2 NA
a-D-Man(1-2)-a-~-ManOpNP a-D-Man( l - 2 ) - ~ - M a n a-D-Man(1 -2)-~-Man a-u-Man( 1-2)-~-Man u-D-Man( I-z)-~-Man
a-D-Man( 1-2)-~-Man a-D-Man(1 -3)-~-Man a-D-Man( 1-3)-~-Man
a-D-Man( 1-3)-~-Man a-D-Man(1- 4 ) - ~ - M a n a-D-Man( 1-4)-~-Man
a-D-Man-OMe apNPMan Man Man Man Man
Man Man Man Man Man Man Man
apNPMan apNPMan Man Man Man Man
Man Man Man Man Man Man Man
69
61
15
67
16
69
67
16
8
a-D-Man(1 -2)-a-~-Man( 1-2)D-Man
Man
Man
61
3.3
a-D-Man( l-l)-a-D-Man
Man
Man
2
8'
2
2
2
i
$ B
3
$-
C
m
b
2
m
r5
2
m
\o
ru
135
134
133
132
131
130
129
128
127
126
125
124
a-D-Man( 1-6)-a-CD a-D-Man( Id)-P-CD a-D-Man( 1-6)-y-CD a - ~ - M a nl-6)-a-u-Glc( ( 1-6)p-CD a-D-Man( I -6)-a-~-Glc( 1-4)a - ~ - G l c1(-6)-a-CD a-D-Man( I-6)-a-~-Glc( 1-4)a - ~ - G l c1-6)-p-CD ( a-D-Man( 1-6)-a-u-Man-OMe
a-CD p-CD ?I-CD a-D-Glc(1-6)-P-CD
I -6)-aa - ~ - G l c1(-4)-a-~-Glc( CD 1-6)-pa-D-Glc( 1-4)-a-~-Glc( CD a-D-Man-OMe
Man Man
a-D-Man-OMe a-u-Man-OMe
a-D-Man( 1-6)-o-Man a-D-Man(1-6)-Man
Man Man
p-D-Gal(1-4)-p-~-Xyl-OpNP a-~-NeuAc(2-3)-P-~-Gal( 14)-P-D-XyI-OpNP
Man aMuNeuAc
Man
33
7.6
9.8
14
15
67
68
14
a - ~ - M a n1-6)-u-Man (
Man
Man
PpNPGal
73
clpNPMan
a-D-Man(l-6)-@-~-Gal-OpNP 5
12
12
72
71
71
71
70
Reference
26
6.1
6.4
5.3
11
10
14
0.3
Yield (%)
4
apNPMan
a-D-Man-OMe
Man
a-D-Man( I-4)-o-Man
Man
Man
a-Mannosidase Rhizopus niveus a-Mannosidase Jack bean a-Mannosidase Jack bean a-Mannosidase Jack bean a-Mannosidase Jack bean a-Mannosidase Jack bean a-Mannosidase Jack bean a-Mannosidase Jack bean a-Mannosidase Jack bean a-Mannosidase Jack bean a-Mannosidase Jack bean a-Mannosidase Aspergillus niyer Sialidase Trypanosoma cruzi
a-D-Man(I -4)-DMan a-D-Man(1-6)-aD-Glc a - ~ - M a nl-6)-a( D-G~c a-D-Man(1-6)-aD-Glc a-D-Man( 1-6)-aD-G~c a - ~ - M a n1-6)-a( D-Glc a-D-Man(1-6)-aD-Glc u-D-Man(l-6)-aD-Man-0 a - ~ - M a n1-6)-8( D-Ga i-0 a-D-Man( 1 - 6 ) - ~ Man a-D-Man(1-6)-uMan a-D-Man(1-6)-0Man a-~-NeuAc(2-3)a-D-Gal
123
Product
Acceptor
Donor
Enzyme Source
Linkage
Entry
Table 1 (continued)
29.6 Recent Developments and New Directions
v,
r-
v,
r-
m 3
x
m
1;3
.;!
ar
v,
W
W
W
v, e
m
v
l
r-
r
-
0
0
r - r - r - r - r -
e
o
W
?
m
w w r - r -
W
r-
W
ar
m
&
m
h
m
747
157
156
155
154
153
152
151
150
149
148
Enzyme Source
a-~-NeuAc(2-3)P-D-GIC-O a-~-NeuAc(2-6)a-D-Gal-0 a-~-NeuAc(2-6)P-D-Gal a-~-NeuAc( 2-6)P-D-Gal a-~-NeuAc(2-6)(3-D-Gal
Sialidase Vibrio cholerae Sialidase Vibrio cholerae Sialidase Vibrio cholerue Sialidase Vibrio cholerae Sialidase Clostridium perfringens
a-~-NeuAc(2-3)- Sialidase Newcastle P-D-G~~ disease virus a-~-NeuAc(2-3)- Sialidase Newcastle P-D-Gal disease virus a-~-NeuAc(2-3)- Sialidase Newcastle P-D-Gal disease virus a-~-NeuAc(2-3)- Sialidase Vibrio cholerae P-D-G~I-O a-~-NeuAc(2-3)- Sialidase Trypanosoma cruzi P-D-Gal-0
Entry Linkage
Table 1 (continued)
P-D-Gal(1-3)-~-GlcNAc
apNPNeu Ac
a-~-NeuAc(2-3)-P-~-Gal( 13)-r,-GlcNAc
3)-D-Gk
a-~-NeuAc(2-3)-P-~-Gal( 1-
3)-D-Gk
a-~-NeuAc(2-3)-P-~-Gal( 1-
Product
12
7.7
2.7
MU)
Yield
apNPNeuAc
P-D-Gal-OMe
a-~-NeuAc(2-3)-P-~-Gal7 OMe NA CH~)~ a-~-NeuAc(2-3)- P - D - G ~ ~ - O - ( C H ~ ) ~ S ~ ( a-~-NeuAc(2-3)-P-D-Gal-Op-D-GaI(1-3)-p(CH2)2Si(CH3)3 D-GalNAc-0(CH2)2 Si(CH313 a-~-NeuAc(2-3)-P-~-Glc4 apNPNeuAc P-D-Glc-OMe OMe apNPNeuAc a-D-Gal-OMe a-~-NeuAc(2-6)-a-~-Gal- 2 1 OMe a-~-NeuAc(2-6)-P-~-Gal( 111 apNPNeuAc P-Lactose-OMe 4)-P-~-Glc-oMe 1a-~-NeuAc(2-6)-P-~-Gal( apNPNeuAc p-D-Gal(1-4)-~-GlcNAc 10 ~)-D-G~cNAc 18.5 a-~-NeuAc(2-6)-P-~-Gal( a-~-NeuAc(2-8)- p-D-Gal(1-3)-~-Gk 3)-D-GIC D-NeuAc
P-D-Gal(1-3)-~-Glc
Acceptor
apNPNeuAc
Donor
78
76
76
76
76
2 23
0,
2.
Tc
9E
g
e
R 5
0 g 7
B
g-
a,
(?
?
0
g
x9
76
78
78
78
Reference
00
P
4
169
168
167
166
165
164
163
162
161
160
159
158
a-~-NeuAc(2-6)- Sialidase Clostridium B-D-Gal perfringens a-~-NeuAc(2-6)- Sialidase P-D-Gal Clostridiuni peyfringens a-~-NeuAc(2-6)- Sialidase P-wGal Clostridium perfringens Sialidase Arthrobucter ureufuciens a-~-NeuAc(2-6)- Sialidase Arthrohurter P-D-Gal ureufuciens a-~-NeuAc( 2-6)- Sialidase Vihrio p-D-Ga1 cholerue a-~-NeuAc(2-6)- Sialidase Vibrio P-D-Gal cholerae a-~-NeuAc(2-6)- Sialidase P-D-Gal Newcastle disease virus a-~-NeuAc(2-6)- Sialidase B-D-Cal Newcastle disease virus a-~-NeuAc(2-6)- Sialidase Vibrio cholerae P-D-Gal-0 a-~-NeuAc(2-6)- Sialidase Vibrio cholerue P-D-Gk-0 a-L-Fuc(1-2)-P- a-Fucosidase Porcine liver D-Gal-0 78
4.1 9.2
a-~-NeuAc(2-6)-P-~-Gal( 13)-~-Glc a-~-NeuAc(2-6)-P-~-Gal( 13)-D-GIc a-~-NeuAc(2-6)-P-~-Gal( 13)-D-Gk
a-~-NeuAc(2-8)- p-D-Gal(1-3)-~-Glc D-NeuAc apNPNeuAc P-D-Gal(1-3)-D-GlC
p-D-Gal(1-3)-D-GlC
P-D-Gal-OMe P-D-Glc-OMe P-D-Gal-OMe
apNPNeuAc
apNPNeuAc apNPNeuAc a-L-Fuc F
2.5
a-~-NeuAc(2-6)-P-~-Gal- 15 OMe a-~-NeuAc(2-6)-P-~-GIc- 12 OMe a-L-Fuc(1-2)-P-~-Gal-OMe 6.5
3)-D-GIC
a-~-NeuAc(2-6)-P-~-Gal( 1-
78
7.4
a-~-NeuAc(2-6)-P-~-Gal( 13)-D-GIC
p-D-Gal(1-3)-~-Glc
apNPNeuAc
0.9
78
5.6
a-~-NeuAc(2-6)-P-~-Gal( 13)-D-GlC
a-~-NeuAc(2-8)- p-D-cal( 1 -3)-~-Glc D-NeuAc
79
76
76
78
78
78
78
18.7 u-~-Ne~uk(2-6)-P-~-Gal( 3)-D-Gk
b-~-Gal( 1-3)-D-Glc
upNPNeuAc
78
2.4
P-D-Gal(1 -3)-~-Glc
{a-~-NeuAc(28)),-~-NeuAc
1a-~-NeuAc(2-6)-P-~-Gal( 3)-D-Gk
78
5.5
a-~-NeuAc(2-6)-P-~-Gal( 13)-~-GlcNAc
a-~-NeuAc(2-8)- p-~-Gal( 1-3)-~-GlcNAc o-NeuAc
2
5g.
b -.
e
2 n
2Q
5
2
Y 3
6
2 n
c:
2
B
5
o\
!c
t u
179
178
177
176
175
174
173
172
a-L-Fuc( 1-2)-~- a-Fucosidase apNPFuc Gdl Corynebacterium
171
a-L-Fuc( 1- 3 ) - ~ - a-L-Fucosidase Glc Penicillium multicolor a-L-Fuc(l-3)-D- a-Fucosidase Aspergillus niger Glc a-L-Fucosidase a-L-FuC(1-3)-DPenicillium GlcNAc multicolor ~-L-FuC(I-~)-D- a-Fucosidase Aspergillus niger GlcNAc a-L-Fuc(l-S)-~- a-Fucosidase Aspergillus niyer GlcNAc a-Fucosidase a-L-Fuc(l-4)-PBovine testes ~-(6-0Bn)GlcN-S a-L-Fuc(l-4)-(3- P-Fucosidase Bovine kidney ~-(6-0Bn)GlcN -S a-Fucosidase a-L-Fuc( 1 -6)-PPorcine liver D-Gal-0
SP.
SP.
49
58 24
a-L-Fuc(1-3)-D-GlcNAc
a-L-Fuc( I -3)-D-GlcNAc a-L-Fuc(1-3)-~-GlcNAc a-L-Fuc(1-4)-P-~-(6-OBn)GlcN-SEt a-L-Fuc( 1-4)-P-~-(6-OBn)GlcN-SEt a-L-Fuc(1-6)-P-~-Gal-0Me
GlcNAc
GlcNAc GlcNAc f3-~-(6-OBn)-GlcN-SEt
p-~-(6-0Bn)-GlcN-SEt
P-D-Gal-OMe
apNPFuc
apNPFuc apNPFuc apNPFuc
apNPFuc
a-L-Fuc F
10
50
33
61
a-L-Fuc(1-3)-~-Glc
Glc
apNPFuc
18
u,-L-Fuc(1-3)-D-Gk
a-L-Fuc(1-2)-~-Gal
79
84
83
82
80
81
80
80
80
Refer(YO) ence
Yield
a-L-Fuc(l-2)-P-~-Gal-OMe 25
Product
Glc
Gal
P-D-Gal-OMe
Acceptor
apNPFuc
apNPFuc
a-Fucosidase Corynehacterium
a-L-Fuc( 1-2)-PD-Gal-0
170
Donor
Enzyme Source
Entry Linkage
Table 1 (continued)
25;.
%$@
g
9
3
r5
0
=: 2
‘p n
tr,
g
tsg CI
v3
ly
3
4 v,
190
189
188
187
186
185
184
183
182
181
I80
SP.
P-~-Fruf(2-l)-a- P-Fructofuranosidase ArthroD-Glc bacter sp. K-1 P-D-Fmf(2- I)-a- P-FructofuranoD-G~c sidase Arthrohacter sp. K-l P-Fructofuranosidase Aspergillus sydowi P-~-Fruf(2-I)+ P-FructofuranoD-Fruf sidase Aspevgillus sydowi P-D-Fruf( 2- 1)-PP-FructofuranoD-FWf sidase Aspergillus oryzae P-Fructofuranosidase Aspergillus oryzae
SP.
P-Glucosidase Agrobacteriuni
Sucrose
Sucrose
Sucrose
Sucrose
Sucrose
Sucrose
Glucal
Glucal
SP.
P-Glucosidase Agrobacterium
Sucrose
Sucrose
Trehalose
Trehalose
a-D-Gk(1-6)-D-GIc
Maltose
P-D-Xyl-SBn
P-D-Glc-SPh
P-D-Xyl-SBn
87
NA
P-~-Fruf’(2-1 )-P-~-Fruf(z-1 )P-~-Fruf(2-l)-a-D-Gk
Glc(l-l)-a-~-Glc P-D-Fruf( 2- 1)- P-D-Fruf( 2- 1)a-D-Glc
17
21
88
88
87
86
86
85
85
85
NA
51
55
~(2-1)-b-~-Fruf(2-6)-a-~-
P-~-Fruf’(2l)-P-D-Fru$P1-I)-a-D~(2-6)-a-~-Glc( Glc P-~-Fruf(2-l)-P-D-Fruf-P-
P-~-Fruf(2-l)-a-D-Glc( 1-6)D-Glc
P-~-Fruf(2-l)-a-~-Glc( 1-4)D-GlC
16
12
2-deoxy-P-~-Glc(1-~)-P-DGlc-SPh 2-deoxy-P-~-Glc(1-~)-P-DXyl-SBn
16
2-deoxy-P-~-Glc(1-3)-P-DXyl-SBn
85
15
1- 3 ) - f i - ~ 2-deoxy-P-~-Gk( Glc-SPh
Glucal
Glucal
80
14
a-L-Fuc(1 -6)-b-~-Gal-OMe
apNPFuc
P-Glucosidase Agrobacterium
SP.
CL-L-FUC(I-~)-P-a-Fucosidase Ampullaria D-Gal-0 P-D-2dGIC(1 4 4 - P-Glucosidase Agrobacterium D-GlC-S
199
198
197
196
195
194
193
192
191
55
7.5
P-~-Fruf(2-I)-D-xyl
P-~-Fruf(2-1)-L-Ara
@-~-Fruf(21)-L-Fuc
P-~-Fruf(2-2)-L-Sor
P-~-FruJ’(2-3)-~-Gal
Gal
XYI L-Ara
L-FUC
L-Sor
Gal
L-Ara
Sucrose
Sucrose
Sucrose
Sucrose
Sucrose
Sucrose
Sucrose
P-~-Fruf(2-4)-L-Ara
27
86
70
86
86
86
86
86
86
86
86
Reference
33
62
52
40
P-~-Fruf(2-l)-D-Ara
D-Ara
Sucrose
P-~-Fruf(2-l)-D-Gal
44
P-~-Fruf(2-1)-P-~-Gal( 1-4)D-G~c
Lactose
Sucrose
P-~-Fruf(2-1)-PD-Gal
P-Fructofuranosidase Arthrobacter sp. K-1 P-D-Fruf( 2- l)-D- P-Fructofuranosidase ArthroAra bacter sp. K-1 P-D-Fruf( 2- 1)-D- P-Fructofuranosidase ArthroGal hacter sp. K-1 P-~-Fruf(2-l)-D- 0-Fructofuranosidase ArthroXYl bacter sp. K-1 P-~-Fruf(2-1)-L- P-Fructofuranosidase ArthroAra bacter sp. K-1 P-~-Fruf(2-l)-L- P-Fructofurdnosidase ArthroFuc hacter sp. K-1 P-D-Fmf(2-2)-L- 0-Fructofuranosidase ArthroSor bacter sp. K-1 P-o-Fruf(2-3)-~- P-Fructofuranosidase ArthroGal bacfer sp. K-1 Pm-FrUf(2-4)-L- P-Fructofuranosidase ArthroAra bacfer sp. K-l
Yield (“%)
Product
Acceptor
Donor
~
Enzyme Source
Entry Linkage
~~
Table 1 (continued)
29.6 Recent Developments and New Directions
t-
oo
00 m
0
m
3
m
W
00
oo Q\
N
m
m
m
m
m
N
a
+
3
A
v
3
3
0 N 0
u 9
-9
ci
ci
w
N N 0
N 0
4 v
6A ci
u
r-
00
0 m
N
N
N
4
-x h
9
u m N 0
d 0 N
u
ci In
0
N
W N 0
0
0
753
219
218
217
216
215
214
213
212
21 1
210
Enzyme Source
P-D-Gal(1-2)-a-D- P-Galactosidase Penicillium Glc multicolor p-D-Gal(1-2)-a-D- P-Galactosidase Bacillus Glc circulans P-Galactosidase p-D-Gal(1-2)-DEscherichiu coli Glc P-Galactosidase P-D-Gal(1-2)-DBacillus Glc circulans 0-Galactosidase P-D-Gal(1-2)-DBacillus Glc circulans p-~-Gal( 1-2)-~- 0-Galactosidase Bovine testes XYl P-Galactosidase P-D-Gal(1-2)-DIntestinal lactase XYl Lamb P-Galactosidase P-D-Gal(1-2)-DAspery illus XYl oryzue 0-Galactosidase P-D-Gal(1-2)-DSuccharomyces XYl fruyilis P-Galactosidase p-D-Gal(1-2)-DEscherichiu coli XYl
Entry Linkage
Table 1 (continued)
40
0.5
1
3
PoNPGal
PoNPGal
95
9
PoNPGal
95
95
95
95
4
PoNPGal
18
94
NA
Lactose
PoNPGal
94
NA
Lactose
Gal
42
42
Reference
17
(“h)
Yield
Lactose
Product
6.7
Acceptor
Lactose
Donor
P-D-GaI(1-2)-LXYl P-D-Gal(1-3)-(60Ac)-Glucal P-,-Gal( 1-3)-a-DGalNAc-0 B-D-Gal(1-3)-a-DGalNAc-0
225
233
232
23 I
230
229
228
221
P-D-Gal(1-3)a-DGalNAc-0 P-D-Gal(1-3)-a-DGalNAc-0 P-D-Gal(1-3)-a-DGalNAc-0 P-D-Gal(1-3)-a-DGalNAc-0 P-D-Gal(1-3)-U-DGalNAc-0
P-D-Gal(1-2)-LXYI
224
226
P-D-Gal( I -2)-LXYI
XYI P-D-Gal(1 -2)-LXYl P-D-Gal(1-2)-LXYl
P-D-Gdl( 1-2)-D-
223
222
22 1
220
P-Galactosidase Escherichia coli P-Galactosidase Bovine testes P-Galactosidase Intestinal lactase Lamb P-Galactosidase Aspery illus oryzae P-Galactosidase Saccharomyces fragilis P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli P-Galactosidase Bovine testes P-Galactosidase Bacillus circulans P-Galactosidase Bovine testes P-Galactosidase Bovine testes P-Galactosidase Porcine testes P-Galactosidase Bovine Testes P-Galactosidase Bovine Testes PpNPGal
PpNPGal
Lactose
PpNPGal
PpNPGal
PpNPGal
PpNPGal
PpNPGal
PoNPGal
apNPGalN Ac
a-D-GalNAc-0-Thr
a-D-GalNAc-0-Ser-(N-Z)OAll
a-D-GalNAc-OEt
L-Xyl
L-Xyl
PoNPCa1 PoNPGal
L-Xyl
L-Xyl
[JoNPGal
PoNPGal
PpNPGal
P-D-Gal(1-2)-L-Xyl
P-D-Gal(1-2)-L-Xyl
P-D-Gal(1-2)-L-Xyl
P-D-Gal(1-2)-L-Xyl
11
3
5
2
95
95
95
95
244
243
242
24 1
240
239
238
237
236
235
p-D-Gal(1-3)-a-D- P-Galactosidase Penicillium Glc multicolor
oryzae
p-D-Gal(1-3)-a-~- P-Galactosidase Bovine Testes GalNAc-0 P-D-Gal(1-3)-a-~- P-Galactosidase GalNAc-0 Bovine Testes P-D-Gal(1-3)-a-D- P-Galactosidase Bovine Testes GalNAc-0 P-D-Gal(1-q-a-D- P-Galactosidase GalNAc-0 Bovine Testes P-D-Gal(1-3)-a-D- P-Galactosidase GalNAc-0 Bovine Testes P-D-Gal(I-3)-a-D- 0-Galactosidase Bovine testes GalNAc-0 p-~-Gal( 1-3)-a-D- P-Galactosidase Aspergillus Gal-0 oryzue p-D-Gal(1-3)-a-D- P-Galactosidase Gal-0 Aspergillus oryzae p-D-Gal(1-3)-a-D- P-Galactosidase Bovine Testes Gal-0 P-D-Gal(1-3)-a-~- P-Galactosidase Aspergillus Glc
234
Enzyme Source
Linkage
Entry
Table 1 (continued)
Lactose
Gal
PpNPGal
PVGal
PoNPGal
Lactose
PpNPGal
PpNPGal
PpNPGal
PpNPGal
PpNPGal
Donor
Trehalose
9.7
1.8
15
a-~-Gal-OAll
p-~-Gal( 1-3)-a-~-Gal-OAll
93
42
62
101
102
101
101
101
101
6
101
Reference
a-~-Gal-OMe
Yield (%)
93
Product
P-D-Gal(1-3)-a-~-GalNAc-O- 35 (CHz)zO(CHz)2N3 P-D-Gal(1-3)-a-~-GalNAc-O- 22 CH2CH(NHz)COOH P-D-Gal(1-3)-a-~-GalNAc- 21 OpNP P-D-Gal(1-3)-u-~-GalNAc- 22 OAll a-D-GalNAc-0p-D-Gal(1-3)-a-~-GalNAc-o- 28 CH(CH3)CH(NH2)COOH CH(CH3)CH(NH2)COOH a-D-GalNAc-0-Ser-(Np-~-Gal( 1-3)-a-~-GalNAc-O- 20 Aloc)-OMe Ser-(N-Aloc)-OMe a-D-Gal-OMe p-D-Gal(1-3)-a-~-Gal-OMe 6 a-D-GalNAc-0(CHz)20(CHz)zN3 a-D-GalNAc-0CH2CH(NH2)COOH apNPGalNAc
Acceptor
258
257
256
255
254
253
252
25 1
250
249
248
247
246
245
p-D-Gal(1-3)-a-D- 0-Galactosidase Bovine Testes GlcNAc-0 P-u-Gal(1-3)-a-D- P-Galactosidase Bovine Testes GlcNAc-0 P-D-Gal(1-3)-a-L- P-Galactosidase Barley FUC-0 p-D-Gal(1-3)-P-D- P-Galactosidase (6-O-Ac)-Gal Escherichia coli p - D - G d ( 1-3)-P-D- P-Galactosidase Barley (6-0Ac)-Gal-0 P-D-Gal(1-3)-0-D- P-Galactosidase (6-OBn)-GlcNBovine testes S P-u-Gal(1-3)-P-D- P-Galactosidase Bijidobacterium Gal bzjidum P-D-Gal(1-3)-P-D- P-Galactosidase Bacillus Gal circulans p-D-Gal(1-3)-P-D- P-Galactosidase Bovine testes GalNAc-0 P-D-Gal(1-3)-P-D- P-Galactosidase GalNAc-0 Porcine testes D-D-Gal(1-3)-8-D- P-Galactosidase Gal-0 Asperg illus oryzae P-u-Gal(1-3)-8-D- P-Galactosidase Escherichia coli Gal-0 P-D-Gal(1-~)-P-D- 0-Galactosidase Escherichia coli Gal-0 P-D-Gal(1-3)-P-D- P-Galactosidase Escherichia coli Gal-0 1-~)-P-DP-~-(6-0-Ac)-Gal( Xyl-OMu P-~-(6-0Ac)-Gal-OMe
PpNPGal
PoNPGal
PoNPGal P-D-Gal-OMe P-D-Gal-OAII
PoNPGal PoNPGal Lactose
a-D-GalNAcOpNP
P-D-GalNAc-0-EtBr
NeuAc
PoNPGal
Lactose
PoNPGal
Lactose
Lactose
PpNPGal
51
28
2.5
30
4
P-D-Gal(I -3)-P-~-Gal-ooNP 14
63
100
45
104
84
51
103
101
32
16
101
I3
P-D-GaI(1-3)-P-o-GalNAc-O- 2.5 EtBr p-D-GaI(1-3)-P-~-GalNAc- 13 OpNP P-D-Gal(l-3)-P-~-Gal-ooNP 11
P-D-Gal(1-3)-P-~-Gal( 1-8)-DNeuAc
a-L-Fuc-OMe
PpNPGal
Lactose
p-D-GaI(1-3)-!3-~-(6-0-Ac)Gal( 1-4)-P-~-Xyl-OMu p-~-Gal( 1-3)-P-~-(6-OAc)Gal-OMe P-D-Gal(1-3)-P-~-(6-OBn)GlcN-SEt
a-D-GlcNAc-OBn
PpNPGal
PpNPGal
P-D-Gal(1-3)-a-~-GlcNAcOAll p-D-Gal(1-3)-a-~-GlcNAcOBn p-D-Gal(1-3)-a-L-Fuc-OMe
a-D-GlcNAc-OAll
PpNPGal
3f
=L
2 a s
3
2
C
g
B
o\
u,
Fu
p-D-Gal(1-3)-P-D- P-Galactosidase Bacillus Gal-0 circulans P-D-GaI(1-3)-p-D- P-Glucosidase A grobacterium Gal-S SP. P-D-Gal(1-3)-P-D- P-Glucosidase A yrobacterium Gal-S SP. p-~-Gal( 1-3)-p-D- P-Galactosidase GIcNAc-0 Bovine testes p-D-Gal(14-P-D- 0-Galactosidase Bovine testes GIcNAc-0 P-D-Gal(1-3)-P-D- P-Galactosidase Bovine Testes GIcNAc-S P-D-Gal(1-3)-P-D- P-Galactosidase Bacillus GIG-0 circulans
269
268
267
266
265
264
P-D-GlcNAc-0-EtSiMe3 P-D-GlcNAc-OMe P-D-GlcNAc-SEt 0-D-Glc-OMe
PpNPGal Lactose Lactose
P-D-Gal(1-3)-P-~-GlcNAc-0- 7 EtSiMe3 p-D-Gal(1-3)-P-~-GlcNAc- about OMe 10 p-D-Gal(1-3)-P-u-GlcNAc17 SEt p-D-Gal(1-3)-P-~-Glc-OMe 19
8.8
P-D-Gal(1-3)-P-~-Gal-SBn
92
108
45
45
85
85
9.5
P-D-Gal-SPh
P-D-Gal(1-3)-D-~-Gal-SPh
2
2 5.
9
n &
s-
0 r5
3 u
0
Q
z is
k
k?
%.
%-
92
PoNPGal
PpNPGal
PpNPGal
Lactose
106
44
Q
h, \n
00
a
5.8
44
Reference
107
Lactose PoNPGal
p-~-Gal( 1-3)-P-~-Gal-oEtSi(Me)3
P-D-Gal-O-EtSi(Me)3
Lactose
263
262
261
260
10
Yield (“A)
wl
4
P-D-Gal-O-Ser-(N-Aloc)P-D-Gal(l-3)-P-D-Gal-O-Ser- 13 (N-Aloc)-OMe OMe monoallyl ether (p-~-Glc-O)- monoallyl ether (P-~-Gal(l- 2 3)-P-~-Glc-0)-1,3,5I , 3,Sbenzene-trirnethanol benzene-trimethanol p-D-Gal-OMe 5.5 P-D-Gal(l-3)-P-~-Gal-OMe
P-D-Gal(l-3)-P-~-Gal-OBn
P-D-Gal-OBn
Lactose
P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli 0-Galactosidase Bovine liver
p-o-Gal(1-3)-P-DGal-0 P-D-Gal(1-3)-P-DGal-0 P-D-GaI(1-3)-P-DGal-0 p-D-Gal(1-3)-P-DGal-0
Product
Acceptor
Donor
Enzyme Source
259
Entry Linkage
Table 1 (continued)
280
279
278
277
276
215
274
273
272
27 1
270
aGalF
PpNPGal
P-D-Gal(1 -~)-P-D- P-Glucosynthase Agrobacterium Xyl-0
P-D-Gal(1-3)-P-D- P-Galactosidase Escherichia coli Xyl-0 p-~-Gal( 1-3)-P-D- 0-Glucosidase A yrobacterium Xyl-s
GalNAc GalNAc
PpNPGal
P-D-Xyl-SBn
Lactose
PpNPGal
P-D-Gal(1-3)-P-D- P-Galactosidase Bacillus Xyl-s circulans P-Galactosidase p-~-Gal( 1-q-DBovine testes GalNAc P-Galactosidase P-D-Gal(1-3)-DBijidobacterium GalNAc blfidurn
SP.
PpNPGdl
P-D-Gal(1-3)-P-D- P-Glucosidase A yrobacterium Xyl-s
SP.
SP.
SP.
PpNPGal
PpNPGal
SP.
p-D-Gal(1-3)-P-D- P-Glucosidase Agrobacterium Xyl-0
38
P-D-Gal(l-3)-P-~-Glc-SPh
P-D-GIG-SPh
PpNPGal
PpNPGal
3.7
b-~-Gal( 1-3)-(3-Glc-OpNP
PpNPGlc
Lactose
p-D-Gal(1-3)-8-D- (3-Glucosidase A yrobacterium Glc-S
SP.
P-Galactosidase Bacillus circulans 1-3)-(3-D- P-Glucosidase (3-~-Gal( Agrobacterium Glc-S
p-~-Gal( 1-3)-(3-DGlc-0 85
109
P-D-GaI(1-3-DGalNAc
P-D-Gal(1-3)-DGalNAc
fl-~-Gal( 1-q-DGlc P-D-Gal(1 -3)-DGlc
282
283
284
b-~-Gal( 1-q-DGlc
p-~-Gal( 1-q-DGlcA
P-D-GaI(1-q-DGlcNAc
b-~-Gal( 1-3)-DGlcNAc
286
287
288
289
285
p-D-Gal(1-3)-DGalNAc
28 1
Entry Linkage
Table 1 (continued)
113
113
104
94
105
113
108
trace
3.7
1.4
0.5 NA
NA
23
4.4
12
P-D-Gal(1-3)-~-GalNAc
p-D-Gal(1-3)-~-GalNAc
b-~-Gal( 1-3)-~-GalNAc
b-D-Gal(1- 3 ) - ~ - G k p-D-Gal( 1- 3 ) - ~ - G k
b-~-Gal( I-~)-D-G~
P-D-Gal(1-3)-~-GlcA
p-D-Gal(1-3)-~-GlcNAc
b-~-Gal( 1-3)-~-GlcNAc
GalNAc
GalNAc
GalNAc
Glc Lactose
Lactose
GlcA
GIcNAc
GlcNAc
PpNPGal
PpNPGal
PpNPGal
Gal Lactose
Lactose
Lactose
PpNPGal
Lactose
P-Galactosidase Penicillium multicolor P-Galactosidase Streptococcus 6646K P-Galactosidase Asperg illus oryzae P-Galactosidase Escherichia coli P-Galactosidase Bijidobacterium bijidum P-Galactosidase Bacillus circuluns P-Galactosidase Bacillus circuluns P-Galactosidase Streptococcus 6646K P-Galactosidase Bovine Testes
40
113
Reference
Yield ("%)
Product
Acceptor
Donor
Enzyme Source
29.6 Recent Developments and New Directions
761
311
310
309
308
307
306
305
304
303
302
Enzyme Source
P-Galactosidase Escherichia coli P-Galactosidase Barley P-Galactosidase Snail P-Galactosidase Bacillus circulans P-D-Gal(1-4)-a-D- P-Galactosidase Bacillus Glc circulans P-D-Gal(1 -4)-a-D- P-Galactosidase Glc Bacillus circulans p-D-Gal(1-4)-a-~- P-Galactosidase Glc Bacillus circulans P-D-Gal(1-4)-a-D- 0-Galactosidase Bacillus Glc circulans P-D-Gal(1-4)-a-D- P-Galactosidase Bacillus Glc circulans P-D-Gal(1-4)-a-D- P-Galactosidase Bacillus Glc circulans
P-D-Gal(1-3)-LXYl P-D-Gal(1-4)-U-DGal-0 p-D-Gal(1-4)-a-~Gal-0 P-D-Gal(1-4)-a-DGlc
Entry Linkage
Table 1 (continued)
22
20
P-D-Gal(1-4)-a-D-Glc(1-4)-a~ - G l c1-6)-a-CD ( 1-6)-Pp-~-Gal( ]-4)-a-~-Glc( CD p-D-Gal(I-4)-a-~-Glc( 1-4)-a~ - G l c1-6)-P-CD (
P-~-Gal(l-4)-a-~-Glc(l-6)-y17 CD P-D-Gal(l-4)-a-D-GIc(1-4)-aD-G~c( 1-6)-y-CD
a-~-Glc( I-4)-a-~-Glc( 1 -6)-aCD u-D-GIc(1-6)-P-CD
a-D-Glc(1-4)-a-~-Glc( 1-6)-8CD a-D-Glc(1-6)-y-CD
1-6)-ya-~-Glc( l-4)-a-~-Glc( CD
Lactose
Lactose
Lactose
Lactose
Lactose
21
22
20
P-~-Gal(1-4)-a-D-Gk( 1-6)-aCD
a-D-Glc(1-6)-a-CD
Lactose
. I
12
Lactose
NA
P-D-Gal(l-4)-{a-~-Glc( 1 4)}4-a-D-Glc-OpNP
a-D-Gal-OMe
PpNPGal
P-D-GaI(l-4)-a-~-Gal-OMe
15
{u-D-GIc(1-4)}4-a-~-GlcOpNP
a-D-Gal-OMe
PpNPGal
P-D-Gal(1-3)-L-Xyl
(oh)
Yield
NA
L-Xyl
PoNPGal
Product
P-D-Gal(1-4)-a-~-Gal-OMe
Acceptor
Donor
115
115
1 15
115
115
115
114
51
51
95
Reference
G.
2
PB
&
e
;s
ff0
0 =: 9 h u
B
g-
y
A
E 3
E
0
s-g.
a N
4
32 1
320
319
318
317
316
315
314
313
312
P-D-Gal(1-4)-a-D- P-Galactosidase Asperg illus Glc oryzae p-D-Gal(1-4)-a-D- P-Galactosidase Bacillus GlcNAc-0 circulans p-~-Gal( 1-4)-a-D- P-Galactosidase Bacillus Glc-S circulans P-wGal(1-~)-P-D- P-Glucosynthase Agrobacterium 2-deoxy-2-ClGlc-0 SP. P-D-GaI(1-4)-P-D- P-Galactosidase Bacillus Gal circulans P-D-Gal(1-4)-P-D- P-Galactosidase Bacillus Gal circulans P-D-Gal(1-4)-P-D- 0-Galactosidase Bacillus Gal circulans p-~-Gal( 1-4)-P-D- 0-Galactosidase Bacillus Gal circulans P-u-Gal(1-~)-P-D- P-Galactosidase Bacillus Gal circulans p-~-Gal( 1-4)-P-D- 0-Galactosidase Bacillus Gal circulans 111
28
105
30
P-~-2-deoxy-2-C1-Glc-O-2,4- p-~-Gal( 1-4)-P-~-2-deoxy-2- 64 CI-Glc-O-2.4-DNP DNP 5
p-~-Gal( 1-4)-u-~-Glc-SEt
a-D-Glc-SEt
aGalF
a-D-Glc(1-6)-y-CD
Lactose
5x 115
a-~-Glc( 1-4)-a-o-Glc(l-6)-PCD
C
115
2
0
3
p-~-Gal( 1-4)-P-~-Ga1(1-4)-a- 5.3 115 D-GIc(1-4)-a-D-GIC(I-6)-PF b CD ? P-D-G~~(~-~)-P-D-G~I(I-~)-~3.2 115 rc ~ - G l c1-6)-y-CD ( 2.
8
z -s
rc
b
1
a-~-Glc( 1-4)-a-~-Glc(l-6)-a- p-~-Gal( 1-4)-P-~-Gal(l-4)-a- 5.3 CD ~ - G l c1-4)-a-~-Glc( ( 1-6)-aCD fl-~-Gal(l-4)-P-~-Gal(l-4)-a-4.2 a-~-Glc( ld)-P-CD ~ - G l cId)-P-CD (
Lactose
Lactose
Lactose
"J
e rc
115
p-~-Gal( 1-4)-p-~-Gal( 1-4)-a~ - G l c1(-6)-a-CD
a - ~ - G l cI-6)-a-CD (
Lactose
b 0
b,
5.7
p-D-Gal(1-4)-P-~-Gal( 1-q-DGlcA
GlcA
Lactose
116
PpNPGal
14
p-~-Gal( I-4)-a-~-GlcNAcOBn
62
a-D-GlcNAc-OBn
D-GIC PpNPGal
P-~-Gal(l-4)-u-~-Glc(l-l)-a1.2
Trehalose
Gal
330
329
328
327
326
325
324
323
322
Enzyme Source
P-Galactosidase Bacillus circulans P-D-Gal(1-4)-P-D- 0-Galactosidase Bacillus Gal circulans P-D-Gal(1-4)-P-D- P-Galactosidase Bacillus Gal circulans p-D-Gal(1-4)-P-D- P-Galactosidase Bacillus GalNAc circulans P-D-Gal(1-4)-P-D- P-Galactosidase Barley Gal-0 P-D-GaI(1-4)-0-D- P-Galactosidase Bacillus Gal-0 circulans P-D-Gal(1-4)-P-D- 0-Galactosidase Gal-S Bacillus circulans P-D-Gal(1-4)-P-~- exo-P-galactanase Aspergillus niyer Gal P-D-Gal(1-4)-P-D- P-Galactosidase Bacillus Glc circulans
Entry Linkage
Table 1 (continued)
Lactose
Galactan
P-D-Gal-{P-D-Gal(I-~)),-DGal Rubusoside
7.3
31
60
52
118
111
92 32
Lactose
P-D-Gal(1-4)-b-~-Gal-SEt
51 3
PpNPGal
PpNPGal
25
PpNPGalNAc
Lactose
17
Lactose
Lactose
117
115
Reference
p-D-Gal( 1-4)-P-~-GalNAcOpNP
Lactose
PpNPGal
p-D-Gal(1-4)-P-~-Gal( 1-4)-a- 8.2 ~ - G l c1-4)-a-~-Glc( ( 1-6)-yCD P-D-Gal(1-4)-P-~-Gal( 1-4)-D- 52 Glu
Yield ("%)
94
a-D-Gk(1-4)-a-D-GIc(1-6)-yCD
Lactose
Product
1- 4 ) - ~ - NA P-D-Gal(1-4)-P-~-Gal( Glc
Acceptor
Donor
P-D-Gal(1-4)-P-DGlcN3-S
P-D-Gal(1-4)-P-DGlcNAc
P-D-Gal(1-4)-P-DGlcNAc
p-D-Gal(1-4)-P-DGlcNAc
P-D-Gal(1-4)-P-DGlcNAc-0
P-D-Gal(1-4)-P-DGlcNAc-0
336
337
338
339
340
P-Galactosidase Bullera singularis P-Galactosidase Bacillus circulans P-Galactosidase Bacillus circulans P-Galactosidase Bacillus circulans P-Galactosidase Diplococcus pneumoniae P-Galactosidase Bullera singularis P-Galactosidase Bacillus circulans
SP.
B-D-Gal(1-4)-P-D- P-Glucosynthase Agrobacterium Glc
SP.
P-D-Gal(1-4)-P-D- P-Galactosidase Bacillus Glc circulans P-D-Gal(1-4)-P-D- P-Glucosynthase Agrobacterium Glc
335
334
333
332
33 1
Lactose
Lactose
PpNPGal
PpNPGal
PpNPGal
PpNPGal
Lactose
13
PpNPGlcNAc
P-D-GlcNAc-0-Ser-(N-Z)OEt
1-6)-~-Man P-D-G~cNAc(
P-D-G~cNAc( 1-2)-~-Man
119
1 17
p-D-Gal(1-4)-P-~-GlcNAcOpNP
Ser-(N-Z)-OEt 21
P-D-Gal(1-4)-P-~-GlcNAc-o- 20
6)-~-Man
25
102
P-~-Gal(l-4)-P-~-GlcNAc(l17 119
2)-~-Man
P-~-Gal(l-4)-P-~-GlcNAc(l4.2
1-~)-P-D-G~cNAc( 1- 48 1-6)-~-GalNAc p-~-Gal( P-D-G~cNAc( 6)-~-GalNAc
z
2
F'
2
2'
b
5P
rL
n 3:
$
(D
B
5
0
(D
P-D-G~~(~-~)-P-D-G~cN~-SE~ 4 116 ru F m
p-D-Gal(1-4)-P-~-GlcN3OMe
83
28
P-D-GlC(1-4)-P-D-GlCOp(0Me)Ph
aGalF
88
1-4)-P- 92 P-D-G~c( 1-4)-P-~-Glc-opNP P-~-Gal(l-4)-P-~-Glc( D-GIc-O~NP
aGalF
P-~-Gal(l-4)-(3-~-Glc( 14-PD-Glc-OpOMePh
28
Rubusoside
Lactose
349
348
347
346
345
344
343
342
341
Enzyme Source
p-D-Gal(1-4)-P-D- 0-Galactosidase Bacillus GlcNAc-0 circulans P-D-Gal(1-4)-B-D- P-Galactosidase GlcNAc-0 Bacillus circulans P-Galactosidase Bacillus circulans p-D-Gal(1-4)-P-D- P-Galactosidase GIcNAc-0 Bovine testes P-D-Gal(1-4)-P-D- P-Galactosidase Bacillus GlcNAc-0 circulans P-D-Gal(1-4)P-D- P-Galactosidase GlcNAc-S Bacillus circulans P-D-Gal(1-4)-P-~- P-Galactosidase Bullera GIcNAc-S singularis P-D-Gal(1-~)-P-D- P-Galactosidase Bacillus GIcNAc-S circulans P-D-Gal(1-4)-P-D- 0-Galactosidase Bullera GlcNPht-S singularis
Entry Linkage
Table 1 (continued)
P-~-GlcNAc-O-6-hydroxymethyl-hexanoate P-D-GlcNAc-OBn
P-D-G~CNAC-O-(CH&CH~
P-D-GlcNAc-OMe
PpNPGal
PpNPGal
PpNPGal
PpNPGal
P-D-GlcNAc-SEt
P-D-GlcNAc-SEt
P-D-GlcNAc-SEt
P-D-GlcNPht-SEt
PpNPGal
Lactose
PpNPGal
Lactose
6'-oxo-P-~-Gal-O- 0-D-GlcNAc-OMe PNP
Acceptor
Donor ("h)
Yield
P-D-Gal(1-4)-P-~-GlcNPhtSEt
P-D-Gal(1-4)-p-~-GlcNAcSEt
p-D-Gal(1-4)-P-~-GlcNAcSEt
P-D-Gal(1-4)-p-~-GlcNAcSEt
B-D-Gal(1-4)-p-~-GlcNAcOMe P-D-Gal(1-4)-b-~-GlcNAcOMe
20
49
11
14
60
10
about
10
P-D-Gal(1-4)-P-~-GlcNAc-0- 57 6-hydroxy-methylhexanoate p-D-Gal(1-4)-p-~-GlcNAc- 14 OBn
Product
84
111
83
116
120
45
116
116
116
Reference
29.6 Recent Developments and New Directions m 00
w m
m
00
00
N
E:
2
2
d
00
N
cx
m
CI
cx
vi
vi
00
00
I-
0
2
E:
3
N
-
w
u3
u3 iD
3 3
m
N
b
9
h
P
t
u
b ci
ci
+ b
4
b
9
@?
t
t
ci
Ci
h
Tt
ci
767
~
369
368
361
P-D-Gal(1-4)-DGalNAc
p-D-Gal(1-4)-DGalNAc
P-Galactosidase Bacillus circulans P-Galactosidase Bifdobacterium bzj5durn P-Galactosidase Penicillium multicolor
SP.
SP.
P-D-Gal(1 -4)-P-D- P-Glucosidase Agrobacterium Xyl-s
365
364
363
366
SP.
P-Galactosidase Escherichia coli P-Galactosidase Escherichiu coli P-Glucosidase Guinea pig liver 0-Glucosidase Agrobacterium
b-~-Gat( 1-4)-P-DXyl-0 p-D-Gal(1-4)-P-DXyl-0 p-~-Gal( 1-4)-P-DXyl-0 P-D-Gal(1-4)-P-DXyl-s
362
85
14 NA
9
11
16
33
1
Lactose PpNPGal PpNPGal
PpNPGal
Lactose
PpNPGal
PpNPGal
113
113
92
85
121
17
PpNPGal
103, 110 74
85
11
PpNPGal
P-D-Gal(I-4)-P-D- P-Glucosidase Xyl-0 A grobacterium
361
SP.
85
Reference
24
Yield (%)
PpNPGal
Product
P-Glucosidase Agrobacterium
Acceptor
P-D-Gd(1-4)P-DMan-S
Donor
~~
Enzyme Source
360
Entry Linkage
~
Table 1 (continued)
29.6 Recent Developments and New Directions
769
p-D-Gal(1-4)-DGlcNAc
p-D-Gal(1-4)-DGlcNAc
p-D-Gal(1-4)-DXYl p-D-Gal(1-4)-DXYI
fi-~-Gal( 1-4)-~XYl
p-D-Gd(1-4)-DXYl
P-D-Gal(1-4)-DXYI
384
385
386
388
389
390
387
P-D-Gd(1-4)-DGlcNAc
P-D-Gal(1-4)-DGlcNAc P-D-Gd(1 -4)-DGlcNAc
383
382
381
Entry Linkage
Table 1 (continued)
P-Galactosidase Escherichiu coli P-Galactosidase Aspergillus oryzae p-Galactosidase Clonezyme libraryTM 0-Galactosidase Bacillus circulans P-Galactosidase Bacillus circuluns P-Galactosidase Bovine testes P-Galactosidase Intestinal lactase Lamb P-Galactosidase Asperg illus oryzue 0-Galactosidase Saccharomy ces fragilis P-Galactosidase Escherichia coli
Enzyme Source
GlcNAc
GlcNAc
XYI XYl
XYl
XYl
XYl
Lactose
PoNPGal PoNPGal
PoNPGal
PoNPGal
SoNPGal
GlcNAc
PoNPGal
Lactose
GlcNAc
Gal
p-~-Gal( 1-4)-D-xyl
p-D-Gal(1-4)-~-GlcNAc
43
42
61
0.9
P-D-Gal(I -4)-~-GlcNAc
GlcNAc
Gal
p-D-Gal(1-4)-~-GlcNAc
Yield ("%)
Product
Acceptor
Donor
95
124, 125
123
21
Reference
4
i?
0 u -
0
4
2 s
0
4.
$
+&
403
402
40 1
400
399
398
397
396
395
394
393
392
39 1
0-Galactosidase Escherichia coli P-Galactosidase Bovine testes P-Galactosidase Intestinal lactase Lamb P-Galactosidase Asperg illus oryzae P-Galactosidase Saccharomyces fragilis p-D-Gal(1-4)-LP-Galactosidase Escherichia coli XYl P-D-Gal(1-5)-a-L- P-Glucosidase Guinea pig liver Araf-0 P-D-Gal(1-6)-a-~- P-Galactosidase GalNAc-0 Porcine testes p-~-Gal( 1-6)-a-D- P-Galactosidase Escherichia coli Gal-0 P-D-Gal(1-6)-a-~- P-Galactosidase Barley Gal-0 1-6)-a-~- 0-Galactosidase p-~-Gal( Snail Gal-0 p-~-Gal( 1-6)-a-~- P-Galactosidase Asperg illus Gal-0 oryzae p-~-Gal( 1-6)-a-~- P-Galactosidase Gal-0 Aspergillus ovyzae 14 NA NA 57 70
P-D-Gal(1-6)-a-~-Gal-OMe P-D-Gal(l-6)-a-~-Gal-OMe P-D-GaI(l-6)-a-~-Gal-OMe p-~-Gal( l-6)-a-~-Gal-OMe
a-~-Gal-OMe a-~-Gal-OMe a-~-Gal-OMe a-D-Gal-OMe
PoNPGa1 PpNPGal PpNPGal PoNPGal
SVGal
I00
P-D-Gal(]-6)-a-~-GalNAcOpNP P-D-GaI(l-6)-a-~-Gal-OMe
apNPGalNAc
Lactose
93
93
51
51
26
121
p-~-Gal( l-5)-a-L-Araf-OpNP NA
a-L-Araf OpNP
PpNPGal
5.6
95
20
P-D-Gal(1-4)-L-xyl
L-Xyl
L-Xyl
PoNPGal
96
PoNPGal
L-Xyl
(3oNPGal
39
L-XyI
L-Xyl
PoNPGal
b-~-Gal( 1-4)-D-xyl
PoNPGal
XYl
PpNPGal
2
6'
2 2
5
35
9%
2 5
C
5
?
5
rp
2J cs
o\
P
b
~
~~
412
41 1
410
409
408
407
406
405
404
Enzyme Source
P-D-Gal(I-~)-cL-D-0-Galactosidase Bacillus Glc circulans P-D-Gal(1-6)-a-~- P-Galactosidase Bacillus Glc circulans P-D-Gal(1-6)-a-~- P-Galactosidase Bacillus Glc circulans P-D-Gal(1-6)-a-~- P-Galactosidase Bacillus Glc circulans P-D-Gd(1-6)-a-~- B-Galactosidase Bacillus Glc circulans P-D-Gal(1-6)-a-~- 0-Galactosidase Penicillium Glc multicolor P-D-Gal(1-6)-a-~- P-Galactosidase Penicillium Glc multicolor P-D-Gal(1-6)-a-~- 0-Galactosidase Penicillium Glc multicolor (I-D-Gal(1-6)-a-~- P-Galactosidase Penicillium Glc multicolor
Entry Linkage
~~
Table 1 (continued)
115
115
1.2
2.3
1.6
6.8
10
14
P-D-Gal(1-6)-a-~-Glc( I -6)-PCD p-D-Gal(1-6)-a-~-GIc( 1-4)-a~ - G l c1 (-6)-P-CD
1-6)-aP-D-GaI(I -6)-a-~-Glc( CD
B-D-Gal(l-6)-a-~-Glc( 1-4)-aD-GIc(1-6)-a-CD P-D-GaI(I-6)-a-D-GIC(1-6)-PCD
P-D-GaI(1-~)-cL-D-GIc( 1-4)-~- 13 D-Gk(1-6)-P-CD
a-D-Glc(ld)-P-CD
a-D-GIC(I-.l)-a-D-GlC(1 -6)-PCD a-~-Glc( 1-4)-a-~-Glc( 1-6)-yCD a-~-Glc( I -6)-a-CD
a-D-Glc(1-4)-a-~-Glc( 1-6)-aCD
Lactose
Lactose
Lactose
Lactose
Lactose
Lactose
Lactose
1-4)-a-D-GlC(1-6)-Pa-~-Glc( CD
115
2.6
P-D-GaI(1-6)-a-D-GIC(I - ~ ) - U D-GIc(1-6)-a-CD
a-D-Gk(1-4)-a-D-Gk(1-6)-aCD
Lactose
115
115
1I5
115
115
115
p-D-Gal(1-6)-a-~-Glc( 1-6)-1~- 1.1 CD
a-~-Glc( 1-6)-a-CD
Reference
Lactose
Yield (%)
Product
Acceptor
Donor
424
423
422
42 1
420
419
418
417
416
415
414
413
P-D-Gal(1-6)-a-~- S-Galactosidase Penicillium Glc multicolor P-D-Gal(1-6)-a-~- P-Galactosidase Penicillium Glc multicolor P-D-Gal(1-6)-U-D- P-Galactosidase Escherichia coli Glc p-D-Gal(1-6)-a-~- P-Galactosidase Asperg illus Glc oryzae S-D-Gal(1-~)-P-D- P-Galactosidase GalNAc-0 Porcine testes P-D-Gal(1-6)-P-D- P-Galactosidase Asperg illus Gal-0 oryzae p-D-Gal(1-~)-P-D- P-Galactosidase Escherichia coli Gal-0 P-D-GaI(1-6)-P-~- 0-Galactosidase Escherichia coli Gal-0 P-D-Gal(1-~)-P-D- P-Galactosidase Escherichia coli Gal-0 P-D-GaI(1-6)-P-D- b-Galactosidase Escherichia coli Gal-0 p-D-Gal(1-6)-p-~- P-Galactosidase Escherichia coli Gal-0 P-D-Gal(1-6)-P-D- P-Galactosidase Bovine liver Gal-0 PoNPGal
Lactose
Lactose
Lactose
PoNPGal
PoNPGal
DoNPGal
Lactose
Gal
ru 9
2
P
44
106 107
3
P-D-GaI(1-6)-P-~-Gal-O-Ser- 6 P-u-Gal-O-Ser-(N-Aloc)(N-A1oc)-OMe OMe 10 monoallyl ether ( P-D-G~c-O)- monoallyl ether (P-D-Gal(16)-P-~-Gal-o)-( P-D-G~c1,3,5-benzene-trimethanol 0)-1,3,5-benzenetrimethanol
S-u-Gal-OBn
W
4 4
2
F'
2
2
s
m
4
3
n
$
2
% 44
2.4
P-D-Gal-OAl1
P-D-Gal(1-6)-P-~-Gal-OA11
C
26
2
o\
3
63
p-D-Gal(1-6)-p-~-Gal-OMe
DoNPGal
P-D-Gal(1-6)-p-~-Gal-OoNP
62
13
floN PGa1
127
NA
P-D-Gal(1-6)-a-~-Glc( 1-4-PD-Fruf 1- 1)-uP-D-Gal(1-6)-a-~-Glc( D-Glc
100
115
8
1-4)-aP-D-Gal(1-6)-a-~-Glc( ~ - G l c1-6)-y-CD (
2.6 p-D-Gal(1-6)-(3-~-GalNAcOpNP P-D-Gal(1-6)-P-~-Gal-ooNP 21
115
9.5
P-D-Gal(1-6)-a-~-Glc( 1-6)-yCD
PpNPGalNAc
Trehalose
Sucrose
a-u-Glc( 1-4)-~-D-Glc( 1-6)-yCD
Lactose
PoNPGal
a-~-Glc( 1-6)-y-CD
Lactose
434
433
432
431
430
429
428
421
426
425
Enzyme Source
P-Galactosidase Bacillus circuluns p-~-Gal( 1-6)-8-~- P-Galactosidase Escherichiu coli GIcNAc-S P-D-GaI(I -6)-P-D- 0-Galactosidase Bovine testes GlcNPth-S P-D-Gal(1-6)-P-D- P-Galactosidase Glc-0 Bacillus circulans p-~-Gal( 1-6)-P-~- P-Galactosidase Bacillus Glc-0 circulans P-D-GaI(I -6)-P-D- 0-Galactosidase Escherichiu coli Glc-0
SP.
1-~)-P-D- P-Glucosidase p-~-Gal( Agrobucterium Gal-S
SP.
p-D-Gal(1-~)-P-D- P-Galactosidase Gal-0 Bucillus circuluns P-D-Gal(1-~)-P-D- 0-Glucosidase Gal-0 Guinea pig liver P-D-Gal(]-~)-P-D- P-Glucosidase Agrobucteriurn Gal-S
Entry Linkage
Table 1 (continued)
Rubusoside
Rubusoside
Rubusoside
Lactose
Lactose
B-D-GlcNPth-SEt
Lactose
Lactose
13-P-~-Gal( 1-6)-P-~-Glc-19P-D-Glc-0-steviol
I~-P-D-GIC-I 9-P-D-Gal(1-6)P-D-Glc-0-steviol
0.5
12
p-D-Gal(1-6)-p-~-GlcNAc- 28 SEt P-D-Gal(1-6)-P-o-GlcNPth10 SEt I~-P-D-G~I( 1-6)-P-~-Glc-l9- 2.6 P-D-Glc-0-steviol
P-D-GlcNAc-SEt
Lactose
26
5.2
p-~-Gal( 1-6)-P-~-GlcNAcOpNP
P-D-Gal(1-6)-p-~-Gal-SBn
PpNPGlcNAc
p-D-Gal-SBn
Lactose
PpNPGal
52
52
52
83
108
25
85
85 8.8
P-D-Gal(1-h)-b-~-Gal-SPh
p-~-Gal-SPh
PpNPGal
121
P-D-Gal(1-6)-P-~-Gal-opNP NA
Reference
PpNPGal
PpNPGal
Yield (“A)
92
Product
1.1
P-D-Gal-OMe
Acceptor
Lactose
Donor
445
444
443
442
441
440
439
438
431
436
435
P-Galactosidase Bacillus circulans p-tA+aI(1-6)-P-D- P-Galactosidase Bacillus Glc-0 circulans p-D-Gal(1-6)-P-D- P-Galactosidase Glc-S Bacillus circulans P-D-Gal(1-6)-~- 0-Galactosidase Escherichia coli (3-OMe)-Glc Pa-Gal( 1-6)-~- P-Galactosidase Kluyueromyces (3-OMe)-Glc fray iris
P-D-Gal(1-6)-P-D- P-Galactosidase Escherichia coli Glc-0 p-D-Gal(1-6)-P-D- P-Galactosidase Aspergillus Glc-0 oryzae P-D-Gal(1-6)-P-D- P-Galactosidase Glc-0 Aspergillus oryzae P-D-Gal(1-6)-P-D- P-Galactosidase Glc-0 Penicillium multicolor p-D-Gal(1-6)-P-~- P-Galactosidase Glc-0 Penicillium multicolor p-~-Gal( 1-6)-P-D- P-Galactosidase Glc-0 Bovine liver Rubusoside
Rubusoside
monoally1 ether ( p-~-Glc-O)- monoally1 ether (p-D-Gd(16)-P-~-Glc-O)-1,3,51,3.5-benzene-trimethanol benzene-trimethanol P-D-Gal(1-6)-P-~-Glc-oMe P-D-Glc-OMe
Lactose
Lactose
PoNPGal
SoNPGal
p-D-Gal(1-6)-~-(3-OMe)-Glc
P-D-Gd(1-6)-P-D-Gk-SEt
P-D-Glc-SEt
PpNPGal
PpNPGal
p-D-Gal(1-6)-P-Glc-OpNP
PpNPGlc
P-D-Glc-0-steviol 3.8
2.3 13-P-~-Gal(l-6)-P-~-Glc-19-
Lactose
Lactose
52
1 ~ - P - D - G ~ c - ~ ~ - P1-6)- D - G ~ ~3.2 (
Rubusoside
Lactose
52
52
52
2.2
Rubusoside
Lactose
P-D-Glc-0-steviol
52
12
Rubusoside
Lactose
116
29 Glycosidase-Catalysed Oliyosaccharide Synthesis
8 0
c 0
3
d
4
W
* d
ci
ci
ci
u
ci
r-
M
o\
0 m
-
d
d
d
d
d
e
d
m
ci N
m
? l
d
d
d
m
b-D-Gal(1- 6 ) - ~ Glc
p-D-Gal(1- 6 ) - ~ Glc
P-D-Gal(1-6)-~Glc
p-D-Gal(1 - 6 ) - ~ Glc3NAc
p-D-Gal(1 - 6 ) - ~ GlcN
P-D-Gal(1-6)-~GlcNAc p-D-Gal(1 - 6 ) - ~ GlcNAc
451
458
459
460
46 1
462
p-D-Gal(1 - 6 ) - ~ GlcNAc
p-D-Gal(1-6)-~GlcNAc
464
465
463
456
P-D-Gal(I - ~ ) - D Glc P-mGal(1 -6)-DGlc
455
P-Calactosidase Eschrvichiu coli j3-Galactosidase BiJidobacterium hiJidum j3-Galactosidase Bijidobacterium bijidum P-Galactosidase Bacillus circulans P-Galactosidase Bacillus circuluns P-Galactosidase Bacillus circulans P-Galactosidase Clonezyme libraryTM P-Galactosidase Eschrrichiu coli P-Galactosidase Kluyveromy ces fragilis P-Galactosidase Bzjidobacterium bijidum P-Galactosidase Eschericliia coli
3.2 NA
NA
NA
NA
9.3
10
30
2.5
9.1
P-u-Gal(1-6)-~-Glc P-D-Gal(1-6)-D-Glc
P-D-Gal(1-6)-[p-~-Gal( 1-4)]D-G~ p-D-Gal(I-6)-u-Glc
p - ~ - G d1-6)-[ ( p-~-Gal( 141~-Glc p-~-Gal( 1-6)-D-Gk3NAC
P-D-Gdl( I-6)-D-GlcN
P - D - G ~1-6)-~-GlcNAc (
p-D-Gal(1-6)-~-GlcNAc
P-D-Gal(1-6)-~-GlcNAc
Glc Lactose
Lactose
Lactose
Lactose
Glc3NAc
GlcN
GlcNAc GlcNAc
GlcNAc
GlcNAc
Gal Lactose
Lactose
Lactose
Lactose
Lactose
Lactose
PoNPGal PoNPGal
PpNPGal
Gal
21
113
20
122
92
94
94
104
104
40
4 4 4
z
2 6'
rh
$.
3F
2
C
B $-
5
rh
? n
ca
P
t u
Table 1 (continued)
b-~-GalNAc( 1-3)-P-o-Glc(1 ~)-P-D-GIc-SE~
1-6)-p-~-Glc-SEt P-D-G~c(
PpNPGalNAc
B-u-GalNAc(13)-P-~-Glc
474
17
6
P-D-GalNAc( 1-3)-P-~-GalOMe
P-o-Gal-OMe
PpNPGalNAc
P-D-GalNAc(13)-p-~-Gal-O
473
8.5
P-D-Gal(1-9)-~-NeuAc
NeuAc
Lactose
P-D-Gal(1-9)aNeuAc
8.5
p-D-Gal(1-8)-~-NeuAc
NeuAc
15
1.9
18
D-D-Gal(1-6)-~-Glucal
Lactose
412
47 1
D-GlUCd
PpNPGal
P-D-Gal(1-6)GlUcdl P-D-Gal(1-8)-~NeuAc
470
S-D-Gal(1-6)-~-GlcNAc
GlcNAc
Lactose
p-~-Gal( 1-6)-~GlcNAc
469
GlcNAc
Lactose
P-D-Gal(l-6)-DGlcNAc
468
129
56
105
105
97
92
126
126
2.3
p-D-Gal( 1-6)-~-GlcNAc
GlcNAc
Lactose
P-D-Gal(1 - 6 ) - ~ GlcNAc
467
40
12
P-D-Gal(1-6)-~-GlcNAc
GlcNAc
Gal
P-Galactosidase Asperg illus oryzae P-Galactosidase Bacillus circulans P-Galactosidase Kluyveromyces lactis P-Galactosidase Bucillus circuluns P-Galactosidase Escherichia coli P-Galactosidase Bacillus circuluns P-Galactosidase Bacillus circulans P-Hexosaminidase Chamelea gallinu P-Hexosaminidase Asperg illus oryzue
p-D-Gal(1-6)-DGlcNAc
466
Reference
("%)
Yield
Product
Acceptor
Donor
Enzyme Source
~~
Linkage
Entry
~
P-D-GalNAc(13)-P-D-GIC-O
P-D-GalNAc(14)-a-D-GIC
P-D-GalNAc(14)-a-DGIcNAc-0 P-D-GalNAc(14)-a-~-GIc-o
P-D-GalNAc(14)-P-~-Glc
P-D-GalNAc(14)-P-D-GIC
P-D-GalNAc(14)-P-DGIcNAc-0 P-D-GalNAc(14)-P-~-Glc-o
P-D-GalNAc(14)-~-GlcNAc
476
477
478
480
48 1
482
484
483
479
P-D-GalNAc(13)-P-D-GIC-O
475
0-Hexosaminidase Aspergillus oryzae P-Hexosaminidase Aspergillus oryzae P-Hexosaminidase Aspergillus oryzae 0-Hexosaminidase Aspergillus oryzae P-Hexosaminidase Aspergillus oryzae P-Hexosaminidase Aspergillus oryzae P-Hexosaminidase Aspergillus oryzae P-Hexosaminidase Aspergillus oryzae P-Hexosaminidase Aspergillus oryzae P-Hexosaminidase Aspergillus oryzae PpNPGalNAc
PpNPGalNAc
PpNPGalNAc
PpNPGalNAc
PpNPGalNAc
PpNPGalNAc
PpNPGalNAc
PpNPGalNAc
PpNPGalNAc
PpNPGalNAc
30
31
P-D-GalNAc(]-4)-a-~-GlcOMe 1P-D-GalNAc(I -4)-P-~-Glc( 4)-p-~-Glc-SEt
a-D-Glc-OMe
GlcNAc
P-D-Glc-OMe
P-D-GlcNAc-OMe
P-D-GIc(1-6)-p-~-Glc-SEt
P-D-GIc(1-4)-p-~-Glc-SEt
10
P-D-GalNAc(1-4)-P-~-GlcOMe
P-D-GalNAc(1-4)-~-GlcNAc 72
20 P-D-GalNAc(1-~)-P-DGlcNAc-OMe
27
129
27
81
P-D-GalNAc(1-4)-a-~GlcNAc-OMe
a-D-GlcNAc-OMe
20
129
29
P-D-GalNAc(1-4)-a-~-Glc( 14)-P-D-Gk-SEt
1-4)-p-~-Glc-SEt a-~-Glc(
P-D-GalNAc(1-4)-P-~-Glc( 16)-P-D-Gk-SEt
130
29
P-D-GalNAc(1-3)-P-~-GlcOMe
P-D-Glc-OMe
129
1P-D-GalNAc(1-3)-P-~-Glc( 6)-p-~-Glc-SEt
41
P-D-GIc(1-4)-P-mGlc-SEt
h
?E
C
$-
B
494
493
492
49 1
490
489
488
487
486
485
Enzyme Source
P-D-GalNAc(16)-a-~GIcNAc-0 P-D-GalNAc(16)-a-~-Glc-O
SP.
P-Hexosaminidase Asperg illus oryzae P-Hexosaminidase Asperg illus oryzae P-Hexosaminidase P-D-GalNAc(1 Asperg illus 6)-~-GalNAc oryzae P-Hexosaminidase P-D-GalNAc(1~)-D-G~cNAc Asperg illus oryzae P-D-Glc(1-2)-P-D- P-Galactosidase Asperg illus Glc-0 oryzae P-D-GIc(1-2)-P-D- 0-Galactosidase Asperg illus Glc-0 oryzae 0-Glucosidase P-D-GlC-(1 -2)-DAlmond Glc P-D-Glc-(1-2)-D- P-Glucosidase Penicillium Glc funiculosum P-Glucosidase P-D-GlC(1 -2)-DAlmond Glc P-D-Glc(14-P-D- P-Glucosynthase Agrobacterium Xyl-0
Entry Linkage
Table 1 (continued)
60, 132 60
133 28
I9
3.1 3.3
5.7 I2
P-D-Gk(1-2)-P-D-GlC-OpNP
P-D-GIc-(I -2)-D-GIC P-D-GIC-(1-2)-D-Gk
P-D-Gk( 1-2)-D-Gk P-D-GlC(I-3)-P-D-Xyl-OpNP
GalNAc
GlcNAc
PpNPGlc
PpNPGlc
Glc Glc
Glc PPNPXYI
PpNPGalNAc
PpNPGalNAc
PpNPGlc
PpNPGlc
Glc Glc
Glc aGlcF
131
P-D-GalNAc(1-6)-~-GlcNAc 33
64
117
130
P-D-GalNAc(1-6)-~-GalNAc 38
OMe
6
P-D-GalNAc(I-6)-a-u-Glc-
a-D-Gk-OMe
PpNPGalNAc
27
8
P-D-GalNAc(1-6)-a-~GlcNAc-OMe
a-D-GlcNAc-OMe
Reference
PpNPGalNAc
Yield (YO)
Product
Acceptor
Donor
2
b rp
3-
h
2
u2
0 =r
il
P
83
%.
%-
0
Q
b
0
00
4
506
505
504
503
502
50 1
500
499
498
491
496
495
SP.
SP.
P-D-Gk(1-4)-P-D- P-Glucosynthase Agrobacterium Glc aGlcF
aClcF
Glc
P-D-GlC(1-4)-P-D- P-Clucosidase Glc Almond P-D-GlC(1-4)-P-D- P-Glucosynthase Agrobacteriurn Glc
SP.
aClcF
aGlcF
aClcF
aGlcF
aClcF
Glc
3
PpNPGlc
PPhGlc
P-D-GlC(1-4)-P-D-Gk(I-4)-PD-GlC-OPh
24
34
P-D-GlC(I-4)-P-D-Gk(1 4 4 - 42 P-~-2-deoxy-2-F-Glc-O-2,4~-2-deoxy-2-F-Glc-O-2,4DNP DNP 4 P-~-2-deoxy-2-F-Glc-O-2,4- P-D-GlC(1-4)-P-D-GlC(1-4)-P~ - G l c1(-4)-P-~-2-deoxy-2DNP F-Glc-O-2,4-DNP NA Cellobiose Glc3
D-D-G~C( 1-4)-P-~-Glc( 1-4)-PD-GlC(1-3)-P-D-Xyl-OpNP
51
28
62
Trehalose
1-4)-a-~-Glc( 1-1)-aP-D-G~c( 0.9 D-GlC P-D-GIC(I -4)-P-~-2-deoxy-2- 38 P-o-2-deoxy-2-F-Glc-0-2,4DNP F-Glc-0-2.4-DNP
Glc
Glc
3.1
P-D-GlC-(1-3)-D-GlC
60, 132 60
133
Glc
Glc
3.4
P-D-GlC-(1-3)-D-GlC
4.2
Glc
Glc
P-D-GlC(1-4)-P-D- P-Glucosynthase Agrobacterium Glc
SP.
P-D-GlC(1-4)-P-D- P-Glucosynthase Agrobacterium Glc
SP.
P-D-GIc(1-4)-P-~- P-Glucosynthase A grobacterium Glc
SP.
P-Glucosidase Almond P-Glucosidase Penicillium funiculosum P-Glucosidase P-D-GlC(1-3)-DAlmond Glc P-D-GlC(1-4)-a-D- P-Glucosidase Almond Glc P-D-GlC(1-4)-P-D- P-Glucosynthase Agrobacterium 2-deoxy-2-FGk-0 SP. P-D-GlC(1-4)-P-D- 0-Glucosynthase Agrobacterium Glc
P-D-GlC-(1-3-DGlc P-D-GlC-(1-3)-DGlc
515
514
513
512
51 1
510
509
508
507
SP.
P-D-GIc(1-4)-P-D- P-Glucosynthase Agrobacterium Glc SP. P-D-GlC(1-4)-P-D- P-Glucosynthase Agrobacteriurn Glc
SP.
p-~-Glc( I -4)-P-D- P-Glucosynthase Glc Agrobacteriurn
SP.
SP.
P-D-GIC(I-4)-P-D-Gk(I-4)-P8
PpOMePhGlc
aGlcF
aGlcF
D-GIc(1-4)-P-~-Glc-opNP
P-D-GIc(1 -4)-P-~-Glc-opNP P-D-GIc(1-4)-P-D-Gk(1-4)-P-
64
79
66
P-D-Glc(1-4)-P-D-GIc(I -4)-PD-Glc-OpOMePh
PpOMePhGlc
aGlcF
~ - G l c1(-4)-P-D-GkOpOMePh I-~)-P-D-GIc( 1-4)-PP-D-GIc(1-4)-P-~-Glc-opNP j3-~-Glc( D-GIc-O~NP
54
P-D-GIc(1-4)-P-~-Glc( 1-4)-P~ - G l c1-4)-P-~-Glc-oMu (
PMuGlc
aGlcF
aGlcF
75
P-D-Glc(I-'I)-P-D-GIc(1-4)-8D-Glc-OMU
PMuGlc
6
aGlcF
P-D-Gk(I -4)-P-D- P-Glucosynthase Glc A yrobacterium SP. P-D-G~c( I -4)-P-D- P-Glucosynthase Glc Agrobacterium SP. P-D-GIc(I-4)-P-D- P-Glucosynthase Agrobacterium Glc
SP.
P-D-GIc(I -4)-P-D-Gk(I -4)-PD-Glc-OoNP
PoN PG1c
P-D-Glc(1-4)-P-D-Gk(1 4 - 0 D-Glc(1-4)-P-D-Glc-OoNP
aGlcF
P-D-Glc(1-4)-P-D- P-Glucosynthase Glc Agrobacterium
10
P-D-Glc(I-'I)-P-D-Gk(I-4)-8D-GlC(I -4)-P-D-Gk-OpNP
PpNPGlc
29
Yield (%)
Product
Acceptor
PoNPGlc
aGlcF
SP.
P-D-Glc(I-4)-P-D- P-Glucosynthase Glc Agrobacterium
Donor
aGlcF
Enzyme Source
P-D-GIc(1-4)-P-D- P-Glucosynthase Glc Agrobacterium
Entry Linkage
Table 1 (continued)
28
28
28
28
28
28
28
28
28
Reference
P-D-G~c-( 1-4)-~- P-Glucosidase Almond Glc
SP.
526
SP.
0-Glucosynthase Agrobacterium
1-4)b-DP-D-G~C( Man-0 Glc
aGlcF
Glc
PpNPMan
P-D-G~c-( 1-4)-D-Gk
5.9
P-~-Glc(l-4)-P-~-Man-opNP 31
OpOMePh
60, 132
28
28
44
P-D-G~c( I-4)-p-~-Glc-
PpOMePhGlc
aGlcF
P-D-G~c( 1-4)-P-u- P-Glucosynthase Agrohacterium Glc-0
SP.
28
P-D-G~c( 1-4)-P-~-Glc-ooNP 41
PoNPGlc
28
aGlcF
38
28
83
83
P-D-GlC(1-4)-P-D- 0-Glucosynthase Agrobacterium Glc-0
SP.
P-D-GlC(1-4)-p-D- 0-Glucosynthase Agrobacterium Glc-0
SP.
b-~-Glc( 1-4)-P-D-Gk-OpNP
48
P-D-G~c( 1-4)-P-D-Glc-OPh
PPhGlc
aGlcF
PpNPGlc
6
1-4)-P-~-GlcNPth P-D-G~c(
P-D-GlcNPth
Ccllobiose
SP.
aGlcF
8
P-D-G~c( 1-4)-P-~-GlcNPthSEt
P-D-GlcNPth-SEt
Cellobiose
83
8
P-D-GlcN3-OMe
Cellobiose
P-D-Glc(I-4)-P-U- 8-Galactosidase Bullera GkN3 singularis 1-4)-P-D- P-Galactosidase P-D-G~c( Bullera GlcNPth singularis P-D-GIc(1-~)-P-D- P-Galactosidase Bullera GlcNPth singularis p-~-Glc( 1-4)-P-D- P-Glucosynthase Agrobacterium Glc-0
28
6
p-D-GlC(I-'I)-P-D-GlC(1-4)-PD-GIc(1-4)-P-~-ManOpNP P-D-GIc(1-4)-P-D-GIcNjOMe
PpNPMan
aGlcF
SP.
b-~-Glc( 1-4)-P-D- P-Glucosynthase Agrohacterium Glc
28
42
p-~-Glc( 1-4)-P-~-Glc( 1-4)-8D-Man-OpNP
PpNPMan
aGlcF
P-D-G~c( 1-4)-P-~- 0-Glucosynthase Agrobarterium Glc
525
524
523
522
521
520
519
518
517
516
s ro
g.
2
Ti
35 9
2.
ro
22
%
C (5 1
B
2
z2
Eu
p-~-GlcNAc( 13)-b-~-Gal
531
0-Hexosaminidase Bucillus circuluns P-Hexosaminidase Nocardia orientalis
P-D-GIcNAc(12)-~-Man
536
535
534
533
532
531
530
529
528
P-Glucosidase Penicillium funiculosum P-D-GIc(1-4)-DP-Glucosidase Almond Glc P-D-GIc(1-6)a-D0-Glucosidase Almond Glc P-D-GlC(1-6)-P-D- P-Glucosidase Sesame Glc P-D-GIc-(I-6)-DP-Glucosidase Almond Glc P-D-GIc-(1-6)-~- P-Glucosidase Penicillium Glc funiculosum 1 - 6 ) - ~ - P-Glucosidase P-D-G~c( Almond Glc b-~-GlcA( 1-3)-P- P-Glucuronidase Bovine liver D-Gal p-~-GlcA( 1-3)-P- P-Glucuronidase Bovine liver D-Gal
Enzyme Source
521
P-D-G~c-( 1-4)-DGlc
Entry Linkage
Table 1 (continued)
GICNAC~
GlcNAc
PpNPGlcA
12
26
p-~-Gal( l-4)-P-~-GlcNAcOpNP
Man
D-XYI-OMU
P-D-GIcNAc(1-3)-P-~-Gal( 14)-fl-D-GlcNAc-OpNP
p-D-Gal(1-4)-P-D-xylOMu 1- 2 ) - ~ - M a n P-D-G~cNAc(
1.5
0.2
P-D-G~~(I-~)-P-D-G~~(~-~)-PP-~-GlcA(1-3)-P-~-Gal(l-4)20
p-~-GlcA( 1-3)-P-~-Gal( 1-4)P-D-XYI-OMU
D-D-Gal( 1-4)-P-~-Xyi-oMu
PpNPGlcA
P-D-GIc(1-6)-~-Glc
Glc
Glc
136
1I9
I03
103
133
17
P-D-GIc-(I - 6 ) - ~ - G k
Glc
Glc
60, 132 60
19
Glc
Glc
62 135
Cellobiose
Cellobiose
5
133
60
Reference
<25
Treh a1ose
Glc
5.1
6.7
Yield ("/)
P-D-Glc(1-6)-a-D-Glc(I -1)-aD-Glc p-~-Glc( 1-6)-fi-~-Glc( 14-PD-Glc P-D-GIc-(1-6)-~-Glc
P-D-G~c( 1-4)-D-Gk
Glc
Glc
~-D-GIc-( 1-4)-~-Glc
Product
Glc
Acceptor
Glc
Donor
548
547
546
545
544
543
542
54 1
540
539
538
P-Hexosaminidase Nocurdiu orientalis P-Hexosaminidase P-D-GIcNAc(1Nocardia 3)-(3-~-Gal orientalis P-Hexosaminidase P-D-GIcNAc(13)-P-~-Gal Nocardiu orientalis P-D-GIcNAc(1P-Hexosaminidase 3)-P-~-Gal Nocurdiu orientalis P-D-GIcNAc(1P-Hexosaminidase 3)-P-~-Gal-o Chamelea gallina P-D-GIcNAc(1P-Hexosaminidase 3)-P-~-Glc Aspergillus oryzae P-D-G~cNAc( 1P-Hexosarninidase 4)-a-DAspergillus oryzae GIcNAc-0 P-D-GIcNAc(10-Hexosaminidase Aspergillus 4)-a-~-Glc-O oryzae P-D-GIcNAc(1P-Hexosaminidase 4)-P-~-Glc Asperg illus oryzae P-D-G~cNAc( 1P-Hexosaminidase ~)-P-D-G~cNAc Aspergillus oryzae P-D-GIcNAc(1P-Hexosaminidase 4)-P-D-GlcNAc Aspergillus oryae
P-D-GlcNAc(13)-P-D-Gal
138
138
56
139
3.3
1.2
4
13
55
b-~-GlcNAc( 1-3)-P-D-Gal(13)-a-~-GalNAc-opNP 1p-~-GlcNAc( ]-3)-P-~-Gal( ~)-P-D-G~INAc-O~NP
P-D-GIcNAc(1-3)-P-~-GalOMe P-D-GlcNAc(1-3)-P-~-GlcOMe P-D-G~cNAc( I -4)-a-~GlcNAc-OMe
P-D-Gal(1-3)-a-~-GalNAcOpNP b-~-Gal( 1-3)-p-~-GalNAcOpNP P-D-Gal-OMe
P-D-Glc-OMe
a-~-GlcNAc-oMe
GIcNAc~
PpNPGlcNAc
PpNPGlcNAc
130
130
140
141
12
10
55
10
P-D-GICNAC( 1-4)-a-~-GlcOMe P-D-GIcNAc(1-4)-P-D-GlCOMe P-D-GIcNAc(1-4)-P-DGlcNAc G 1cN Ac5
a-D-Glc-OMe
P-D-Glc-OMe
GlcNAc
GICNAC~
PpNPGlcNAc
PpNPGlcNAc
PpNPGlcNAc
G1cN Ac3
PpNPGlcNAc
GICNAC~
130
137
1.9
1-4)-b-~-Glc-OpNP P-D-GkNAc(1-3)-P-D-GaI(1p-~-Gal( ~)-P-D-GIC-OPNP
GlcNAcz
137
3.4 ~-~-GkNAc(l-3)-fi-~-Gal( 14)-P-~-Glc-oMe
P-D-Gal(1-4)-fi-~-Glc-OMe
GlcNAc2
558
551
556
555
554
553
552
551
550
P-Hexosaminidase 1P-D-GICNAC( ~)-P-D-G~cNAc Asperg illus oryzae P-Hexosaminidase P-D-GlcNAc(1~)-P-D-G~cNAc Asperg illus oryzae 0-Hexosaminidase b-~-GlcNAc( 1~)-P-D-G~cNAc Asperg illus oryzae 1P-D-G~cNAc( Chitinase Nocardia ~)-P-D-G~cNAc orientalis P-D-GIcNAc(1P-Hexosaminidase ~)-P-D-GIcNAc Nocardia orientalis 1b-~-GlcNAc( P-Hexosaminidase Asperyi1lu.c 4)-P-Doryzae GlcNAc-0 Chitobiase Bacillus P-D-GIcNAc(14)-D-GkNAc SP. P-D-G~cNAc( IP-Hexosaminidase 4)-D-GlcNAc Asperg illus oryzae P-D-GIcNAc(10-Hexosaminidase Asperg illus 4)-D-GkNAc oryzae P-Hexosaminidase P-D-GICNAC( 1Nocardia 6)-a-~orientalis GalNAc-0
549
Enzyme Source
Linkage
Entry
Table 1 (continued)
23
1.5
GlcNAc3
G1cN Ac4
p-~-GlcNAc( 1-6)-[P-o-Gal(13)1-a-~-GalNAc-OpNP
G 1cN Ac2
GlcNAcz
P-D-Gal(1 -3)-a-~-GalNAcOpNP
GlcNAc?
GlcNAc?
6.2
43
P-D-GlcNAc(1-4)-D-GlcNAc
GlcNAc
GlcNAcoxazoline GlcNAc?
I38
145
145
144
139
24
P-D-G~cNAc( 1-~)-P-DGlcNAc-OMe
0-D-GlcNAc-OMe
PpNPGlcNAc
143
13
GIcNAc~
GlcNAcz
GkNAc:!
142
G1cN Ac6
G1cN Ac4
GlCNAc4
141
141
141
Reference
34
1
GlcNAch
GlcNAc4 GlcNAc4
GlcNAc4
GICNAC~
Yield (%)
20
GlCNAcs
GICNAC~
Product
GlcNAC5
Acceptor
Donor
29.6 Recent Developments and New Directions
m W
0
m
4
W
m
m
w
* r = I
cici
r-
r-
00
3
3
3
m
m
m
M
m 3
00
m 3
v, \D
787
579
578
577
576
575
574
573
572
57 1
570
Enzyme Source
Donor
P-D-GIcNAc(16)-P-~-Gal-o P-D-GIcNAc(16)-P-DGlcNAc-0 P-D-GIcNAc(1 6)-P-DGlcNAc-0
P-Hexosaminidase PpNPGlcNAc Jack bean P-Hexosaminidase GlcNAcz Nocardia orientalis P-Galactosidase &NPGlcNAc Asperg illus oryzae (Impurity?) P-Hexosaminidase GICNAC~ P-D-GIcNAc(16)-P-D-GlC-O Nocardia orientalis P-D-GIcNAc(10-Hexosaminidase GIcNAcz Nocardia 6)-b-D-Gk-O orientalis P-Hexosaminidase PpNPGlcNAc 1b-~-GlcNAc( 6)-~-GalNAc Asperg illus oryzae P-Hexosaminidase PpNPGlcNAc P-D-GIcNAc(1Aspergillus 6)-D-GlcNAc oryzae P-Hexosaminidase GlcNAc P-D-GIcNAc(1~)-D-GICNAC Asperg illus oryzae P-Hexosaminidase GlcNAcz P-D-GlcNAc(1Nocardia 6)-D-GlcNAC orientalis P-Hexosaminidase GlcNAc P-D-GlcNAc(1Bacillus 6)-~-Man circulans
Entry Linkage
Table 1 (continued)
143
119
P-D-GlcNAc(~ - ~ ) - D - G ~ c N A15c
P-D-GlcNAc(1 -~)-D-GIcNAc 25
P-D-GIcNAc(1-6)-~-Man
GlcNAc
GICNAC~
Man
1.7
146
140
P-D-GkNAc(1-6)-P-DGlcNAc
GlcNAc
22
117
1-6)-~-GalNAc 26 p-~-GlcNAc(
137
137
GalNAc
2.1
10
1P-u-GlcNAc(1-6)-[p-~-Gal( 4)]-P-u-Gk-OMe
p-~-Gal( 1-4)-P-~-Glc-OMe
I -6)-[b-~-Gal( 1P-D-Gal(1-4)-P-~-Glc-opNP p-~-GlcNAc( 4)I-P-D-GIC-OpNP
4
j3-~-GlcNAc( 1-6)-P-DGICNAc-OpNP
PpNPGlcNAc
64
136
0.8
P-D-Gal(1-4)-p-~-GlcNAcOpNP
45
14
P-D-GIcNAc(I -6)-P-~-GalOMe p-~-GlcNAc( 1-6)-[P-~-Gal( 1~)]-P-D-GICNAC-OPNP
P-D-Gal-OMe
Reference
Yield ("/')
Product
Acceptor
593
592
59 1
590
589
588
587
586
585
584
583
582
581
580
SP.
P-Glucosidase Agrobacterium
SP.
P-Mannosidase Guinea pig liver P-Mannosidase Guinea pig liver 0-Galactosidase Aspergillus oryzae P-Glucosidase A yrobacterium
0-Mannanase Aspergillus niger P-Mannosidase Aspergillus oryzae D-~-Man(l4)-P- P-Galactosidase Aspergillus D-Man-0 oryzae P-D-Man(1-6)-P- P-Mannosidase D-Man-0 Guinea pig liver P-D-Man(1-6)-P- P-Mannosidase D-Man-0 Guinea pig liver p - ~ - X y l ( l - l ) - P - ~ -P-Xylosidase Aspergillus niger XYl P-D-XYl(1-4)-DP-Xylosidase Aspergillus niyer Man p-~-Xyl( 1-6)-a-~- P-Xylosidase Glc Aspergillus awamori K4 P-Xylosidase P-D-xyl(1-6)-DGlcNAc Aspergillus niger
P-D-Man(1-4)-PD-GlcNAc P-D-Man(1-4)-PD-GIcNAc
P-D-Man(1-2)-PD-Man-0 P-D-Man(1-2)-PD-Man-0 P-D-Man(1-3)-aD-Man-0 a/PpNPMan (mixture)
P-D-GIG-SPh
a/PpNPMan (mixture) PManF
64
85
7
7
147 150 150 151
152
3.7 NA NA 10
36
P-D-XYI(I -4)-~-Man
PpNPMan
Man
PpNPMan
GlcNAc
Trehalose
1-6)-~-GlcNAc p-~-Xyl(
147 0.7 P-D-Man(1-6)-P-~-ManOpClP P-D-Man(1-6)-P-~-ManOpNP p-~-Xyl( l-l)-P-~-Xyl
PpClPMan
PpCIPMan
XYl
64
13
P-D-Man(1-4)-P-D-ManOpNP
149
148 26
3.7
85
147
1.3
9
147
0.9
a/PpNPMan (mixture)
P-D-Man(1-4)-P-DGlcNAc( 1-4)-~-GlcNAc P-D-Man(1-4)-P-DGlcNAc( 1-4)-~-GlcNAc
P-D-Man(1-3)-P-~-Glc-sPh
P-D-Man(1-2)-p-D-ManOpClP P-D-Man(1-2)-P-~-ManOpNP P-D-Man(]-3)-a-~-ManOpNP
a/ppNPMan (mixture)
PpNPMan
Man3
P-D-Xyl-SPh
PpNPMan
PpNPMan
SManF
PpClPMan
PpCIPMan
p-~-Xyl( 1-6)-~Man p-~-Xyl( 1-6)Mannitol
p-~-Xyl( 1-6)Sorbitol
594
596
595
Linkage
Entry
Table 1 (continued)
Asperg illus awamori K4 P-Xylosidase Asperg illus awamori K4
P-Xylosidase Aspergillus niger P-Xylosidase
Enzyme Source
XYl2
XYl2
XYl2
Donor
Sorbitol
Mannitol
Man
Acceptor
P-D-XYI(1-6)Sorbitol
P-D-xyl(l-6)-Mannitol
fb~-Xyl( 1-6)-~-Man
Product
<10
<10
NA
(%)
Yield
151
151
150
~~
Reference
4.
g
P s
3
zg
F
Q
\o
0
v3
4
7
6
5
4
3
2
1
EnzymeJSource
(l-?)-D-
a-D-GalNAc-
Endo-a-N-acetylgalactosaminidase Diplococcus (6-0Me)pneumoniae Gal Endo-a-N-acetyla-D-GalNAc(1-?)-D-Fuc galactosaminidase Diplococcus pneumoniae Endo-a-N-acetyla-D-GalNAcgalactosaminidase ( 1-?)-D-Gal Diplococcus pneumoniae Endo-a-N-acetyla-D-GalNAc(1-?)-D-GIC galactosaminidase Diplococcus pneumoniae Endo-a-N-acetyla-D-GalNAcgalactosaminidase (1 -1)-0 Diplococcus pneumoniae a-D-GalNAc-0 Endo-a-N-acetylgalactosaminidase Diplococcus pneumoniae a-D-GalNAc-0 Endo-a-N-acetylgalactosaminidase Diplococcus pneumoniue
Entry Linkage
~~
D-FUC
Gal
Gk
Glycerol
Ser
Thr
p-D-Gal(1-3)-C(-DGalNAcasialoglycoprotein p-D-Gal(1-3)-a-DGalNAcasialoglycoprotein p-u-Gal(1-3)-a-DGalNAcasialoglycoprotein p-D-Gal(1-3)-a-~GalNAcasialoglycoprotein P-D-Gal(1-3)-a-DGalNAcasialoglycoprotein
(6-OMe)-~-Gal
Acceptor
p-D-Gal(1 -3)-a-DGalNAcasialoglycoprotein
p-~-Gal( 1-3)-a-DGa1N Acasialoglycoprotein
Donor
Table 2. Enzymatically synthesized oligosaccharides using endo-glycosidases. (9%)
153
153
153
153
153
153
20
26
22
69
17
12
p-D-Gal(1-3)-a-DGalNAc(1-?)-D-Fuc
p-~-Gal( 1-3)-a-DGalNAc(1-?)+Gal
p-D-Gal(1-3)-a-~GalNAc( l-?)-~-Glc
D-D-Gal(1-3)-a-~-GalNAc(1-1)-Glyceryl
p-~-Gal( 1-3)-a-~-GalNAc0-Ser
p-D-Gal(1-3)-a-~-GalNAc0-Thr
153
Yield Reference
17 p-D-Gal(1-3)-a-~GalNAc(1-?)-~-(6-0Me)Gal
Product
4
9
a
OpNP a-D-GlC(I-4)-a-D-Gk(1-4)a-~-Glc( 1-3)-P-D-GlCOClNP { a-~-Glc( 1-4))B-D-GIc
18
17
16
15
14
13
12
a-~-Glc( 1-4)-a- Cyclomaltodextrinase y-CD Glc 20 D-G~c Bacillus sphaericus E-244 a-~-Glc( 1-4)-a- Amylase Klebsiella {a-D-Glc(M)),~-D- apNPGlc { a-~-Glc( 1-4)) ,j-a-~-Glc13 D-G~c-O pneumoniue Glc OpNP a-~-Glc( 1-4)-a- a-Amylase Aspera-Maltosyl-F {~-D-G~C(~-~))~-P-D-G~C{a-~-Gk(l-4))2-{a-~-Glc(l59 D-G~c gillus oryzae 0-indoyl ethanol 3))2 - P-~-Glc-O-indoyl ethanol a-D-Glc(1-4)-a- Cyclomaltodextrinase p-CD Glc { ~-D-GIC( 1-4))7 - ~ - G l ~ 39 D-G~c Bacillus sphuericus E-244 u-D-G~c( 1-4)-a- Pullulanase Klebsiella Pullulan Stevioside 13-{a-D-GlC(1-4))l-p-DNA D-G~c SP. Glc(1-2)-P-D-Glc-19-P-~Glc-0-steviol u-D-G~c( 1-4)-a- Pullulanase Klebsiella Pullulan Stevioside 13-a-D-GlC(l-4)-P-D-Gl~(lNA D-G~c SP. 2)-P-D-GlC-19-P-D-GlC-0steviol a-~-Glc( 1-4)-a- Pullulanase Klebsiella Pullulan Stevioside 13-P-D-GlC(1-2)-P-D-Glc-19- NA D-G~c SP. {a-~-Glc( 1-4))2-P-~-Glc0-steviol
PClNPGlc
a-D-Glc(1-3)-P-~-Glc-
61
61
61
155
157
156
155
154
154
1.5 154
11
Glc
{ a-D-Glc(1-4))3-~-
I./“(
Yield Reference
U-D-GIC(l-4)-a-D-GlC(1-4)- 16
1Y.-D-Gk(l-4)-a-D-Gk(1-4)a-~-Glc( I -3)-U-D-GlCOpNP
Product
u-~-Glc( 1-3)-P- a-Amylase StreptoD-Glc-0 myces griseus
Glc
PpNPGlc
apNPGlc
(U-D-GIC( 1-4))3-~Glc { a-~-Glc( 1-4))3 - ~ -
Acceptor
Donor
10
myces griseus
a-~-Glc( 1-3)-P- a-Amylase Strepto-
9
D-Glc-0
a-~-Glc( 1-3)-a- a-Amylase StreptoD-Glc-0 myces griseus
Enzyme/Source
8
Entry Linkage
Table 2 (continued)
2F’
9 s
ff2
803-
2
2 u 2
0
B
6 E 8
4
s E?
%0
m
0
Eu
4 \o E3
a-Maltosyl-F
a-~-Glc( 1-6)-a- Isoamylase D-G~ Pseudomonas umyloderamosa a-~-Glc( 1-6)-a- Pullulanase Klebsiella D-G~c aerogenes a-~-Glc( 1-6)-a- Pullulanase Aerobacter aerogenes D-GIc a-D-Glc(1-6)-a- Pullulanase AeroD-GIc bacter aerogenes a-D-Glc(1-6)-a- Pullulanase Bacillus D-G~c acidopullulyticus a-D-Glc(1-6)-a- Pullulanase Bacillus D-G~c acidopullulyticus a-~-Glc( 1-6)-a- Isoamylase D-G~c Pseudomonas arnyloderamosa Pullulanase Bacillus acidopullulyticus Isoamylase Pseudornonas amyloderamosa
24
32
31
30
29
28
21
26
25
a-Maltosyl-F
a-Maltosyl-F
a-Maltosyl-F
a-Maltosyl-F
a-Maltosyl-F
a-Maltosyl-F
a-Maltosyl-F
Maltose
((r-D-Gk(1-4)}3-~- PpNPGlc Glc
a-CD
y-CD
y-CD
a-CD
b-CD
p-CD
1-4)-a-~-Glc( 1 a-~-Glc( 6)-y-CD a-CD
p-CD
PClNPGlc
apNPGlc
apNPGlc
apNPGlc
apNPGlc
1 -4)-p- a-Amylase Streptoa-~-Glc( D-Glc-0 myces griseus
{a-D-GlC(l-4)}4-~Glc {a-D-GlC(1-4)}4-DGlc { a-~-Glc( 1-4))3 - ~ Glc {a-D-Gk(I-4)}s-DGlc {a-D-GlC(l-4)}3-DGlc
23
22
21
20
19
Amylase Pseudomonas stutzeri Amylase Pseudomonas stutzeri a-Amylase Streptomyces griseus Amylase Bacillus licheniformis a-Amylase Streptomyces griseus
a-~-Glc( 1-4)-aD-Glc-0 a-~-Glc( 1-4)-aD-G~c-O a-D-Glc(1-4)-aD-Glc-0 a-D-Glc(1+aD-G 1c-0 a-~-Glc( 1-4)-pD-Glc-0
18
60
25
NA
NA
62
NA
60
11
13
13
15
54
12
a-D-Glc(1-4)-a-~-Glc( 1-6)-y- 43 CD a-D-Glc(1-4)-a-~-Gk( 1-6)60 a-CD
6))2-y-CD a-D-Glc(]-4)-a-~-Glc( 1-6)a-CD a-~-Glc( 1-4)-a-~-GIc( 1-6)p-CD a-~-Glc( 1-4)-a-~-Glc( 1-6)p-CD a-~-Glc( 1-4)-a-~-Glc( 1-6)a-CD a-~-Glc( 1-4)-a-~-Glc( 1-6)-yCD
{ a-o-Glc( 1-4)-a-~-Glc( 1-
{a-D-Gk(l-4)}~-a-D-GkOpNP (a-~-Gk(l-4)}~-a-~-GlcOpNP { a-D-Glc(1-4)}3-a-D-GlcOpNP {a-D-Glc(1-4)}s-a-~-GlcOpNP a-D-GlC(1-4)-a-D-GIC(1-4)a-D-Glc(1-4)-P-~-GlcOClNP 1-4)a-D-Glc(1-4)-a-~-Glc( a-D-Glc(1-4)-b-D-GkOpNP a-D-Glc(1-4)-a-~-Glc( 1-6)p-CD
161
161
161
161
161
161
161
162
161
154
154
160
154
159
158
w
W
4
46
45
44
43
42
41
40
39
38
37
36
35
34
33
Pullulanase Aerobarter aerogenes Pullulanase Klebsiellu uerogenes Pullulanase Klebsiellu uerogenes Pullulanase Klebsiellu uerogenes Pullulanase Klebsiella aerogenes Pullulanase Klebsiellu uerogenes Isoamylase Pseudomonus
Emy me/Source
~-D-GIC( 1-6)-aD-Gk a-D-Glc(1-6)-aD-Glc 1-6)-aa-~-Glc( D-Glc a-D-GIc(1-6)-aD-Glc a-~-Glc( 1-6)-aD-Glc
Isoamylase Pseudomonas Isoamylase Pseudomonus Isoamylase Pseudomonus Isoamylase Pseudomonas Isoamylase Pseudomonas
a-~-Glc( 1-6)-a- Isoamylase Pseudomonas D-Glc a-~-Glc( 1-6)-a- Isoamylase D-Glc Pseudomonus
a-D-Glc(1-6)-aD-Glc a-D-Glc(1-6)-aD-Glc a-~-Glc( 1-6)-aD-Glc a-~-Glc( 1-6)-aD-Glc a-~-Glc( 1-6)-aD-G~c 1-6)-a~-D-GIC( D-Glc a-u-Glc(l-6)-aD-Glc
Entry Linkage
Table 2 (continued)
y-CD a-~-Glc( 1-4)-a-~-Glc( 16)-a-CD p-CD
Maltose Maltose
a-CD
{a-D-Glc(1-4))~-DGlc {a-~-Glc(I-4))2-DGlc
p-CD
y-CD
p-CD
Maltose Maltose
a-CD
{a-~-Glc( 1-4)}*-D-Glc
Maltose
{a-D-GIC(I-4))2-DGlc { a-D-Glc(1-4))2-DGlc
{a-D-Glc(1-4))~-DGlc
Maltose {a-D-Gk(1-4))2-~-Glc
a-D-Gk(1-4)-a-~-Glc( 16)-P-CD a-CD
Maltose Maltose
y-CD
Acceptor
a-Maltosyl-F
Donor
("w
NA
163
163
163
163
163
163
163
163
6. 162 NA
C'
52
R @
2.
;s
0
R
2
9
z
P g-
?
162
162
162
0
$
2
%0
NA
39
NA
GIG(1 -6)I-D-Gk {a-~-Glc(l-4)}2-a-~-Glc(lNA 6)-{a-D-Ck(1-4))z-D-Gk a-D-Glc(1-4)-a-~-Gk(l-4)- NA a - ~ - G k1(-6)-[a-~-Gk( 1.I)]-a-D-GIC(1-4)-D-Gk 1-4)-a-~-Glc( 1-6)- NA a-~-Glc( a-CD a-D-Glc( 1-4)-a-~-Glc( 1-6)- NA p-CD 1-6)-y- NA a-D-Glc(1-4)-a-~-Gk( CD {a-~-Gk(l-4))~-a-~-Glc(lNA 6)-a-CD {a-D-GIC(I-4)}2-a-D-GIC(I-NA 6)-P-CD
a-D-Glc(1-4)-a-D-Glc(1-6)a-CD a-D-Glc(1-4)-a-~-Gk( 1-6)-yCD { ~-D-GIC( 1-4)-a-~-Glc( 16))z-a-CD a-D-GIc(1-4)-a-~-Gk( 1-6)p-CD a-D-GlC(1-4)-a-D-Gk(1-4)[ {a-~-Glc( 1-4)}px-~-
162
161
Yield Reference
a-D-Glc( 1-4)-a-~-GIc( 1-6)-y- N A CD { a-D-Gk( I-'I)-a-D-GIC(1NA 6)Iz-P-CD
Product
iD P
4
59
58
57
56
55
54
53
52
51
50
49
48
47
a-D-Glc(1-6)-a- Isoamylase Pseudomonas D-Glc a-~-Glc( 1- 6 ) - ~ - Neopullulanase Bacillus subtilus Glc ~-D-GIC( I - 6 ) - ~ -Neopullulanase Bacillus subtilus Glc 1- 6 ) - ~ - Neopullulanase ~-D-GIC( Bacillus subtilus Glc a-D-Glc(1- 6 ) - ~ - Neopullulanase Bacillus subtilus Glc Rice debranching a-~-Glc-O enzyme P-~-3-deoxy-3- Cellulase Trichoderma F-Glc viridae P-Galactanase Penicillium citrinum P-Galactanase Penicillium citrinum P-Galactanase Penicillium citrinum P-Galactanase Penicillium citrinum P-Galactanase Penicillium citrinum P-Galactanase Penicillium citrinum
{a-~-Glc( 1-4)}*-a-~-Glc( 16)-y-CD a-D-GlC(1-6)-D-GlC 164
10
17
18
1-3)p-D-Gal(1-3)-~-GalNAc P-D-Gal(I-~)-P-D-G~I( D-GalNAc 1-6)P-D-Gal(1-h)-~-GlcNAc p-D-Gal(1-4)-P-~-Gal( D-GIcNAc
Arabinogalactan
Arabinogalactan
19
P-D-Gal(1-4)-a-~-Gal-OMe
16
3.5
p-D-GaI(1-4)-b-~-Gal( 1-4)a-D-Gal-OMe
p-D-Gal(1-4)-~-GlcNAc P-~-Gal(l-4)-P-o-Gal( 1-4)D-GIcNAc
a-D-Gal-OMe
166, 167
165
164
164
164
163
NA
5.7 1-6)1-4)-a-~-Gk( { a - ~ - G l c ( l - 4 ) } ~ - ~ - ~-D-GIC( ~1~ a-D-Gk(1-4)-~-Gk a-~-Glc( 1-4)-a-~-Glc( 1-6)- NA {a-~-Glc( 1-4)}2-D-Glc D-G~c ~-D-GIC( l-6)-a-~-Glc( 1-4)- NA a-D-Glc NA {a-~-Glc( 1-4))2-a-D-GlcMeOH OMe P-D-Gal(I-'I)-P-D-GIC(1-4)18 P-~-3-deoxy-3-F-GlcP-~-3-deoxy-3-F-GkOMe OMe I P-D-GaI(1-3)-P-~-Gal-OMe
{a-D-GlC(I-4)}2-D-Glc
y-CD
Arabinogalactan
Arabinogalactan
Arabinogalactan
Arabinogalactan
P-Lac-F
(a-D-GlC(1-4))~-DGlc (a-D-GIC(I-4)}2-DGlc {a-D-GlC(l-4)}2-DGlc {a-D-GIC(1-4)}2-DGlc { a-D-GlC(1-4)}2-DGlc Pullulan
o\
P
b
69
68
67
66
65
64
63
62
61
p-~-Glc( 1-6)DNJ
Glycerol Glycerol [ 1-’4C]-Glc
{ p-D-Gal(1-4))~-D-
Gal Arabinogalactan Laminarin
Cellooligosaccharides DNJ
P-D-Gal-OMe
Arabinogalactan
P-D-Glc(1-4)-P-D-GIc(1-6)DNJ
P-D-Gal(1-4)-P-D-Gal-OGIyceryl p-D-Gal(1-4)-P-D-Gal-OGlyceryl {P-D-GlC(l-3)}!1-5,-D-[ Ii4C]-G1~
P-D-Gal(1-4)-o-~-Gal-OMe
PpNPGal
Arabinogalactan
I68
168
168
168
NA
171
40 to 169 75 40 to 169 75 26 170
16
P-D-Gal(1-4)-p-~-Gal-OpNP 14
PpNPGal
Arabinogalactan
11
P-D-Gal(1-3)-~-GlcNAc P-D-Gal(1-4)-P-u-Gal(1-3)D-GlcNAc
Arabinogalactan
Arabinogalactan
D-G~c
p-~-Gal(1-4)-P-~-Gal(l-4)11
P-D-Gal(1-4)-~-Glc
Arabinogalactan
P-Galactanase Penicillium citrinum P-Galactanase Penicillium citrinum 0-Galactanase Penicillium citrinum P-Galactanase Penicillium citrinum P-Galactanase Penicillium citrinum P-Galactanase Penicillium citrinum P-Galactanase Bacillus subtilus P-Galactanase Bacillus subtilus Endo-P(1-3)glucanase L-IV Spisula sachalinensis Cellulase Trichoderma sp.
(“A)
60
Yield Reference
Product
Acceptor
Donor
Enzyme/Source
~
Entry Linkage
~
Table 2 (continued)
~~~
80
79
p-~-Glc( 1-4)-8- Cellulase TrichoD-Glc derma viridae
P-D-GlC(I 4 - P - (I-3),(l-'I)-PD-Glc Glucanosynthase Bacillus licheniformis p-~-Glc( 1-4)-P- Cellulase TrichoD-G~c derma viridae
78
77
76
75
74
73
72
71
Cellulase P-D-GIc(1-4)Trichoderma sp. DNJ P-D-GIc(1-4)-P- Cellulase Trichoderma ~-(3-0Me)viridae Glc-0 P-D-G~c( 1-4)-8- (I-3),(1-4)-PGlucanase Bacillus D-G~c lichenformis P-D-GlC(1 4 - P - (l-3),(l-4)-PD-GIc Glucanase Bacillus licheniformis P-D-GIc(1-4)-P- (1-3),(1-4)+ Glucanase Bacillus D-Glc licheniformis 1-4)-P- (1-3),(1-4)-0P-D-G~c( Glucanase Bacillus D-Glc licheniformis (l-3),(1-4)-PGlucanosynthase Bacillus lichenformis Endoglucanase I Humicola insolens
70
P-D-GIc(1-4)-P-D-GICOMe
P-Lac-F
I -4)-a-D-GICP-D-G~c( OMe
P-D-GlC(1-4)-P-D-GICOMu
P-D-G~c( 1-3)-a-DGlc-F
0-Lac-F
P-D-G~c( 1 -4)-P-D-Glc-oindoyl ethanol
1-3)-P-D-GkP-D-G~c( OMu
P-D-G~c( 1-3)-U-DGlc-F
P-Lac-F
P-D-GIc(1-3)-O-D-GICOMe
P-D-Glc(I -3)-p-D-Glc-F
p-~-Glc( 1-3)-p-~-Glc-F
/3-~-(3-OMe)-Glc-OMe
1-3)-P-DP-D-G~c( Glc-F
P-D-GlC(I-3)-P-DGlc-F
P-D-GIc(1-3)-P-DGlc-F
P-D-GlC(1-4)P-DG k (1-3)-P-D-Gk-F
P-Lac-F
Cellooligosaccharides DNJ
173
29
174
40
NA
60
173
173
10
5
172
20
166, 167
171
1P-D-Gal(1-4)-{ P-D-G~c( 4))2-a-D-Glc-OMe
4))2-f3-D-GIC-OMU
60
36
166, 175, 167, 176 166, 167
NA 29 P-D-G~c(~-~)-{P-D-G~c(~-
1p-D-Gal(1-4)-{P-D-G~c( 4)}2-P-~-Glc-O-indoy1 ethanol
P-D-GlC(I-3)-P-D-GIC(1-4)P-D-GIc(1-3)-(3-~-GkOMe P-D-GIc(1-3)-P-~-Glc( 1-4)P-D-GIc(1-3)-P-D-GkOMu
4)}2-O-D-G1C(1-3)-D-Gk
{ P-D-GIC(1-3)-P-D-GlC(I-
P-D-Glc(1-4)-P-D-Gk(1-3){P-D-GlC(I-4))~-P-DGlc(1-3)-D-Gk P-D-G~c( 1-3)-P-D-Gk(1-4)P-D-GIc(1- 3 ) - ~ - G k
P-D-GIc(~-~)-P-D-GIC( 1-4)- NA DNJ P-D-Gal(1-4)-P-~-Glc(l-4)- 8 P-~-(3-0Me)-Glc-OMe
91
90
89
88
B-Lac-F
P-D-GlC(1-3)-a-DGlc-F
P-D-GlC(1-3)-P-DGk-F
P-Lac-F
p-D-Gal(I -4)-P-D-GIC(1-4)P-D-Glc-OMe
176
13
P-D-Glc-OMU
167
23
P-D-Gal(1-4)-{P-D-GIc(14)}2-P-~-Glc-SMe P-D-Gal(I -‘I)-{P-D-GIc(1~)}~-P-D-GIc-OAII { p-~-Glc( I-~)-P-D-G~C( 14)}2-P-D-Gk(1-3)-P-DGlc-OMe P-D-GIc(I-~)-P-D-GIC( 1-4)P-D-GIc-OMU P-D-GIc(1-4)-P-D-GkSMe P-D-GlC(1-4)-fl-D-GICOAll P-D-GlC(1-3)-P-D-GICOMe
P-Lx-F
87
29
166, 167, 176
88
51
0.6 173
177, 167
25 Cellulose
P-Cellobiosyl-F
P-Cellobiosyl-F
86
8
P-Cellobiosyl-F
P-Cellobiosyl-F
85
177, 167, 178 177; 167
166, 167, 176
166, 167
166, 167
Cellulose
P-Cellobiosyl-F
P-Cellobiosyl-F
Cellulase Trichodermu viridae Cellulase Aspergillus niger Cellulase Polyporus tulipiyerue Cellulase Trichoderma viridue Cellulase Trichodermu viridue (I-3),(1-4)-PGlucanase Bacillus lichenijormis P-D-GIc(1-4)-P- (1-3),(1-4)-PGlucanosynthase D-Glc-0 Bacillus licheniformis P-D-GIc(1-4)-P- Cellulase Trichodermu viridue D-Glc-0
84
P-D-GIC(I 4 - P D-GIC P-D-GIc(1-4)-PD-Glc P-D-GIc(1-4)-PD-Glc P-D-GIc(1-4)-PV-Glc fi-D-GIC(14-PD-GIC P-D-GlC(1 4 4 D-Glc
P-D-GIc(]-4)-P-~-Glc( 14)-P-~-Glc-oMe
P-Lac-F
P-D-GIc(1 -6)-P-~-GlcOMe
P-Lac-F
P-D-GIc(1-4)-P- Cellulase TrichoD-Glc derma viridue
82
83
23 P-D-Gal(l-4)-P-~-Glc( 1-4)P-D-GlC(I -3)-P-D-GICOMe 26 p-D-Gal(I - ~ ) - O - D - G 1-4)~( P-D-GIc(1-6)-P-~-GlcOMe P - D - G ~ I ( ~ - ~ ) - { P - D - G ~ ( 27 I4)}2-B-D-GIC(1-4)-P-DGlc-OMe 54 Cellulose
P-D-GIc(I -3)-P-D-GlCOMe
P-Lac-F
(“w
Yield Reference
Product
Acceptor
Donor
b-~-Glc( 1-4)-P- Cellulase D-Glc Trichoderma viridue p-~-Glc(1-4)-8- Cehlase TrichoD-Glc derma viridue
EnzymeJSource
81
Entry Linkage
Table 2 (continued)
Lo
-1 00
Cellulase Trichoderma viridae Endoglycoceramidase Cornybacterium sp. Endoglycoceramidase Cornybacterium sp. Endoglycoceramidase Cornybacterium sp. Endoglycoceramidase Cornybacterium sp. Endoglycoceramidase Cornybacterium sp. Endoglycoceramidase Cornybacterium sp. Endoglycoceramidase Macrobdella decora Endoglycoceramidase Cornybacterium sp.
P-D-GlC-0
P-D-GIc-O
P-D-Glc-0
102
103
104
106
P-D-GlC-0
P-D-Gk-0
101
105
P-D-GlC-0
100
99
97
98
Cellulase Trichoderma viridae Cellulase Trichoderma viridae Cellulase Trichoderma viridae Cehlase Trichoderma viridae
Cellulase Trichoderma viridae Xylanase Trichoderma viridae
P-D-GlC(1-4)-PD-G1C-S P-D-GlC(1-4)-Pu-Glc-S P-D-GlC(1 4 4 D-GlC-S P-D-GlC(1-4)-PD-G1C-S
96
95
94
93
92
166, 167 180
166, 167 181
52 58
25 22
P-D-Man-OMe P-D-xyl(I -4)-D-D-Glc-F
P-Lac-F p-u-Xyl(1-4)-8-uGlc-F
181 181 181 181 182
181
22 25 30 18 NA
35
2.7 181
179
41
P-D-GlC-S(1-4)-P-D-GIC-F
P-D-Glc-S(1-4)-P-DGlc-F
167
36
P-D-GlC-S-Ph
P-Lac-F
167
30
P-D-G~C-S-CI~H~~
P-Lac-F
167
43
P-u-Glc-S-CH2CONHPr
P-Lac-F
Q ..
5
a
P-D-GlC-0
0-D-GlC-0
fl-D-Gk-0
p-D-GlC-0
109
110
111
112
116
115
114
113
HO-(CH2)sCH3
Asialo-GM1
P-D-Gk-0
108
Endoglycoceramidase Macrobdella decora Endoglycoceramidase B-D-Gk-0 Macrobdella decora Endoglycoceramidase p-D-G1C-0 Macrobdella decora Endoglycoceramidase B-D-G~c-O Macrobdella decora (2-deoxy-21-4)- Lysozyme Hen egg D-D-GICN( white monochloroP-D-GkN acetamido-a-DGlc),
GM1
(2-deoxy-2chloroacetamido-fl-~Glc)3
(HO-CH2)3CNHCO(CH&COOMe
IV3NeuAcPGalNAc- HO-(CH2)5CH3 GbOse4-0-Cer Ceramide LacSer-support
MeOH
GMI
Endoglycoceramidase Cornybacterium sp. Endoglycoceramidase Cornybacterium sp. Endoglycoceramidase Cornybacterium sp. Endoglycoceramidase Leech Endoglycoceramidase Macrobdella decora
p-u-Glc-0
107
Acceptor
Donor
Enzyme/Source
Entry Linkage
Table 2 (continued)
17
GlcN
{ (P-D-GkN(1-4))(343)-D-
3NeuAcCgOse4-O-(CH2)gOH
3NeuAcCgOse4-O(CH2)6NHCOCO(CH3)3
3NeuAcCgOse4-OCH2(HOCH2)2CNHCO(CH2)4COOMe 3NeuAcCgOse4-O(CH2)6NHCOCF3
33
NA
NA
184
182
182
IV3NeuAcpGalNAc9.6 181 G~OS~~-O-(CH~)~CH~ GM3 61 18, 183
181
181
Yield Reference (%)
C ~ O S ~ ~ - O - ( C H ~ ) ~ C H33~
Product
m
g.
9 6 $
&?
0
Q
G-
l u v,
0
0
125
124
123
122
121
120
1 I9
1I8
117
Endo- P-N-acetylglucosaminidase Arthrohucter protophormiue P-D-GIcNAc(1- Endo-P-N-acetyl?)-a-Dglucosaminidase GIcNAc-0 Arthrohacter protophormiue P-D-GIcNAc(1- Endo- P-N-acetyl?)-~-2dGlc glucosaminidase Arthrobucter protophormiue 1- Endo-P-N-acetylP-D-G~cNAc( ?)-~-6-deoxy- glucosaminidase Glc Arthrohucter protophormiae P-D-GIcNAc(1- Endo-P-N-acetyl?)-D-F~ glucosaminidase Arthrohacter protophormiue P-D-GIcNAc(1- Endo-P-N-acetyl?)-D-Gk glucosaminidase Arthrobacter protophormiue P-D-GIcNAc(1- Endo-b-N-acetyl?)-D-xyl glucosaminidase Arthrobacter protophormiae 1- Endo-P-N-acetylP-D-G~cNAc( ~)-P-L-Fucglucosaminidase OMe Arthrohactrr protophormiue P-D-GIcNAc(1- Lysozyme Hen egg 3)-a-~-Glc white
P-D-GIcNAc(1?)-(3-OMe)D-Gk
36
36
Man&D-GlcNAc( 1-?)-~-6- 67 deoxy -G1c
GICNAC~
Man9GlcNAczAsn
Man9GlcNAc~Asn
Man9GlcNAc2Asn
Man9GlcNAc.1Asn
Man9GlcNAc2Asn
{a-D-Gk( I-4)}4-a-D-GkOpNP
0-L-Fuc-OMe
XYl
Glc
Fru
6-deoxy-Glc
36
36
185
61
16
34
5
Mans-P-D-GlcNAc( 1-?)-DClc
1-?)-DMan9-P-~-GlcNAc( XYl
Mans-P-D-GlcNAc(1-2)-0L-Fuc-OMe
p-~-GlcNAc(I-3)-{ a-DGIC(1-4))4-a-D-GIC-OpNP
186
36 4.7
Mans-P-~-GlcNAc(1-?)-DFru
Mang-P-~-GlcNAc( 1-?)-D2dGlc
61
2dGlc
36
l-?)-a-~-66 Mang-P-~-GlcNAc( GlcNAc-OMe
a-D-GlcNAc-OMe
ManqGlcNAczAsn
Mans GlcNAc2Asn
36
30
Man&~-GlcNAc( 1-?)-(3OMe)-D-Glc
(3-OMe)a-Glc
Man9GlcNAc2Asn
135
134
133
132
131
130
129
128
127
126
Man&~-GlcNAc( 1-4)-aD-GIc-O~NP
apNPGlc
Endo-J3-N-acetylglucosaminidase Arthrobacter protophormiae Lysozyme Hen eggwhite
GIcNAc(1-4)}3-PA
{ P-D-GkA(1-3)-P-D-
Hyaluronic acid
NA
P-D-GIc(1-6)-~-Glc
PpNPGlc
39
TIN(Glc)AS
MansGlcNAcz Asn
GICNAC~
188
P-D-GIcNAc(I -4)-D-D-GIC49 189 OpNP 72 191 { P-D-GlCA(I-3)-P-DGICNAC(~-~)}(~-~~)-PA
30, 31
190
3.4 189
75
PpNP2dGlc
Man6-P-~-GlcNAc( 1-4)-PD-Glc(1-6)-D-Glc
187 0.6 187
4.2
1.9 187
8.5 187
Yield Reference (“A)
GICNAC~
P-D-GIcNAc(1-4)-P-D2dGlc-OpNP
P-D-GIcNAc(1-4)-a-DGlc( 1-4)-a-~-Glc-OpNP P-D-GIcNAc(1-3)-a-~GlcNAc-OpNP
a-~-Glc( 1-4)-a-~-GlcOpNP apNPGlcNAc
Lysozyme Hen eggwhite Lysozyme Hen eggwhite
Product P-D-GlcNAC(1-3)-a-DG k (1 -4)-a-D-Glc-OpNP P-D-G~cNAc( 1-3)-a-nGlcNAc-OpNP
Acceptor ~-D-GIC( I-4)-a-D-GICOpNP apNPGlcNAc
Donor
Lysozyme Hen eggwhite Lysozyme Hen eggwhite
Enzyme/Source
Endo-P-N-acetylglucosaminidase Arthrobacter protophormiae P-D-GIcNAc(1- Endo-0-N-acetylglucosaminidase 4)-P-D-GIC Arthrobacter protophormiae P-D-GIcNAc(1- Lysozyme Hen eggwhite 4)-P-D-GIC-O P-D-GIcNAc(1- Hyaluronidase Bovine testes ~)-P-D-GIcA
P-D-GIcNAc(14)-P-D2dG 1c-0 P-D-GIcNAc(14)-P-D-GIC
P-D-GIcNAc(13)-a-D-GIC P-D-GIcNAc(13)-a-DGIcNAc-0 P-D-G~cNAc( 14)-a-~-Gk P-D-GIcNAc(14)-a-nGIcNAc-0 1S-D-GICNAC( 4)-a-n-Glc-O
Entry Linkage
Table 2 (continued)
144
143
CSN[GlcNAc]LST
GlcNAc2-0xazo1ine
GlcNAc2-0xazoline
MansGlcNAcz Asn
Endo-j3-N-acetylglucosaminidase Arthrobacter protophormiae P-D-GIcNAc(1- Chitinase Nocardia orientalis 4)-P-DGlcNAc 1- Endo-P-N-acetylP-D-G~cNAc( 4)-p-Dglucosarninidase GlcNAc Mucor hiemalis 1- Chitinase Bacillus sp P-D-G~cNAc( 4)-p-DGlcNAc
141
STF-GP
Chondroitin
P-D-G~cNAc( 1- Hyaluronidase 4)-p-~-GlcA Bovine testes
140
GlcNAcs
Chondroitin 4-0SO3H
P-D-GIcNAc(1- Hyaluronidase 4)-p-~-GlcA Bovine testes
139
100
19
8.5 193 CSN[(NeuAcGalG1cNAcMan)zManGlcNAcz]LST Chitin
192
23
32
191
191
191
191
191
G1cNAc.l
2.1 P-D-GIcA(1-3)-B-~-(6-0S03H)-GalNAc(1-4)-{0D-GIcA(1-3)-p-~GlcNAc)3-PA 45 { P-D-GlcA( I-3)-p-D-(4-O- { P-D-GIcA(1-3)-8-~GlcNAc(l-4)}(1-4)-{p-DS03H)-GalNAc(1-4))3 GkA( 1-3)-p-~-(4-0PA SO3H)-GalNAc)3-PA 35 1-3)-8-~{ p-D-GlcA(1-3)-8-D-(6-O- { p-~-GlcA( GICNAC(1-4))i 1-31 - { p-DS03H)-GalNAc(1-4))3 GlcA( 1-3)-p-~-(6-OPA S03H)-GalNAc)3-PA 10 { P-D-GlcA(1-3)-p-D-(4-O{ P-D-GlCA( 1-3)-p-DS03H)-GalNAc(1GIcNAc( 1-4))3-PA 4)) (1-21-tp-~-GlcA( 1-3)-pD-GIcNAc)~-PA 20 { p-~-GlcA( 1-3)-8-~{ p-D-GlCA( 1-3)-8-Dp-DGalNAc(1-4)}(1-5)-{ GIcNAc( 1-4)}3-PA GlcA( 1-3)-p-~GICNAC)~-PA NA EEKYN[Man6EEKYN[GlcGlcNAc21LTSVL NAcILTSVL GlcNAc( 1-4))3-PA
{ P-D-G~cA( 1-3)-p-D-
GlcNAcs
Hyaluronic acid
p-~-GlcNAc( 1- Hyaluronidase 4)-p-~-GlcA Bovine testes
138
142
Hyaluronic acid
p-~-GlcNAc( 1- Hyaluronidase 4)-p-~-GlcA Bovine testes
137
P-D-GICNAC( 14)-P-DGlcNAc
Chondroitin 6-0SO?H
P-D-GIcNAc(1 - Hyaluronidase 4)-p-~-GlcA Bovine testes
136
1P-D-G~cNAc( 4)-P-DGlcNAc
P-D-GIcNAc(14)-P-DGlcNAc
P-D-GIcNAc(14)-P-DGlcNAc
P-D-G~cNAc( 14)- P-DGlcNAc
P-D-GIcNAc(14)P-DGlcNAc P-D-GIcNAc(14)-P-DGlcNAc P-D-GIcNAc(14)-P-DGlcNAc
146
147
148
149
1so
152
151
P-D-GIcNAc(14)-P-DGlcNAc
145
Entry Linkage
Table 2 (continued)
Endo-P-N-acetylglucosaminidase Arthrobacter protophormiae Endo-P-N-acetylglucosaminidase Arthrobacter protophormiae Endo-P-N-acetylglucosaminidase Arthrobacter protophormiae Endo-P-N-acetylglucosaminidase Arthrobacter protophormiae Endo-P-N-acetylglucosaminidase Arthrobacter protophormiae Endo-P-N-acetylglucosaminidase Mucor hiemalis Endo-P-N-acetylglucosaminidase Mucor hiemalis Chitinase Nocardia orientalis
Enzyme/Source
26
NA
TIN[Man9GlcNAczCHz-IAS
ManhGlcNAcz-RNAseB
Man6GlcNAczAsn
NA Man6GlcNAc-p-DGIcNAc(1-4)-P-DGICNAC( 1-~)-D-GIcNAc
TIN[GIcNAc-CH~-]AS
GlcNAc-RNAse B
P-D-GlcNAc-Asn
GlcNAcz
Man9GlcNAczAsn
Man6GlcNAc2Asn
Man6GlcNAcz
Man6GlcNAczAsn
193
193
192
NA
NA
21
CSN[Man6GlcNAcz]LST
CSN[(GalG1cNAcMan)zManGlcNAcz]LST
CSN[GlcNAcILST
CSN[GlcNAc]LST
GICNAC~
Asn-GlcNAczMan6
ASTF-GP
GlcNAca
NA
2s
Po)
TIN[GlcNAc]AS
Yield Reference
Man9GlcNAczAsn
Product
Acceptor
Donor
52
00
162
161
160
159
158
157
156
155
154
153
P-D-GIcNAc(1- Lysozyme Hen egg white 4)-P-DGlcNAc P-D-G~cNAc( 1- Chitinase 4)-P-DTrichoderma reesei GlcNAc KDR-11 1- Lysozyme Hen egg ~-D-G~CNAC( 4)P-Dwhite GlcNAc S-D-GICNAC( 1- Lysozyme Hen egg 4)-P-Dwhite GlcNAc 1- Endo-0-N-acetylP-D-G~cNAc( 4)-P-Dglucosaminidase Arthrohactev GIcNAcprotophormiae CH21- Lysozyme Hen egg B-D-GICNAC( white 4)-P-DGlcNAc-0 1- Lysozyme Hen egg P-D-G~cNAc( 4)-P-Dwhite GlcNAc-0 P-D-GIcNAc(1- Lysozyme Hen egg4)-P-Dwhite GlcNAc-0 1- Lysozyme Hen eggP-D-G~cNAc( 4)-P-Dwhite GlcNAc-0 P-D-GIcNAc(1- Endo-P-N-acetyl4)- P-Dglucosaminidase GlcNAc-0 Arthrohacter protophormiae PpNPGlcNAc
PpNPGlcNAc
P-D-GlcNAc-0(CH2)3CH=CH2
GlcNAcz
MansGlcNAczAsn
OpNPGlcNAc
GlcNAcs
G~cNAc~
PpNPGlcNAc
TIN(GlcNAc-CHz-)AS
GlcNAc4
GIcNAc~
GIcNAc~
MangGlcNAc2Asn
GlcNAcl
GIcNAc~
GIcNAc~
GlcNAcz
14
14
35
17
3.3
26
33
84
Man&D-GlcNAc( 1-4)-PD-GlcNAc-0(CH2)3CH=CH2
189
195, 196
6.5 187
56
26
195, 196
14
40
18
14
12
P-D-G~cNAc( 1-4)-P-DGlcNAc-OpNP
P-D-GIcNAc(1-4)-P-DG k NAc-OpNP
GlcNAc-OpNP
{ P-D-G~cNAc( 1-4)}4-P-~-
GlcNAc-OpNP
{ P-D-G~cNAc( 1-4)}3-fi-D-
GlcNAcS
GICNAC~
GlcNAc7
169
168
167
166
165
164
163
Enzyme/Source
P-D-G~cNAc( 1- Endo-P-N-acetylglucosaminidase 4)-P-DArthrobacter GIcNAc-0 protophormiae P-D-GIcNAc(1- Endo-P-N-acetyl4)-P-Dglucosaminidase GlcNAc-0 Arthrohacter protophormiae P-D-GIcNAc(1- Endo-P-N-acetyl4)p-Dglucosaminidase GlcNAc-0 Arthrohacter protophormiae P-D-GIcNAc(1- Endo-P-N-acetyl4)-p-Dglucosaminidase GlcNAc-0 Arthrobacter protophormiae 1- Endo-P-N-acetylP-D-G~cNAc( glucosaminidase 4)-P-DArthrobacter GIcNAc-0 pro tophormiae P-D-G~cNAc( 1- Endo-P-N-acetylglucosaminidase 4)-P-DArthrobacter GlcNAc-0 protophormiae P-D-GIcNAc(1 - Endo-P-N-acetylglucosaminidase 4)-P-DArthrobacter GICNAC-S protophormiae
Entry Linkage
Table 2 (continued)
Man9-P-~-GlcNAc(1-4)-PD-GIcNAc-OMU
PMuGlcNAc
PpNPGlcNAc
Man9GlcNAczAsn
Man9GlcNAc2Asn
81
81
81
Man9-P-o-GlcNAc(1-4)-PD-GIcNAc-0CH2CH=CH2
P-D-GkNAc-0CH2CH=CH2
P-D-GIcNAc-S-CH~-Mans-P-~-GlcNAc( 1-4)-PCONHCH2CH(OMe)* D-GIcNAc-SCH2CONHCH2CH(OMe)2
Man9GlcNAc2Asn
Man9GlcNAczAsn
33
66
67
70
33
33
Yield Reference (“10)
Man9GlcNAc2Asn
Man9-P-~-GlcNAc(1-4)-PD-GlcNAc-OpNP
Man9-P-~-GlcNAc(1-4)-0D-GlcNAc-OBn
P-o-GlcNAc-OBn
MansGlcNAc2Asn
Product
P-D-G~cNAc-O-(CH~)~-Man9-P-~-GlcNAc(1-4)-pNHCOCH=CH2 D-G~cN Ac-O(CH2)3NHCOCH=CH2
Acceptor
Man9GlcNAczAsn
Donor
h,
\o
a
0
00
179
178
177
176
175
174
173
172
171
170
Endo-P-N-acetylglucosaminidase A rthrobacter protophormiue P-D-GICNAC( 1- Endo-0-N-acetylglucosaminidase 4)-P-DArthrobacter GlcNAc-S protophormiae P-D-GIcNAc(1- Lysozyme Hen egg4)-P-~-Man- white 0 b-~-GlcNAc( 1- Endo-P-N-acetylglucosaminidase 4)-~-Glc Arthrobacter protophormiae p-~-GlcNAc( 1- Endo-P-N-acetyl4)-~-GlcNAc glucosaminidase Mucor hiemalis P-D-GICNAC( 1- Endo-P-N-acetyl4)-~-GlcNAc glucosaminidase Mucor hiemalis 1 - Endo-P-N-acetylb-~-GlcNAc( glucosaminidase 4)-D-GlcNAc Mucor hiemalis P-D-G~cNAc( 1- Endo-P-N-acetyl4)-D-GlcNAc glucosaminidase Mucor hiemalis P-D-G~cNAc( 1- Endo-P-N-acetyl4)-D-GlcNAc glucosaminidase Mucor hiemalis P-D-GICNAC( 1- Endo-P-N-acetyl4)-~-GlcNAc glucosaminidase Arthrobacter protophormiae
P-D-G~cNAc( 14)p-DGlcNAc-S
GlcNAc-Asn-FMOC
GlcNAc
1-4)-Dp-~-GlcNAc( GlcNAc-PA
ASTTTN[GlcNAcIYT
p-~-GlcNAc( 1-4)-DGlcNAc-PA GlcNAc
ASTF-GP
ASTP-GP
STF-GP
MansGlcNAc2Asn
Man9GlcNAc2Asn
PpNPMan
STF-GP
GlcNAc2
30, 31
197
Man6-P-~-GlcNAc(l-4)-~-NA Glc
(NeuAcGalGlcNAcMan)2- 20 ManGlcNAcz-AsnFMOC (Ga1GlcNAcMan)zNA
NA
9
NA 91
(GalGlcNAcMan)2ManGlcNAc3-PA ASTTTN[( NeuAcGalG1cNAcMan)zManGlcNAczlYT Man6GlcNAc3-PA Man9GlcNAc2
ManGlcNAc2
187
b-~-GlcNAc( 1-4)-P-~-Man- 10 OpNP
185
198
199
198
198
33
33
83
P-D-GICNAC-S-CH~CN Man9-P-~-GlcNAc(1-4)-PD-G~cNAc-S-CH~CN
MangGlcNAc2Asn
Man9-P-~-GlcNAc(l-4)-p- 78 D-GIcNAc-S-(CH~)~CH~
P-D-GlcNAc-S(CH2)3CH3
Man9GlcNAclAsn
s
0 4
co
2
2.
2
b -.
2 co
24
ro
22
'h
%
2 c
2,
'5
? co
o\
9
ru
~~~~
187
186
185
184
IN[GlcNAc]ATL
IN[GlcNAc]ATL
DansylASTTTN[GlcNAc]YT
GlcNAc-Asn-FMOC
DansylASTTTN[GlcNAc]YT
STF-GP
183
P-D-GIcNAc(1- Endo-0-N-acetyl~)-D-G~cNAc glucosaminidase Mucor hiemalis P-D-G~cNAc(1- Endo-a-N-acetyl~)-D-GIcNAc glucosaminidase Mucor hiemalis D-D-GICNAC( 1- Endo-0-N-acetyl~)-D-G~cNAc glucosaminidase Mucor hiemalis P-D-GIcNAc(1- Endo-P-N-acety l~)-D-GICNAC glucosaminidase Mucor hiemalis
GlcNAc-Asn-FMOC
ASTF-GP
182
D-GIcNAc
Mans GlcNAcz Asn
CSN[GlcNAc]LSTCVL GKSNELHKLNTYPRT DVGAGTP
Acceptor
P-D-GIcNAc(1- Endo-P-N-acetyl4)-~-GlcNAc glucosaminidase A rthrobacter protophormiae P-D-GIcNAc(1- Endo-P-N-acetyl4)-~-GlcNAc glucosaminidase Mucor hiemalis P-D-G~cNAc( 1- Endo-P-N-acetyl4)-~-GlcNAc glucosaminidase Mucor hiemalis
Donor
181
~
STF-GP
~~
P-D-G~cNAc( 1- Endo-P-N-acetyl4)-~-GlcNAc glucosaminidase Mucor hiemalis
EnzymeJSource
180
Entry Linkage
~
Table 2 (continued)
~~~
IN[( NeuAcCalG1cNAcMan)zManGlcNAcz]ATL
Dansyl-ASTTTN[(GalGlcNAcMan)2ManClcNAc2]YT IN[Man6GlcNAc2lATL
(GalGlcNAcMan)2ManGlcNAc2-AsnFMOC DansylASTTTN[( NeuAcGalGlcNAcMan) 2 ManGlcNAcz]YT Man6GlcNAc2-Asn-FMOC
CSN[(NeuAcGalGlcNAcMan)zManGlcNAczILSTCVLGKSNELHKLNTYPRTD-VGAGTP Mans-P-~-GlcNAc(1- 4 ) - ~ GlcNAc
Product
-
197
4
197
199
15
10
197
199
197
33
8
16
15
85
8.5 200
Yield Reference
196
195
194
193
192
191
190
189
188
1- Endo- P-N-acetylb-~-GlcNAc( 4)-~-GlcNAc glucosaminidase Mucor hiemalis P-D-G~cNAc(1- Endo-P-N-acetylglucosaminidase 4)-~-Man Arthrobucter protophormiue P-D-GICNAC( 1- Endo-P-N-acetyl4)-~-Man glucosaminidase Arthrobacter protophormiae 1- Lysozyme Hen eggP-D-G~cNAc( 4)-~-Man white P-D-GIcNAc(1- Endo-P-N-acetyl~)-L-Fuc glucosaminidase Arthrobacter protophormiae P-D-GIcNAc(1 - Endo-P-N-acetylglucosaminidase 4)-L-Gal Arthrobucter protophormiue D-D-GICNAC-O Endo- P-N-acetylglucosaminidase Arthrobucter protophormiue P-D-GlcNAc-0 Endo-P-N-acetylglucosaminidase Arthrobacter protophormiue Endo-P-N-acetylglucosaminidase Arthrobucter protophormiue
185
33
33
33
83
21 26
19
64
8
47
I-4-DMan9-P-~-GlcNAc( Man
p-~-GlcNAc(1-4)-~-Man Mang-P-~-GlcNAc(l-2)-LFuc
Mans-P-~-GlcNAc(l-2)-LGal
Mans GlcNAc-OMe
Mang GlcNAc-OPr
Mans GlcNAc-OEt
Man
Man L-Fuc
L-Gal
MeOH
PrOH
EtOH
GlcNAcz Man9GlcNAc2Asn
Mans GlcNAczAsn
Mans GlcNAcz Asn
Man9GlcNAczAsn
Man9GlcNAc2Asn
185
187
185
30, 31
Man&b~-GlcNAc(I-~)-D- NA Man
197
Man
I
ManhGlcNAczAsn
IN[(GalGlcNAcMan)zManGlcNAcz 1ATL
IN[GlcNAc]ATL
ASTF-GP
199
198
197
Enzyme/Source
glucosaminidase Arthrobacter protophormiae P-D-GlcNAc-0 Endo-0-N-acetylglucosaminidase Arthrobacter protophormiae P-D-xyl( 1-4)-P- Xylanase TrichoD-xyl derma viridae
P-D-GlcNAc-0 Endo-P-N-acetyl-
Entry Linkage
Table 2 (continued)
P-D-xyl(1-4)-P-DXyl-F
MangGlcNAczAsn
Man9GlcNAc2Asn
Donor
P-D-Xyl(1-4)-P-D-Xyl-F
Glycerol
iPrOH
Acceptor
{ p-D-xyl(1-4))(2-12)-D-x)’l
Man9GlcNAc-(1-?)Glyceryl
Mans GlcNAc-OiPr
Product
72
56
9.6
(XI
201
33
33
Yield Reference
L? 0
2
0
R
$
-
?
9
0
&
Q
%-
0
b
0
+
00
a-D-Gal-0
a-D-Gal-0
a-D-Gal-0
a-D-Gal-0
a-D-Gal-0
a-D-Gal-0
a-D-Gal-0
a-D-Gal-0
a-D-Gal-0
a-D-Gal-0
1
2
3
4
5
6
I
8
9
10
Entry Linkage apNPGal
a-Galactosidase Asperg illus oryzae a-Galactosidase Asperg illus oryzae a-Galactosidase Coffee bean a-Galactosidase Trichoderma reesei a-Galactosidase Trichoderma reesei a-Galactosidase Trichoderma reesei a-Galactosidase Trichoderma reesei a-Galactosidase Trichoderma reesei a-Galactosidase Trichoderma reesei a-Galactosidase Asperg illus niger Gal
apNPGal
apNPGal
apNPGal
apNPGal
apNPGal
apNPGal
Raffinose
apNPGal
Donor
Enzyme/Source
Table 3. Enzymatically synthesised alkyl glycosides.
202
44 203
203
203
203
23
NA NA
NA
NA
NA
a-D-Gal-O-elymoclavine
a-D-Gal-OAll a-D-Gal-OMe
a-D-Gal-OEt
a-D-Gal-OPr
Elymoclavine
HOAll MeOH
EtOH
203
203
204
NA
NA
41
a-D-Gal-O-(CH2)3CH3
a-D-Gal-O-CH(CH3)CH*CH3
a-D-Gal-O(CH2)60H
HO-(CHz)jCH3
HO-CH(CH3)CHlCH3
HO-(CH2)60H
iPrOH
a-D-Gal-OiPr
202
24
a-D-Gal-0-chanoclavine
Chanoclavine
PrOH
Reference
Yield (%)
Product
Acceptor
3s
a-D-Gal-0
a-D-Gal-0
a-D-Gal-0
a-D-Gal-0
a-D-Gal-0
a-D-GalNAc-0
a-D-GalNAc-0
a-D-GalNAc-0
a-D-GalNAc-0
11
12
13
14
15
16
17
18
19
Entry Linkage
~~
Table 3 (continued)
204
205
205
206 206 207
207
207
207
~ - D - G ~ ~ - O ( C H ~ ) ~ C H = C H ~37
6
11
3
8 10
NA
NA
NA
a-D-Gal-O-lysergol
a-~-Gal-O-9,lO-dihydrolysergol
a-D-Gal-O-Ser-(N-Boc)-OMe a-D-Gal-O-Ser-(N-A1oc)-OMe a-D-GalNAc-0-L-Ser
a-D-GalNAc-0-L-Thr
a-D-GalNAc-0-D-Thr
HO-(CH2)3CH=CH2
Lysergol
9,lO-Dihydrolysergol
Ser-(N-Boc)-OMe Ser-(N-A1oc)-OMe L-Ser
D-Ser
L-Thr
D-Thr
Gal
apNPGal
apNPGal
Raffinose Raffinose GalNac
GalNac
GalNac
GalNac
a-Galactosidase Aspergillus niger a-Galactosidase Aspergillus oryzae a-Galactosidase Aspergillus oryzae a-Galactosidase Coffee bean a- Galactosidase Coffee bean a-N-acetylgalactosaminidase Bovine liver a-N-acetylgalactosaminidase Bovine liver a-N-acetylgalactosaminidase Bovine liver a-N-acetylgalactosaminidase Bovine liver
Yield (YO) Reference
Product
Acceptor
Donor
Enzyme/Source
30
29
28
27
26
25
24
23
22
21
20
a-D-GalNAc-0 a-Galactosaminidase Aspergillus oryzae a-D-GalNAc-0 a-Galactosaminidase Aspergillus oryzae a-D-GalNAc-0 a-Galactosaminidase Aspergillus oryzae a-D-GalNAc-0 a-Galactosaminidase Aspergillus oryzae a-D-GalNAc-0 a-Galactosaminidase Aspergillus oryzae a-D-GalNAc-0 a-Galactosaminidase Aspergillus oryzae Glucoamylase a-D-Glc-0 Rhizopus oryzae Glucoamylase a-D-Glc-0 Rhizopus oryzae a-Glucosidase a-D-Gk-0 Talaromyces duponti a-Glucosidase a-D-Glc-0 Talaromyces duponti a-D-Glc-0 a-Glucosidase Talarornyces duponti 208
10-50
a-D-GalNAc-0-Ser-(N-COCH3)OMe
210
21 1
21 1
NA
23
18
Maltose
Maltose
Maltodextrins
Glc
Glc
209
208
2
a-D-GalNAc-0-Ser-(N-CO(CH3)3)- 10-~50 OMe
a-D-GalNAc-0-Ser-(NCOCH*C(C1)3)-OMe
209
Ser-(N-COC(CH3)3)OMe
Ser-(N-COCH2C(C1)3)OMe
208
208
10-50
a-D-GalNAc-0-Ser-(NCOCH2CH=CH2)-OMe
25
apNPGalNAc
apNPGalNAc
apNPGalN Ac Ser-(NCOCH2CH=CH2)OMe apNPGalNAc Ser-(N-COCH3)-OMe
10-50
208
50
a-D-GalNAc-0-Ser-(N-CO0Me)OMe
Ser-(N-CO0Me)-OMe
apNPGalNAc
208
29
a-D-GalNAc-0-Thr-(N-Ac)-OMe
apNPGalNAc Thr-(N-Ac)-OMe
w
co
3F
Q
3
n
8 14
29 Glycosiduse-Cutulysed Oligosaccharide Synthesis
r0 N
2
+ L
$
$ c
Q
Q L
Y C
5
58
57
56
55
54
53
52
51
50
49
48
47
46
45
44
a-Mannosidase Jack bean a-Mannosidase a-D-Man-0 Almond a-Mannosidase a-D-Man-0 Jack bean a-Mannosidase a-D-Man-0 Almond a-Mannosidase a-D-Man-0 Almond a-D-Man-0 a-Mannosidase Almond a-L- Ara-O P-Galactosidase Bovine liver a-L-Fuc-0 a-L-Fucosidase Limpet Invertase P-D-Fruf-0 Bakers yeast b-D-Fruf-0 Invertase Bakers yeast P-D-Fruf-0 Invertase Succharomyces cervisiae P-D-Gal( 1-2)- P-Galactosidase Bacillus DNJ circulans P-Galactosidase P-D-Gal(1-3)DNJ Bacillus circuluns p-D-Gal(1-4)- P-Galactosidase DNJ Bacillus circuluns p-~-Gal( 1-4)-0 Rhodotorulu luctosa cells
a-D-Man-0
205 205 107 202 214 214 215
216
216
3 2 3 2 3 1
40
6
20
a-D-Man-0-lysergol
a-~-Man-O-9,lO-dihydrolysergol
p-~-F:ruf-O-(CH2)3CH3
p-~-Gal( 1-3)-DNJ
9,lO-Dihydrolysergol Monobenzyl ether 1,3,5- monobenzyl ether 1,3-(a-L-Ara-0)1,3,5-benzene-trimethanol benzene-trimethanol a-L-Fuc-0-chanoclavine Chanoclavine !~-D-FIx~-O-(CH~)~CH~
Lysergol
apNPMan a-L-pNPAra
216
217
26
10
P-D-Gal(1 -4)-DNJ
S-D-Gal(1-4)-O-calystegineB2
HO-(CH2)3CH3 HO-(CH2)3CH3
DNJ
DNJ
DNJ
CalystegineB2
Sucrose Sucrose
Lactose
Lactose
Lactose
Lactose
P-D-FN~-O-(CH~)~CH~
HO-(CH2)3CH3
Fru
apNPFuc
apNPMan
205
3
a-D-Man-0-elymoclavine
Elymoclavine
apNPMan
213
68
a-~-Man-O-5-phenyl1-pentyl
5-phenyl-1-pentanol
apNPMan
202
18
a-D-Man-0-chanoclavine
Chanoclavine
apNPMan
202
13
a-D-Man-0-ergometrine
Ergometrine
Man
ul
L
00
3F
a
3
R
68
67
66
65
64
63
62
61
60
59
P-D-Gal(1-6)-
Entry Linkage
Table 3 (continued)
P-Galactosidase Bacillus circulans P-Galactosidase Aspergillus oryzae P-Galactosidase Aspergillus oryzae P-Galactosidase Aspergillus oryzae P-Galactosidase Asperg illus oryzae P-Hexosaminidase Aspergillus oryzae P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli
Enzyme/Source
212
63
13
10
P-D-Gal-0-p-bis(hydroxymethyl) benzene P-~-Gal-o-Ser
p-bis(hydroxymethy1) benzene Serine
PpNPGal
PoNPGal
Lactose
Propane-] ,2-diol
26 (R: 8 ee)
219
219 P-D-G~~-O-CH(CH~)CH~OH 20 (R: 13 ee)
P-D-Gal-O-CH2CHOHCH3
219
32 ( R)-P-D-G~I-O-CH(CH~)CH~CH~
(R)-(-)-Butan-2-01
Lactose
Lactose
219 P-D-G~I-O-CH(CH~)CH~CH~ 32 (R: 6 ee)
Butan-2-01
Lactose
218
212 12
P-~-Gal-O-pentaerythritol
Pentaerythritol
Pp NPGa1
23 P-D-GIcNAc-O-~-COOCH~-~-NO~ OH BpNPGlcNAc p-COOCH3-m-NO2-Bn-
212
20
P-D-Gal-0-cyanuricacid
Cyanuricacid
PpNPGal
Reference 216
(“/o)
7.2
Yield
P-D-Gal(1-6)-DNJ
Product
DNJ
Acceptor
Lactose
Donor
b v3
a
03
P-D-Gal-0
P-D-Gal-0
P-D-Gal-0
p-D-Gal-0
p-D-Ga1-0
P-D-Gal-0
P-D-Gal-0
P-D-Gal-0
P-D-Gal-0
P-D-Gal-0
B-D-Gal-0
69
70
71
72
73
74
75
76
77
78
79
P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli p-Galactosidase Escherichia coli P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli B-Galactosidase Escherichia coli P-Galactosidase Asperg illus oryzae 219
219
13
219 8 (R: 33 ee)
@)-(+)-Propane-1,2-diol
Butane-l,3-diol
Butane-I ,3-diol
Lactose
Lactose
Lactose
219
219
44
44
44
220
7
42
NA
NA
NA
20
(S)-(+)-Butane-l,3-diol
Propane-l,3-diol
HOAll
HOBn
Lactose
Lactose
HO-(CH2)2Si(CH3)3
HO-(CH2)2C6H5
Lactose
PPhGal
Lactose
Lactose
219 54
(S)-(+)-Butane-l13-diol
Lactose
219
38
(S)-(+)-Propane-l,2-diol
Lactose
3F
4
Donor
89
88
87
86
85
84
83
82
81
P-Galactosidase Lactose Escherichiu coli 0-Galactosidase Lactose Escherichiu coli P-Galactosidase Lactose Escherichia coli P-Galactosidase Lactose Escherichiu coli P-Galactosidase Lactose Escherichiu coli P-Galactosidase Lactose Asperg illus oryzue P-Galactosidase Lactose Asperg illus oryzue P-Galactosidase PPhGal Asperg illus oryzae P-Galactosidase PPhGal Aspergillus oryzue P-Galactosidase PPhGal Asperg illus oryzue
~~
80
~
EnzymeJSource
Entry Linkage
~~~~
Table 3 (continued) ~
P-D-Gal-O-3-digitoxigenin
P-~-Gal-0-3-16B,17P-epoxy-170.digitoxigenin
16B,17P-EPOXY-170.digitoxigenin
P-~-Gal-O-3-gitoxigenin
~~
13 (70 de)
36 (50 de)
28 (63 de)
24 (75 de)
10 (90 de)
30 (89 de)
64
38
26
222, 223
222, 223
222, 223
22 1
22 1
22 1
22 1
22 1
22 1
22 1
Yield (“4)Reference
P-D-Gal-O-cis-l,2-~ycIohexanediol 20 (38 de)
P-D-Gal-0-cis- 1,2-cyclopentanediol
P-D-Gal-0-cis- 1,2-cyclopentanediol
P-D-Gal-0-cis-norbornanediol
0-D-Gal-0-cis-norbornenediol
P-D-Gal-o-cis- 1,2-cyclohexanediol
P-D-Gal-0-cis-1,2-cyclopentanediol
Product
Digitoxigenin
Gitoxigenin
cis- 1,2-CycIohexanediol
cis-I ,2-Cyclopentanediol
4-Cyclopentane- 1,4-diol
cis-Norbornanediol
cis-Norbornenediol
cis- 1,2-Cyclohexanediol
cis- 1,2-Cyclopentanediol
Acceptor
819
29.6 Recent Developments and New Directions
d
N N
d
N N
d
N N
d
,?i
N N
N
-
0 A
0
59
6n
U
U
ci
0
d
0
9 e,
-
c u
0
4
Q
c
9
Q
2i
0 h
9
0
U
a
.e
63
4
b
ci
t3
e
110
109
108
107
106
105
104
103
102
coli
P-Galactosidase Escherichia coli P-Galactosidase Kluyveromyces lactis P-Galactosidase Kluyverornyces lactis P-Galactosidase Kluyveromyces lactis P-Galactosidase Kluy veromyces lactis P-Galactosidase Kluyveromyces lactis P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli
P-D-Gal-08 224 C H ( C H ~ ) ( C H ~ ) Z C O O ( C H ~ )(R: ~ C11 H ~ee)
CH3CHOH (CH2)zCOO(CH2)3CH3 HO-(CHz)60H
PoNPGal
Lactose
P-D-Gal-0-1-Glyceryl
Glycerol
HOBn
EtOH
Lactose
Lactose
Lactose
~-D-G~I-O-(CH~)~OH
P-D-G~~-O-(CH~)~OH
PoNPGal
Lactose
P-D-Gal-OEt
P-D-Gal-OBn
P-D-G~I-O(CH~)~OH
Lactose
P-D-G~I-O-(CH~)~OH
P-D-Gal-0CH(CH3)(CH2)2COOCH2CH3
CH3CHOH(CH2)2COOCH2CH3
50
92
36
21
60
40
49
226
226
225
225
225
225
225
37 224 (R: 55 ee)
46 224 (R: 65 ee)
P-D-Gal-0CH(CH3)(CH2)2COOCH3
Reference
CH3CHOH(CH2)2COOCH3
P-Galactosidase PoNPGal Escherichia coli P-Galactosidase PoNPGal Escherichia
101
Yield (%)
Product
Acceptor
Donor
Enzyme/Source
Entry Linkage
Table 3 (continued)
121
120
119
118
117
116
115
114
113
112
111
P-Galactosidase Escherichia coli P-Galactosidase Kluyveromyces lactis P-Galactosidase Kluyveromyces lactis 0-Galactosidase Asperg illus oryzae P-Galactosidase Asperg illus oryzae P-Galactosidase Escherichia coli P-Galactosidase Escherichiu coli P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli
coli
C
2
sa
3s
214
227 228, 229
228, 229
228, 230
228,230
23
14
52
23
66
82
Phenylethanol
HO-CH(CH3)CH2CH3
HO-C(CH3)3
5-Phenylpentanol
HO-(CH2)7CH3
Lactose
PpNPGal
PpNPGal
PpNPGal
PpNPGal
Lactose
g. 0 5
9 2
s.
8
2
5
c.i rp
5
o\
HO-(CH2)3CH3
\o
ru
214 23
HO-(CH2)3CH3
Lactose
214
18
HO-(CHZ)~CH~
226
27
Gal
226
50
HO-(CH2)7CH3
HO-(CHz)gCH3
226
78
HO-(CH2)loOH
PoNPGal
P-Galactosidase PoNPGal Escherichiu coli P-Galactosidase PoNPGal Escherichia
0-Galactosidase Escherichia coli P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli P-Galactosidase Bacillus circulans P-Galactosidase Bucillus circulans 0-Galactosidase Bacillus circulans P-Galactosidase Bacillus circulans 0-Galactosidase Escherichia coli
122
131
130
129
128
127
126
125
124
123
Enzyme/Source
Entry Linkage
Table 3 (continued) ~
228, 231
41 16
36
<5
67
P-D-Gal-0-cholesteryl
P-D-Gal-O-(R)-1-phenylethyl P-D-Gal-o-(S)-I-phenylethyl
P-D-Gal-O-(R)-1-methyloctyl
P-D-Gal-O-(S)-I-methyloctyl
P-D-G~~-O-(CH~)~CH~
Cholesterol
(R)-1-Phenylethanol
(S)-I-Phenylethanol
(R)-I-Methyloctanol
(S)- 1-Methyloctanol
HO-(CH&CHj
PpNPGal
PpNPGal
PpNPGal
PpNPGal
PpNPGal
PpNPGal
228, 230
228, 230
228
228
228
228
P-D-Gal-0-1,2-di-O-dodecylglyceryl 52
1,2-di-0Dodecylglycerol
PpNPGal
7.2
228
P-D-Gal-O-1,3-di-O-dodecylglyceryl9
1,3-di-0Dodecy lglycerol
PpNPGal
%,
G.
2
9 E
P
5.
5
0, 8g
6.
+ -
Ba
f3
8-?
13
228, 230
15
P-D-Gal-O-(CHl)II C H ~
PpNPGal
2 228, 230
Yield (“h) Reference 13
Product P-D-G~~-O-(CH~)~CH~
Acceptor
PpNPGal
Donor
00 h,
w
29.6 Recent Developments and New Directions
z
rn 0
N
c\I
WN
00-
N
N
r J
N m
0 rn N
0
m
0 m N
0
N
w
00-
00-
w-
N
N
N N
00
m
3
N
w
N
v,
m
N N
m
N
N
2 N
m g r . 4N gg hl N
m
rN
W W
m
N
N
0
d
3
3
N
N
m
s-
rT)
N
N
d
s
0
cu
-
0
a ci
n u
0
d
+ 0
4
U
b
0
z
Ci
Ci
d
b
ru
m
m
0-
b
v
823
N
d
0-D-Gal-0
P-D-Gab0
p-D-Gal-0
P-D-Gal-0
p-D-Gal-0
P-D-Gal-0
0-D-Gal-0
P-D-Gal-0
P-D-Gal-0
B-D-Gal-0
144
145
146
147
148
149
150
151
I52
153
Entry Linkage
Table 3 (continued)
P-Galactosidase Bacillus circulans P-Galactosidase Escherichia coli P-Galactosidase Aspergillus oryzae P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli 0-Galactosidase Escherichia coli P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli 0-Galactosidase Aspergillus oryzae P-Galactosidase Aspergillus oryzae
Enzyme/Source
106
106
106
106
13
9
11
P-D-Gal-O-Ser-(N-Boc)-Gly-OMe
P-D-Gal-O-Ser-(N-Boc)-Ala-OMe
P-D-Gal-O-Ser-(N-Boc)-Ser-OMe
Gly-(N-BOG)-[ P-~-Gal]-0-Ser-OMe 7
5
P-o-Gal-O-but-3-en-2-yl
But-3-en-2-01
Ser-(N-Boc)-Gly-OMe
Ser-(N-Boc)-Ah-OMe
Ser-(N-Boc)-Ser-OMe
Gly-(N-Boc)-Ser-OMe
Ala-(N-Boc)-Ser-OMe
HOAll
HOAll
Gal
Lactose
Lactose
Lactose
Lactose
Lactose
PoNPGal
PVGal
93
93
62
50 /3-~-Gal-OAll
213
P-D-Gal-OAlI
Ala-(N-BOG)-[ P-~-Gal]-0-Ser-OMe
21 233 (R: 40 ee) 106
62
P-~-Gal-O-5-phenyl-1-pentyl
5-Phenyl-1-pentanol
PpNPGal
230, 231
35
P-D-Gal-0-pNP
PNP
PpNPGal
Yield (“YO) Reference
Acceptor
Product
Donor
P-u-Gal-0
0-D-Gal-0
P-D-Gal-0
0-u-Gal-0
P-r>-Gal-O
P-D-Gal-0
P-u-Gal-0
158
159
160
161
162
163
164
157
234
204
204
48
22
fi-D-Gal-O(CH2)60H
HO-(CH2)60H
Lactose
Gal
Gal
236
17
15
P-D-Gal-OAI1
HOAll
Gal
38
235
4
P-D-Gal-0-Thr-(N-Boc)
Thr-(N-Boc)
P-D-Gdl-OAll
235
4
P-D-Gal-0-Ser-(N-Boc)
Ser-(N-Boc)
Lactose
HOAll
234
28
P-D-Gal-O-Hyp-(N-Z)-OMe
Hyp-(N-Z)-OMe
Lactose
Lactose
234
10
P-D-Gal-0-Thr-(N-Z)-OMe
Thr-(N-Z)-OMe
107
107
Monoallyl ether (4,6-iso- monoally1 ether (P-~-Gal-0)-(4,6- 17 isopropylidine-P-D-Glc-0)-1,3,5propyhdine-P-D-Glcbenzene-trimethanol 0)-1,3,5-benzenetrimethanol monoally1 ether (p-D-Gal-O)-(p-D- 10 Monoallyl ether (0-DGlc-O)-l,3,5-benzeneGlc-0)-1,3,5-benzene-trimethanol trimethanol 35 P-u-Gal-0-Ser-(N-Z)-OMe Ser-(N-Z)-OMe
Lactose
Lactose
P-Galactosidase Achatina uchatinu P-Galactosidase Achatina achatina P-Galactosidase Achatina achatina 0-Galactosidase Asperg illus oryzae P-Galactosidase Asperg illus oryzae P-Glucosidase Almond P-Galactosidase Asperg illus oryzae P-Galactosidase Asperg illus oryzae P-Galactosidase Streptococcus therrnophilus
P-D-Gal-0
156
P-Galactosidase PoNPGal Bovine liver
P-Galactosidase PoNPGal Bovine liver
P-u-Gal-0
155
154
174
173
172
171
170
169
168
167
166
165
Entry Linkage
Table 3 (continued)
P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli P-Galactosidase Escherichia coli 0-Galactosidase Achatina achatina P-Galactosidase Achatina achatina P-Galactosidase Achatina achatina P-Galactosidase Achatina achatina P-Galactosidase Achatina achatina P-Galactosidase Achatina achatina Rhodotorula lactosa cells
P-D-Gal-O-Ser-(N-Boc)-OMe
P-D-Gal-O-Ser-(N-A1oc)-OMe
P-D-Gal-O-Ser-(N-Z)-OMe
P-D-Gal-0-Ser-(N-Z)-Gly-OEt
P-D-Gal-0-Ser-(N-Z)-Ala-OEt
Gly-(N-2)-[P-~-Gal-o]-Ser-OEt
Ala-(N-Z)-[P-~-Gal-0]-Ser-OEt
Ala-(N-Z)-[P-~-Gal-0]-Ser-OMe
P-D-Gal-0-Thr-(N-Z)-Gly-OEt
Ser-(N-Boc)-OMe
Ser-(N-Aloc)-OMe
Ser-(N-Z)-OMe
Ser-(N-Z)-Gly-OEt
Ser-(N-Z)-Ala-OEt
Gly-(N-Z)-Ser-OEt
Ala-(N-Z)-Ser-OEt
Ala-(N-Z)-Ser-OMe
Thr-(N-Z)-Gly-OEt
CalystegineB 1
Lactose
Lactose
Lactose
Lactose
Lactose
Lactose
Lactose
Lactose
Lactose
Cellobiose
P-D-GIc(1-3)-O-calystegineB1
Product
Acceptor
Enzyme/Source Donor
0.9
9
7
8
29
15
16
9
15
15
Yield (“!o)
217
237
206
Reference b v3
m
N
03
827
29.6 Recent Developments and New Directions m m
m
2 N
% N
0
N
N
0 N N
oom "
m
m
N
m
N
N
00-
"
m
N
00-
m
N
m
m
N
oom
N
r
%
4
w-
m
%
%
00-
00-
N
m
N
m
m m
N
m m
N
d
z
5 ? P
82 a
P
sz
v
v
?
-8
?
'1 a
u
5a
u
L 8
v 3
?
?
h
h
2
-t
3
5
2
v
e:Pe
L
8 G
8
b ci
c i u
8
8
8 8
8 8 8 8
ci
& I
u
&L
Bn
5n
5a 59
c?
5a 5n 59 ci
cl
9
cl
8 8 59 5a a
&L
240
240 240 24 1
2 1.9 1.6 1.1
1 22
P-D-Glc-0-mHOBn P-D-Glc-0-pHOBn P-D-Glc-0-oHOBn P-D-Glc-0-mHOBn P-D-Glc-0-pHOBn P-D-Glc-OMe
P-D-Gk-OEt
P-D-Glc-OPr
P-~-Glc-O-3-gitoxigenin
P-~-Glc-O-3-digitoxigenin
mHOBnOH pHOBnOH oHOBnOH mHOBnOH pHOBnOH MeOH
EtOH
PrOH
Gitoxigenin
Digitoxigenin
Cellobiose Cellobiose Glc Glc Glc Cellobiose
Cellobiose
Cellobiose
PPhGlc
PPhGlc
P-D-G~c-O
P-D-G~c-O
P-D-G~c-O
P-D-G~c-O
P-D-Glc-0
P-D-G~c-O
P-D-G~c-O
P-D-GIc-O
P-D-G~c-O
P-D-GIc-O
191
192
193
194
195
196
197
198
199
200
222, 223
222, 223
74
24 1
24 1
240
240
43
33
10
240
Cellobiose
P-Glucosidase Almond P-Glucosidase Almond 0-Glucosidase Almond P-Glucosidase Almond P-Glucosidase Almond P-Glucosidase Almond P-Glucosidase Fusarium oxysporum P-Glucosidase Fusarium oxysporum P-Glucosidase Fusarium oxysporum P-Galactosidase Aspergillus oryzae 0-Galactosidase Aspergillus oryzae
P-D-GIc-O
190
239
P-D-G~c-O-CH~CH(CH~)NHAC 39 HOCH~CH(CH~)NHAC (R) ( R) P-D-Glc-0-oHOBn 3 oHOBnOH
PoNPGlc
P-Glucosidase Almond
P-D-G~c-O
Yield (“A) Reference
189
Product
Acceptor
Donor
Enzyme/Source
Entry Linkage
Table 3 (continued)
829
29.6 Recent Developments and New Directions
m
e
a"
b ci
9 B9 U
9
9
Bn
B9 Ba
0
ci
U
0
z
ci
Geraniol
3.4
246
246
Cellobiose
P-D-GIc-O
227
1.2
Geraniol
Cellobiose
P-D-GIC-O
226
214
44
HO-(CH2)3CH3
Glc
P-D-G~c-O
225
245
41
HO-(CH2)60H
Glc
P-D-Gk-0
224
245
40
HO-(CH2)50H
Glc
P-D-Glc-0
223
245
47
HO-(CH2)40H
Glc
P-D-G~c-O
222
24 5 55
HO-(CH2)30H
Glc
P-D-GIc-O
22 1
244
7.4
Cellobiose
P-D-Glc-0
220
244
12
Cellobiose
P-D-GIc-O
219
243
2.1
243
42
Glc
P-D-Glc-0
2 18
Reference
Yield ('%)
243
P-D-Glc-0
217
Product
3.2
PPhGlc
P-Glucosidase Sulfolobus solfataricus P-Glucosidase Almond P-Glucosidase Sulfolobus solfataricus P-Glucosidase Trichoderma viridae J3-Glucosidase Trichoderma viridae P-Glucosidase Almond P-Glucosidase Almond J3-Glucosidase Almond P-Glucosidase Almond P-Glucosidase Almond 0-Glucosidase Trichoderma reesei 0-Glucosidase Asperg illus niger
P-D-G~c-O
216
Acceptor
Glc
Donor
Enzyme/Source
Entry Linkage
Table 3 (continued)
0
w
00
P-D-GIc-O
P-D-GIC-O
P-D-Glc-0
P-D-G~c-O
P-D-G~C-O
P-D-GIc-O
p-D-G1c-0
P-D-GIC-O
P-D-G~c-O
P-D-G~c-O
P-D-GIc-O
P-D-G~c-O
229
230
23 1
232
233
234
235
236
237
238
239
240
228
P-Glucosidase Cundidu molischiana P-Glucosidase Almond P-Glucosidase Trichoderma reesei P-Glucosidase Asperg illus niger 0-Glucosidase Cundida molischiana P-Glucosidase Almond P-Glucosidase Trichoderrna reesei P-Glucosidase Asperg illus niger P-Glucosidase Almond P-Glucosidase Almond P-Glucosidase Almond P-Glucosidase Asperg illus niger 0-Glucosidase Agrobacterium tumejaciens 213
248
23
80
P-~-Glc-O-5-phenyl-1 -pentyl
5-Phenyl-1-pentanol
PpNPGlc
PpNPGlc
247 8 P-D-GIc-O-(CH~)~CH~
HO-(CH2)7CH3
247 17
2-Hydroxybenzylalcohol P-~-Glc-O-2-hydroxybenzyl
Glc Glc
246 0.1
246 0.3
P-D-Glc-0-citronellyl
246 0.3
Citronellol
246 0.1
Cellobiose
246
3.3
246
246
0.8
0.6
246
0.6
P-D-Glc-0-citronellyl
246
0.1
Citronellol
P-u-Glc-0-neryl
P-D-Glc-0-geranyl
Cellobiose
Citronellol
Nerol
Cellobiose Cellobiose
Nerol
Cellobiose
Nerol
Cellobiose
Nerol
Geraniol
Cellobiose
Cellobiose
Geraniol
Cellobiose
c
w
co
P-Glucosidase Almond
P-Glucosidase Almond P-Glucosidase Almond P-Glucosidase Almond P-Glucosidase Almond P-Glucosidase Almond P-Glucosidase Sulfolobus solfataricus
P-D-Glc-0
P-D-G1c-0
P-D-GIc-O
P-D-G~c-O
P-D-Glc-0
P-D-GIc-O
P-D-GIc-O
P-D-GIc-O
P-D-G~c-O
P-D-G1c-0
P-D-Glc-0
242
243
244
245
246
247
248
249
250
25 1
252
P-Glucosidase Almond P-Glucosidase Almond P-Glucosidase Almond P-Glucosidase Almond P-Glucosidase Almond
P-D-Gk-0
Enzyme/Source
241
Entry Linkage
Table 3 (continued)
Hex-1-en-3-01 Pent-3-en-2-01
Glc Glc
HO-CH2CHOHCH3
Pent-1-en-3-01
Glc
PPhGlc
But-3-en-2-01
HO-(CH&jOH
10
Glc
249
7
HO-(CH2)60H
1,2-3,5-DiphenylboronateGlcf 1,2-3,5-DiphenylboronateGlcf Glc
77
12 (R: 40 ee) 5 (R: 80 ee) 17 (R: 37 ee) 17
(R: 20 ee)
250
250
233
233
233
233
249
62
HOAll
Glc
27
249
17
HS-(CH& SH
249
249
7
HO-(CH2)6NHCOCF?
Glc Glc
249
Reference
Yield (%) 61
Product
HO-(CH2)60H
Acceptor
Glc
Donor
w
CO W
265
264
263
262
26 1
260
259
258
257
256
255
254
253
P-Glucosidase Sulfolobus souataricus P-Glucosidase P-D-GIGO Pyrococcus furiosus P-D-GIc-O P-Glucosidase Pyrococcus jiuriosus P-D-GIC-O P-Glucosidase Almond P-D-GIGO 0-Glucosidase Almond P-D-Glc-0 P-Glucosidase Almond P-D-Glc-0 P-Glucosidase Almond P-D-Glc-0 P-Glucosidase Almond P-D-G~c-O P-Glucosidase Almond P-D-G~cNAc-O P-Hexosaminidase Aspergillus oryzae P-D-GIcNAc-0 P-Hexosaminidase Aspergillus oryzae P-D-GIcNAc-0 0-Hexosaminidase Aspergillus oryzae B-D-GIcNAc-0 P-Hexosaminidase Aspergillus oryzae
P-D-G~c-O
250
250
63
15
Cellobiose
Cellobiose
17 204 204 204 212
212
208
208
40 61 50
11 29
12
10
10-50
HOBn HO-(CH2)60H HO-(CH2)3CH=CH2 HO-(CH2)2Si(CH3)3 Cyanuricacid
Glc Glc Glc Glc PpNPGlcNAc
PpNPGlcNAc Ser-(N-COCHzCH= CH2)-OMe
PpNPGlcNAc Ser-(N-CO0Me)-OMe
P-D-GlcNAc-0-Ser-(NCOCH2CH=CH2)-OMe
17 65
HOAll
Glc
PpNPGlcNAc p-bis(hydroxymethy1) benzene
25 1
NA
HO-(CH2)7CH3
Glc
P-D-GIcNAc-O-~ bis(hydroxymethy1) benzene P-D-GlcNAc-0-Ser-(N-CO0Me)OMe
250
HO-CH2CHOHCH3
20
PPhGlc
b
w w
cb
E
F:
(D
211
276
215
214
273
212
271
210
269
268
261
266
~~
Enzyme/Source
P-D-G~cNAc-O P-Hexosaminidase Bovine kidney P-Glucosidase P-D-GIc-O Almond 0-Mannosidase P-D-Man-0 Rhizopus niveus P-Mannosidase 0-D-Man-0 Rhizopus niveus P-Mannosidase P-D-Man-0 Rhizopus niveus P-Mannosidase P-D-Man-0 Snail 0-Mannosidase P-D-Man-0 Snail P-D-Man-0 P-Mannosidase Snail P-Mannosidase P-D-Man-0 Snail 0-Mannosidase P-D-Man-0 Snail 0-Mannosidase 0-D-Man-0 Snail P-Mannosidase P-D-Man-0 Snail
Entry Linkage
____
Table 3 (continued)
~~
Acceptor
206
8
76
45
P-D-Man-OMe P-D-Man-OEt P-D-Man-O-CH(CH3)l
P-D-Man-O-(CH2)20H P-D-Man-O-(CH2)40H P-D-Man-O-(CH&OH
MeOH EtOH HO-CH(CH3)2 HO-(CH2)3CH3 HO-(CH2)20H HO-(CH2)40H HO-(CH2)60H
PpNPMan PpNPMan PpNPMan PpNPMan PpNPMan PpNPMan PpNPMan
P-D-M~~-O-(CH~)~CH~
P-D-Man-OBn
P-D-Man(1-4)- HOBn D-Man
36
19
9
15
61
15
252
252
252
252
252
252
252
70
70
4
P-D-Man-O-(CH2)20H
70
1
P-D-Man(1-4)D-Man
P-D-Glc-Ser-(N-Aloc)-OMe P-D-Man-O-(CH2)3CH3
Ser-(N-A1oc)-OMe
Reference
213
Yield (%)
P-~-GlcNAc-O-5-phenyl-l-pentyl 33
Product
b-D-Man( 1-4)- HO-(CH2)3CH3 D-Man
Cellobiose
PpNPGlcNAc 5-Phenyl-1-pentanol
Donor
29 1
290
289
288
287
286
285
284
283
282
28 1
280
279
278
P-Mannosidase Snail 0-Mannosidase Snail P-Mannosidase Snail P-Mannosidase Snail P-Mannosidase Snail P-Mannosidase Snail P-Mannosidase Snail P-Mannosidase Snail P-Mannosidase Snail P-Mannosidase Snail P-Galactosidase Asperyillus oryzae (Impurity?) P-Galactosidase Aspergillus oryzae (Impurity?) P-Mannosidase Helix pomatia P-Xylosidase Aspergillus niger MeOH
HO-(CH2)60H
Man
XYh
0-D-Man-0-Thr-( N-Boc)
Thr-( N-Boc)
Man
253, 254
235
8
84
235
14
64 2
PpNPMan
P-D-Man-O-Ser-(N-Boc)
64 15
PpNPMan
Ser-(N-Boc)
64
1
PpNPMan
Man
64
5
PpNPMan
64
64
20
PpNPMan
3
64
50
PpNPMan
P-D-Man-0-cyclohexanol
64
75
PpNPMan
PpNPMan
64
65
PpNPMan
CH(CH3)(CH2)2CH3 Cyclohexanol
252
26
PpNPMan
UI
w
00
836
29 Glycosidase-Catalysed Oligosaccharide Synthesis
s N
W-
vr
N
00 N
-9u,z? u
0
v
39
U
5
u,
h
g u 0 z
v
-x"
N
m N N
W
m N
3N
vr
m
N
W
r-
00
N
N
N
m
Q\
m
m
N m
0
O m
-
X
29.6 Recent Developments and New Directions
z N
3 N
m
m"
m
N
N
N
m
m
m
m m
N
m m
N
m m N
Q
z
N
d
?
U
N
m 0
m m 0
Bm
omm
\o 0
m
r-
4
00
4
837
838
29 Glycosidase-Catalysed Oligosaccharide Synthesis
References E. Bourquelot, Ann. Chim., 1913, 29, 145. G. M. Watt, P. A. S. Lowden, and S. L. Flitsch, Curr. Opin. Struct. Biol., 1997, 7, 652-660. D. H. G. Crout and G. Vic, Curr. Opin. Struct. Biol., 1998, 2, 98-111. V. Kren and J. Thiern, Chem. SOC.Rev., 1997,26,463-473. C. H. Wong, R. L. Halcomb, Y. Ichikawa, and T. Kajimoto, Angew. Chem. Znt. Ed. Engl., 1995,34, 521-546. 6. J. Thiem, FEMS Microbiol. Rev., 1995, 16, 193-211. 7. K. G. I. Nilsson, Tibtech, 1988, 6, 256-264. 8. B. Henrissat and G. Davies, Curr. Opin. Struct. Biol., 1997, 7, 637-644. 9. D. E. Koshland, Biol. Reu., 1953,28, 416-436. 10. M. L. Sinnott, Chem. Rev., 1990, 90, 1171-1202. 11. J. D. McCarter and S. G. Withers, Curr. Opin. Struct. Biol., 1994, 4 , 885-892. 12. G . Davies and B. Henrissat, Structure, 1995, 3, 853-859. 13. G. Davies, M. L. Sinnott, and S. G. Withers, in Comprehensive Biological Catalysis, (Ed. M. L. Sinnott), Academic Press, London, Vol. I , 1997, pp. 119-208. 14. T. Usui, H. Matsui, and K. Isobe, Curbohydr. R e x , 1990, 203, 65-77. 15. K. Ajisaka, I. Matsuo, M. Isornura, H. Fujirnoto, M. Shirakabe, and M. Okawa, Curbohydr. Rex, 1995, 270, 123-130. 16. S. Suwasono and R. A. Rastall, Biotechnol. Lett., 1996, 18, 851-856. 17. G. Vic and D. H. G. Crout, Carbohydr. Rex, 1995,279, 315-319. 18. S. Nishimura and K. Yamada, J. Am. Chem. Soc., 1997, 119, 10555-10556. 19. S. Kobayashi, T. Kiyosada, and S. Shoda, J. Am. Chem. Soc., 1996, 118, 13113-13114. 20. P. Finch and J. H. Yoon, Carbohydr. Rex, 1997, 303, 339-345. 21. K. Ajisaka, H. Nishida, and H. Fujirnoto, Biotechnol. Lett., 1987, 9, 387-392. 22. G. F. Herrmann, Y. Ichikdwa, C. Wandrey, F. C. A. Gaeta, J. C. Paulson, and C.-H. Wong, Tetrahedron Lett., 1993, 34, 3091-3094. 23. Y. Ito and J. C. Paulson, J. Am. Chem. Soc., 1993,115, 7862-7863. 24. V. Kren and J. Thiem, Angew. Chem. Znt. Ed. Engl., 1995,34, 893-895. 25. T. Usui, S. Kubota, and H. Ohi, Curbohydr. R e x , 1993, 244, 315-323. 26. K. G. I. Nilsson, Carbohydr. R e x , 1987, 167, 95-103. 27. D. H. G. Crout, S. Singh, B. E. P. Swoboda, P. Critchley, and W. T. Gibson, J. Chem. Soc., Chem. Commun., 1992, 704-705. 28. L. F. Mackenzie, Q. P. Wang, R. A. J. Warren, and S. G. Withers, J. Am. Chem. Soc., 1998, 120, 5583-5584. 29. C. Malet and A. Planas, FEBS Lett., 1998, 440, 208-212. 30. K. Takegawa, S. Yamaguchi, A. Kondo, I. Kato, and S. Iwahara, Biochem. Znt., 1991, 25, 829-835. 31 K. Takegawa, S. Yamaguchi, A. Kondo, H. Iwamoto, M. Nakoshi, I. Kato, and S. Iwahara, Biochem. Znt., 1991,24, 849--855. 32 K. Takegawa, M. Tabuchi, S. Yamaguchi, A. Kondo, I. Kato, and S. Iwahara, J. Biol. Chem., 1995,270, 3094-3099. 33 J. Q. Fan, M. S. Quesenberry, K. Takegawa, S. Iwahara, A. Kondo, I. Kato, and Y. C. Lee, J. Biol. Chem., 1995,270, 17730-17735. 34 K. Yarnamoto and K. Takegawa, Trends Glycoscience Glycotechnol., 1997, 9, 339-354. 35 L. X. Wang, J. Q. Fan, and Y. C. Lee, Tetrahedron Lett., 1996, 37, 1975-1978. 36 J. Q. Fan, K. Takegawa, S. Iwahara, A. Kondo, I. Kato, C. Abeygunawardana, and Y. C. Lee, J. Biol. Chem., 1995,270, 17723-17729. 37 H. Nakano, M. Moriwaki, T. Washino, and S. Kitahata, Biosci. Biotechnol. Biochem., 1994, 58, 1435-1438. 38 H. Nakano, K.-I. Hamayasu, K. Fujita, K. Hara, M. Ohi, H. Yoshizumi, and S. Kitahata, Biosci. Biotechnol. Biochem., 1995, 59, 1732-1736. 39 H. Hashimoto, C . Katayama, M. Goto, T. Okinaga, and S. Kitahata, Biosci. Biotechnol. Biochem., 1995,59, 179-183.
I. 2. 3. 4. 5.
References
839
40. K. Ajisaka, H. Fujimoto, and H. Nishida, Curbohydr. Rex, 1988, 180, 35-42. 41. K. Koizumi, T. Tanimoto, Y. Okada, K. Ham, K. Fujita, H. Hashimoto, and S. Kitahata, Curbohydr. Rex, 1995, 278, 129-142. 42. K. Koizumi, T. Tanimoto, Y. Kubota, and S. Kitahata, Curbohydr. Rex, 1998,305, 393-400. 43. K. G. I. Nilsson and A. Fernandez-Mayoralas, Biotechnol. Lett., 1991, 13, 715-720. 44. K. G. I. Nilsson, Carbohydr. Rex, 1988, 180, 53-59. 45. K. G. I. Nilsson, Curbohydr. Rex, 1989, 188, 9-17. 46. A. Naundorf, M. Caussette, and K. Ajisaka, Biosci. Biotechnol. Biochem., 1998, 62, 13131317. 47. I. Matsuo, H. Fujimoto, M. Isomura, and K. Ajisaka, Bioorg. Med. Chem. Lett., 1997, 7, 255-258. 48. G. Vic, C. H. Tran, M. Scigelova, and D. H. G. Crout, J. Chem. Soc., Chem. Commun., 1997, 169- 170. 49. K. G. I. Nilsson, Tetrahedron Lett., 1997, 38, 4527-4527. 50. G. Vic, M. Scigelova, J. J. Hastings, 0. W. Howarth, and D. H. G. Crout, J. Chem. Soc., Chem. Commun., 1996, 1473-1474. 51. A. Millqvist-Fureby, D. A. MacManus, S. Davies, and E. N. Vulfson, Biotechnol. Bioeng., 1998, 60, 197-203. 52. S. Kitahata, H. Ishikawa, T. Miyata, and 0. Tanaka, Agric. Biol. Chem., 1989, 53, 29292934. 53. K. Hara, K. Fujita, N. Kuwahara, T. Tanimoto, H. Hashimoto, K. Koizumi, and S. Kitahata, Biosci. Biotechnol. Biochem., 1994, 58, 652-659. 54. S. Kitahata, K. Hara, K. Fujita, N. Kuwahara, and K. Koizumi, Biosci. Biotechnol. Biochem., 1992,56, 1518-1519. 55. K. Ajisaka and H. Fujimoto, Curbohydr. Rex, 1989, 185, 139-146. 56. K. G. I. Nilsson, Curbohydr. Rex, 1990, 204, 79-83. 57. K. Dax, M. Ebner, R. Peinsipp, and A. E. Stutz, Tetruhedron Lett., 1997, 38, 225-226. 58. T. Kasumi, C. F. Brewer, E. T. Reese, and E. J. Hehre, Curbohydr. Rex, 1986, 146, 39-49. 59. H. Fujimoto and K. Ajisaka, Biotechnol. Lett., 1988, 10, 107-112. 60. H. Fujimoto, H. Nishida, and K. Ajisaka, Agric. Biol. Chem., 1988, 52, 1345-1351. 61. S. V. Lobov, R. Kasai, K. Ohtani, 0. Tanaka, and K. Yamasaki, Agric. Biol. Chem., 1991, 55,2959-2965. 62. K. Ajisaka and H. Fujimoto, Curbohydr. Res., 1990, 199, 227-234. 63. B. Sauerbrei and J. Thiem, Tetrahedron Lett., 1992, 33, 201-204. 64. N. Taubken, B. Sauerbrei, and J. Thiem, J. Curbohydr. Chem., 1993,12, 651-667. 65. V. Laroute and R. M. Willemot, Enzyme Microb. Technol., 1992, 14, 528-534. 66. V. Laroute and R.-M. Willemot, Biotechnol. Lett., 1989, 11, 249-254. 67. R. A. Rastall, N. H. Rees, R. Wait, M. W. Adlard, and C. Bucke, Enzyme Microb. Technol., 1992,14, 53-57. 68. S. Suwasono and R. A. Rastall, Biotechnol. Lett., 1998, 20, 15-17. 69. E. Johansson, L. Hedbys, and P.-0. Larsson, Biotechnol. Lett., 1986, 8, 421-424. 70. H. Fujimoto, M. Isomura, and K. Ajisaka, Biosci. Biotechnol. Biochem., 1997, 61, 164-165. 71. K. Hamayasu, K. Hara, K. Fujita, Y. Kondo, H. Hashimoto, T. Tanimoto, K. Koizumi, H. Nakano, and S. Kitahata, Biosci. Biotechnol. Biochem., 1997, 61, 825-829. 72. K. Hara, K. Fujita, H. Nakano, N. Kuwahara, T. Tanimoto, H. Hashimoto, K. Koizumi, and S. Kitahata, Biosci. Biotechnol. Biochem., 1994, 58, 60-63. 73. T. J. Bartlett, R. A. Rastall, N. H. Rees, M. W. Adlard, and C. Bucke, J. Chem. Technol. Biotechnol., 1992, 55, 73-78. 74. A. Vetere, S. Ferro, M. Bosco, P. Cescutti, and S. Paoletti, Eur. J. Biochem., 1997, 247, 10831090. 75. Y. Makimura, H. Ishida, A. Kondo, A. Hasegawa, and M. Kiso, J. Curbohydr. Chem., 1998, 17, 975-979. 76. J. Thiem and B. Sauerbrei, Angew. Chem. Int. Ed. Engl., 1991, 30, 1503-1505. 77. A. Vetere and S. Paoletti, FEBS Lett., 1996, 399, 203-206. 78. K. Ajisaka, H. Fujimoto, and M. Isomura, Carbohydr. Rex, 1994, 259, 103-115. 79. S. C. T. Svensson and J. Thiem, Curbohydr. Rex, 1990, 200, 391-402.
840
29 Glycosiduse-Cutalysed Oligosucckuride Synthesis
K. Ajisaka and M. Shirakabe, Carbohydr. Rex, 1992, 224, 291-299. K. Ajisaka, H. Fujimoto, and M. Miyasato, Curbohydr. Rex, 1998, 309, 125-129. A. Vetere, C. Galateo, and S. Paoletti, Biochem. Biophys. Res. Commun., 1997,234, 358-361. K. G. I. Nilsson, H. F. Pan, and U. Larsson-Lorek, J. Curbohydr. Chem., 1997, 16, 459477. 84. K. G. I. Nilsson, A. Eliasson, and U. Larsson-Lorek, Biotechnol. Lett., 1995, 17, 717-722. 85. H. Prade, L. F. MacKenzie, and S. G. Withers, Carbohydr. Rex, 1998,305, 371-381. 86. K. Fujita, N. Kuwahara, T. Tanimoto. K. Koizumi, M. Iizuka, N. Minamiura, K. Furuichi, and S. Kitahata, Biosci. Biotechnol. Biochem., 1994, 58, 239-243. 87. M. Muramatsu and T. Nakakuki, Biosci. Biotechnol. Biochem., 1995, 59, 208-212. 88. M. Kurakake, T. Onoue, and T. Komaki, Appl. Microbiol. Biotechnol., 1996, 45, 236-239. 89. A. J. J. Straathof, A. P. G. Kieboom, and H. van Bekkum, Carbohydr. Rex, 1986, 146, 154159. 90. H. Ishikawa, S. Kitahata, K. Ohtani, and 0. Tanaka, Chem. Pharm. Bull., 1991, 39, 20432045. 91. H. Ishikawa, S. Kitahata, K. Ohtani, C. Ikuhara, and 0. Tanaka, Agric. Biol. Chem., 1990, 54, 3137-3143. 92. T. Usui, S. Morimoto, Y. Hayakawa, M. Kawaguchi, T. Murata, Y. Matahira, and Y. Nishida, Curbohydr. Res., 1996,285, 29-39. 93. V. Chiffoleau-Giraud, P. Spangenberg, and C. Rabiller, Tetrahedron: Asymmetry, 1997, 8, 20 17-2023. 94. S. Yanahira, T. Kobayashi, T. Suguri, M. Nakakoshi, S. Miura, H. Ishikawa, and I. Nakajima, Biosci. Biotechnol. Biochem., 1995, 59, 1021-1026. 95. E. Montero, J. Alonso, F. J. Canada, A. Fernandez-Mayorales, and M. Martin-Loma, Cavbohydr. Res., 1998,305, 383-391. 96. J. J. Aragon, F. J. Canada, A. Fernandez-Mayoralas, R. Lopez, M. Martinlomas, and D. Villanueva, Curbohydr. Rex, 1996,290, 209-216. 97. G. C. Look and C. H. Wona. Tetrahedron Lett., 1992, ?, 4253-4256. 98. K. Suzuki, H. Fujimoto, Y. ko, T. Sasaki, and K. Ajisaka, Tetrahedron Lett., 1997,38, 12111214. 99. U. Gambert and J. Thiem, Carbohydr. Rex, 1997,299, 85-89. 100. T. Murata, T. Itoh, Y. Hayakawa, and T. Usui, J. Biochem., 1996, 120, 851-855. 101. U. Gambert, R. G. Lio, E. Farkas, J. Thiem, V. V. Bencomo, and A. Liptak, Bioorg. Med. Chem., 1997, 5, 1285-1291. 102. K. G. I. Nilsson, Biotechnol. Lett., 1996, 18, 791-794. 103. T. Yasukochi, K. Fukase, Y. Suda, K. Takagaki, M. Endo, and S. Kusumoto, Bull. Chem. Soc. Jpn., 1997, 70, 2719-2725. 104. V. Dumortier, J. Montreuil, and S. Bouquelet, Carbohydr. Res., 1990, 201, 115-123. 105. S. Yanahira, Y. Yabe, M. Nakakoshi, S. Miura, N. Matsubara, and I. Nakajima, Biosci. Biotechnol. Biochem., 1998, 62, 1791-1794. 106. S. Attal, S. Bay, and D. Cantacuzene, Tetrahedron, 1992, 48, 9251-9260. 107. S. Menzler, H. Seker, M. Gschrey, and M. Wiessler, Biotechnol. Lett., 1997, 19, 269-272. 108. L. Hedbys, E. Johansson, K. Mosbach, P.-0. Larsson, A. Gunnarson, S. Swensson, and H. Lohn, GlycoconjugateJ., 1989, 6, 161-168. 109. T. Murata, S. Akimoto, M. Horimoto, and T. Usui, Biosci. Biotechnol. Biochem., 1997, 61, 1118-1120. 110. K. Fukase, T. Yasukochi, Y. Suda, M. Yoshida, and S. Kusumoto, Tetrahedron Lett., 1996, 37. 6763-6766. 111. G. Vie, J. J. Hastings, 0. W. Howarth, and D. H. Crout, Tetrahedron: Asymmetry, 1996, 7, 709-720. 112. L. Hedbys, E. Johansson, K. Mosbach, and P.-0. Larsson, Carbohydr. Res., 1989, 186, 217223. 113. J. H. Yoon and K. Ajisaka, Carbohydr. Res., 1996,292, 153-163. 114. T. Usui, K. Ogawa, H. Nagai, and H. Matsui, Anal. Biochem., 1992,202, 61-67. 115. K. Koizumi, T. Tanimoto, K. Fujita, K. Hara, N. Kuwahara, and S. Kitahata, Carbohydr. Res., 1993,238, 75-91. 80. 81. 82. 83.
References
841
116. S. Takayama, M. Shimazaki, L. Qiao, and C. H. Wong, Bioorg. Med. Chem. Lett., 1996, 6 , 1 123-1 126. 117. S. Singh, M. Scigelova, G. Vic, and D. Crout, J. Chem. SOC.,Perkin Trans. I , 1996,21, 19211926. 118. E. Bonnin and J. F. Thibault, Enzyme Microb. Techno/., 1996, 19, 99-106. 119. H. Fujimoto, M. Isomura, T. Miyazaki, I. Matsuo, R. Walton, T. Sakakibara, and K. Ajisaka, Glycoconjugate J., 1997, 14, 75-80. 120. T. Kimura, S. Takayama, H. M. Huang, and C. H. Wong, Angew. Chem. Znt. Ed. Engl., 1996, 35, 2348-2350. 121. W. S. Hays, D. J. VanderJagt, B. Bose, A. S. Serianni, and R. H. Clew, J. Biol. Chem., 1998, 273, 34941-34948. 122. J. W. Fang, W. H. Xie, J. Li, and P. G. Wang, Tetrahedron Lett., 1998, 39, 919-922. 123. J. Li and P. G. Wang, Tetrahedron Lett., 1997, 38, 7967-7970. 124. A. Vetere and S. Paoletti, Biochem. Biophys. Res. Commun., 1996, 219, 6-13. 125. G. F. Herrmann, U. Kragl, and C. Wandrey, Angew. Chem. Znt. Ed. Engl., 1993, 32, 13421343. 126. K. Sakai, R. Katsumi, H. Ohi, T. Usui, and Y. Ishido, J. Carbohydr. Chem., 1992, 11, 553565. 127. K. Suyama, S. Adachi, T. Toba, T. Sohma, C.-J. Hwang, and T. Itoh, Agric. Biol. Chem., 1986, 50, 2069-2075. 128. L. Hedbys, P.-0. Larsson, and K. Mosbach, Biochem. Biophys. Res. Commun., 1984, 123, 815. 129. S. Singh, M. Scigelova, P. Critchley, and D. H. G. Crout, Carbohydr. Rex, 1998, 305, 363370. 130. D. H. G. Crout, 0. W. Howarth, S. Singh, B. E. P. Swoboda, P. Critchley, and W. T. Gibson, J. Chem. SOC.,Chem. Commun., 1991, 1550-1551. 131. S. Singh, D. H. G. Crout, and J. Packwood, Carbohydr. Rex, 1995,279, 321-325. 132. C. Ravet, D. Thomas, and M. D. Legoy, Biotechnol. Bioeng., 1993, 42, 303-308. 133. K. Ajisaka, H. Nishida, and H. Fujimoto, Biotechnol. Lett., 1987, 9, 243-248. 134. T. Tanaka and S. Oi, Agric. Biol. Chem., 1985,49, 1267-1273. 135. K. Kuriyama, K. Tsuchiya, and T. Murui, Biosci. Biotechnol. Biochem., 1995,59, 1142-1143. 136. T. Murata, A. Tashiro, T. Itoh, and T. Usui, Biochim. Biophys. Acta G., 1997, 1335, 326-334. 137. Y. Matahira, A. Tashiro, T. Sato, H. Kawagishi, and T. Usui, Glycoconjugate J., 1995, 12, 664-671. 138. T. Murata, T. Itoh, and T. Usui, Glycoconjugate J., 1998, 15, 575-582. 139. S. Singh, J. Packwood, andD. H. G. Crout, J. Chem. SOC.,Chem. Commun., 1994,2227-2228. 140. S. Singh, J. Packwood, C. J. Samuel, P. Critchley, and D. H. G. Crout, Carbohydr. Res., 1995, 279, 293-305. 141. S. Singh, R. Gallagher, P. J. Derrick, and D. H. G. Crout, Tetrahedron: Asymmetry, 1995, 6 , 2803-28 10. 142. F. Nanjo, K. Sakai, M. Ishikawa, K. Isobe, and T. Usui, Agric. Biol. Chem., 1989, 53, 21892195. 143. F. Nanjo, M. Ishikawa, R. Katsumi, and K. Sakai, Agric. Biol. Chem., 1990, 54, 899-906. 144. S. Kobayashi, T. Kiyosada, and S. Shoda, Tetrahedron Lett., 1997, 38, 2111-2112. 145. S. Singh, J. Packwood, C. J. Samuel, P. Critchley, and D. H. G. Crout, Carbohydr. Rex, 1995, 279, 293-305. 146. E. Rajnochova, J. Dvorakova, Z. Hunkova, and V. Kren, Biotechnol. Lett., 1997, 19, 869872. 147. S. Kyosaka, S. Murata, Y. Tsuda, and M. Tanaka, Chem. Pharm. Bull., 1986, 34, 51405143. 148. T. Usui, M. Suzuki, T. Sato, H. Kawagishi, K. Adachi, and H. Sano, GlycoconjugateJ . , 1994, 11, 105-110. 149. S. Singh, M. Scigelova, and D. H. G. Crout, J. Chem. SOC.,Chem. Commun., 1996,993-994. 150. H. Kizawa, H. Shinoyama, and T. Yasui, Agric. Biol. Chem., 1991, 55, 671-678. 151. M. Kurakake, S. Osada, and T. Komaki, Biosci. Biotechnol. Biochem., 1997, 61, 2010-2014. 152. A. Vetere, M. Bosco, and S. Paoletti, Carbohydr. Rex, 1998, 311, 79-83.
842
29 Glycosiduse-Cutulysed Oligosucchuvide Synthesis
153. R. M. Bardales and V. P. Bhavanandan, J. Biol. Chem., 1989,264, 19893-19897. 154. T. Usui, T. Murata, Y. Yabuuchi, and K. Ogawa, Carbohydr. Res., 1993,250, 57-66. 155. R. Uchida, A. Nasu, K. Tobe, T. Oguma, and N. Yamaji, Carbohydr. Res., 1996,287, 271274. 156. K. Ogawa, 0. Uejima, T. Nakakuki, T. Usui, and K. Kainuma, Agric. Biol. Chem., 1990, 54, 581-586. 157. N. Payre, S. Cottaz, and H. Driguez, Angew. Chem. Znt. Ed. Engl., 1995, 34, 1239-1241. 158. T. Usui and T. Murata, J. Biochem., 1988, 103, 969-972. 159. K. Ogawa, T. Murata, and T. Usui, Curbohydr. Rex, 1991, 212, 289-294. 160. K. Ogawa, T. Nakakuki, andT. Usui, J. Curbohydr. Chem., 1991,10, 877-886. 161. Y. Yoshimura, S. Kitahata, and S. Okada, Agric. Biol. Chem., 1988, 52, 1655-1659. 162. S. Hizukuri, S. Kawano, J.-I. Abe, K. Koizumi, and T. Tanimoto, Biotechnol. Appl. Biochem., 1989,11, 60-73. 163. J.-I. Abe, N. Mizowaki, S. Hizukuri, K. Koizumi, and T. Utamura, Curbohydr. Rex, 1986, 154, 81-92. 164. T. Kuriki, M. Yanase, H. Takata, Y. Takesada, T. Imanaka, and S. Okada, Appl. Enoiron. Microbiol., 1993, 59, 953-959. 165. N. Shiomi and J. Yamada, Agric. Biol. Chem., 1988,52, 1861-1862. 166. S. I. Shoda, K. Obata, 0. Karthaus, and S. Kobyashi, J. Chem. Soc., Chem. Commun., 1993, 18, 1402-1404. 167. 0. Karthus, S. Shoda, H. Takano, K. Obata, and S. Kobayashi, J. Chem. Soc., Perkin Trans. I , 1994, 1851-1857. 168. H. Fujimoto, H. Nakano, M. Isomura, S. Kitahata, and K. Ajisaka, Biosci. Biotechnol. Biochem., 1997, 61, 1258-1261. 169. H. Nakano, S. Kitahata, H. Kinugasa, Y. Watanabe, H. Fujimoto, K. Ajisaka, and S. Takenishi, Agric. Biol. Chem., 1991, 55, 2075-2082. 170. P. W. Bezukladnikov and L. A. Elyakova, Carbohydr. Res., 1988, 184, 268-270. 171. T. Kawaguchi, K. Sugimoto, H. Hayashi, and M. Arai, Biosci. Biotechnol. Biochem., 1996, 60, 3444346. 172. J. L. Viladot, B. Stone, H. Driguez, and A. Planas, Curbohydr. Res., 1998, 311, 95-99. 173. J. L. Viladot, V. Moreau, A. Planas, and H. Driguez, J. Chem. Soc., Perkin Trans. 1, 1997, 2383-2387. 174. S. Armand, S. Drouillard, M. Schulein, B. Henrissat, and H. Driguez, J. Biol. Chem., 1997, 272, 2709-2713. 175. S. Kobayashi, T. Kawasaki, K. Obata, and S. I. Shoda, Chem. Lett., 1993, 685-686. 176. S. Shoda, T. Kawasaki, K. Obata, and S. Kobayashi, Carbohydr. Res., 1993,249, 127-137. 177. S. Kobayashi, K. Kashiwa, T. Kawasaki, and S. Shoda, J. Am. Chem. Sac., 1991,113, 30793084. 178. J. H. Lee, R. M. Brown, S. Kuga, S. Shoda, and S. Kobayashi, Proc. Nat. Acud. Sci. USA, 1994, 91, 7425-7429. 179. V. Moreau and H. Driguez, J. Chem. Soc., Perkin Trans. I, 1996, 525-527. 180. M. Fujita, S. Shoda, and S. Kobayashi, J. Am. Chem. Soc., 1998,120,6411-6412. 181. H. Ashida, Y. Tsuji, K. Yamamoto, H. Kumagai, and T. Tochikura, Arch. Biochem. Biophys., 1993,305, 559-562. 182. Y. T. Li, B. Z. Carter, B. N. N. Rao, H. Schweingruber, and S. C. Li, J. Biol. Chem., 1991, 266, 10723--10726. 183. K. Yamada, E. Fujita, and S. I. Nishimura, Curbohydr. Rex, 1998, 305, 443-461. 184. K. Akiyama, K. Kawazu, and A. Kobayashi, Curbohydr. Rex, 1995, 279, 151-160. 185. J. Q. Fan, L. H. Huynh, B. B. Reinhold, V. N. Reinhold, K. Takegawa, S. Iwahara, A. Kondo, I. Kato, and Y. C. Lee, Glycoconjugute J., 1996, 13, 643-652. 186. H. Matsui, H. Kawagishi, and T. Usui, Biochim. Biophys. Actu, 1990, 1035, 90-96. 187. Y. Matahira, K. Ohno, M. Kawaguchi, H. Kawagishi, and T. Usui, J. Curbohydr. Chem., 1995,14,213-225. 188. K. Takegawa, K. Fujita, J. Q. Fan, M. Tabuchi, N. Tanaka, A. Kondo, H. Iwamoto, I. Kato, Y. C. Lee, and S. Iwahara, Anal. Biochem., 1998,257, 218-223. 189. T. Rand-Meir, F. W. Dahlquist, and M. A. Raftery, Biochemistry, 1969, 8, 4206-4214.
References
843
190. I. L. Deras, K. Takegawa, A. Kondo, I. Kato, and Y. C. Lee, Bioorg. Med. Chem. Lett., 1998, 8,1763-1766. 191. H. Saitoh, K. Takagaki, M. Majima, T. Nakamura, A. Matsuki, M. Kasai, H. Narita, and M. Endo, J. Biol. Chem., 1995,270,3741-3747. 192. T. Usui, Y. Hayashi, F. Nanjo, K. Sakai, and Y. Ishido, Biochim. Biophys. Acta, 1987,923, 302-309. 193. K. Haneda, T. Inazu, M. Mizuno, R. Iguchi, K. Yamamoto, H. Kumagai, S. Aimoto, H. Suzuki, and T. Noda, Bioory. Med. Chem. Lett., 1998,8, 1303-1306. 194. L.X. Wang, M. Tang, T. Suzuki, K. Kitajima, Y. Inoue, S. Inoue, J. Q. Fan, andY. C. Lee, J. Am. Chem. Soc., 1997,119,11137-11146. 195. T. Usui, Y. Hayashi, F. Nanjo, and Y. Ishido, Biochim. Biophys. Acta, 1988,953,179-184. 196. T. Usui and H. Matsui, Agric. Biol. Chem., 1989,53, 383-388. 197. K. Haneda, T. Inazu, K. Yamamoto, H. Kumagai, Y. Nakahara, and A.Kobata, Carbohydr. Res., 1996,292,61-70. 198. K. J. Yamamoto, S. Kadowaki, J. Watdnabe, and H. Kumagai, Biochem. Biophys. Res Commun., 1994,203,244-252. 199. K. Yamamoto, K. Fujimori, K. Haneda, M. Mizuno, T. Inazu, and H. Kumagai, Carbohydr. Res., 1998,305,415-422. 200. M. Mizuno, K. Haneda, R. Iguchi, I. Muramoto, T. Kawakami, S. Aimoto, K. Yamamoto, andT. Inazu, J. Am. Chem. SOC.,1999,121,284-290. 201. S.Kobayashi, X.Wen, and S.-I. Shoda, Macromolecules, 1996,29,2698-2700. 202. M. Scigelova, V. Kren, and K. G. I. Nilsson, Biotechnol. Lett., 1994,16, 683-688. 203. E. V. Eneyskaya, A. M. Golubev, A. M. Kachurin, A. N. Savel’ev, and K. N. Neustroev, Carbohydr. Rex, 1998,305,83-91. 204. G. Vic, J. J. Hastings, and D. H. G. Crout, Tetrahedron: Asymmetry, 1996, 7, 1973-1984. 205. M. Scigelova, P. Sedmera, V. Havlicek, V. Prikrylova, and V. Kren, J. Carbohydr. Chem., 1998,17,981-986. 206. D. Cantacuzene, S. Attal, and S. Bay, Biomed. Biochim. Acta, 1991,50, S231LS236. 207. E. Johansson, L.Hedbys, and P. 0. Larsson, Enzyme Microb. Technol., 1991,13,781-787. 208. K. G. I. Nilsson, G. Ljunger, and P. M. Melin, Biotechnol. Lett., 1997,19,889-892. 209. V.Laroute and R.-M. Willemot, Biotechnol. Lett., 1992,14, 169-174. 210. M.-P. Bousquet, R.-M. Willemot, P. Monsan, and E. Boures, Enzyme Microb. Technol., 1998, 23, 83-90. 21 1. M.-P. Bousquet, R.-M. Willemot, P. Monsan, and E. Boures, Appl. Microbiol. Biotechnol., 1998,50, 167-173. 212. C.Kieburg, T.K . Lindhorst, andV. Kren, J. Carbohydr. Chem., 1998,17, 1239-1247. 213. T. Mori and Y. Okahata, Tetrahedron Lett., 1997,38,1971-1974. 214. A. Ismail and M. Ghoul, Biotechnol. Lett., 1996,18, 1199-1204. 215. A. J. J. Straathof, J. P. Vrijenhoef, E. P. A. T. Sprangers, H. van Bekkum, and A. P. G. Kieboom, J. Carbohydr. Chem., 1988,7, 223-238. 216. M. Kojima, T.Seto, Y. Kyotani, H. Ogawa, S. Kitazawa, K. Mori, S. Maruo, T. Ohgi, and Y. Ezure, Biosci. Biotechnol. Biochem., 1996,60, 694-696. 217. N. Asdno, A. Kato, H. Kim, K. Matsui, R. C. Griffiths, M. G. Jones, A. A. Watson, and R. J. Nash, Carbohydr. Res., 1997,304, 173-178. 218. T. Wiemann, N. Taubken, U. Zehavi, and J. Thiem, Carbohydr. R e x , 1994,257,CI-C6. 219. D.H.G. Crout, D. A. Macmanus, and P. Critchley, J. Chem. Soc., Perkin Trans. 1, 1990, 1865-1 868. 220. Y.Ooi, N.Mitsuo, and T. Satoh, Chem. Pharm. Bull., 1985,33,5547-5550. 221. H.-J. Gais, A. Zeissler, and P. Maidonis, Tetrahedron Lett., 1988,29, 5743-5744. 222. Y.Ooi, T. Hashimoto, N. Mitsuo, and T. Satoh, Chem. Phurm. Bull., 1985,33,1808-1814. 223. Y.Ooi, T. Hashimoto, N. Mitsuo, and T. Satoh, Tetrahedron Lett., 1984,25,2241-2244. 224. S. Matsumura, H. Yamazaki, and K. Toshima, Biotechnol. Lett., 1997,19,583-586. 225. D. E. Stevenson, R. A. Stanley, and R. H. Furneaux, Biotechnol. Bioeng., 1993,42, 657-666. 226. S. Matsurnura, H.Kubokawa, and S. Yoshikawa, Chem. Lett., 1991,945-948. 227. Y. Fortun and B. Colas, Biotechnol. Lett., 1991,13,863-866. 228. Y.Okahata and T. Mori, J. Chem. Soc., Perkin Trans. 1, 1996,2861-2866.
844 229. 230. 231. 232. 233. 234. 235. 236. 237. 238. 239. 240. 241. 242. 243. 244. 245. 246. 247. 248. 249. 250. 251. 252. 253. 254. 255. 256.
29 Glycosiduse-Cutulysed Oligosucchuride Synthesis T. Mori, S. Fujita, and Y. Okahata, Chem. Lett., 1997, 73-74. T. Mori, S. Fujita, and Y. Okahata, Carbohydr. Rex, 1997, 298, 65-73. Y. Okahata and T. Mori, J. Mol. Cat. B Enzym., 1998, 5, 119-123. T. MoriandY. Okahata, J. Chem. Soc., Chem. Commun., 1998, 2215-2216. R. R. Gibson, R. P. Dickinson, and G. J. Boons, J. Chem. Soc., Perkin Trans. I , 1997, 33573360. S. Leparoux, Y. Fortun, and B. Colas, Biotechnol. Lett., 1994, 16, 677-682. K. G. I. Nilsson and M. Scigelova, Biotechnol. Lett., 1994, 16, 671-676. D. E. Stevenson and R. H. Furneaux, Carbohydr. Rex, 1996,284, 279-283. S. Leparoux, M. Padrines, Y. Fortun, and B. Colas, Biotechnol. Lett., 1996, 18, 135-138. N. J. Turner and M. C. Webberley, J. Chem. Soc., Chem. Commun., 1991, 1349-1350. A. Baker, N. J. Turner, and M. C. Webberley, Tetrahedron: Assymetry, 1994, 5, 2517-2522. G. Vic and D. Thomas, Tetrahedron Lett., 1992, 33, 4567-4570. P. Christakopoulos, M. K. Bhat, D. Kekos, and B. J. Macris, Znt. J. Biol. Macromol., 1994, 16, 331-334. M. Gelo-Pujic, E. Guibe-Jampel, and A. Loupy, Tetrahedron, 1997, 53, 17247-17252. A. Trincone and E. Pagnotta, Biotechnol. Lett., 1995, 17, 45-48. H. Shinoyama, K. Takel, A. Ando, T. Fujii, M. Sasaki, Y. Doi, and T. Yasui, Agric. Biol. Chem., 1991, 55, 1679-1681. A. Millqvist-Fureby, 1. S. Gill, and E. N. Vulfson, Biotechnol. Bioeng., 1998, 60, 190-196. Z. Gunata, M. J. Vallier, J. C. Sapis, R. Baumes, and C. Bayonove, Enzyme Microb. Technol., 1994, 16, 1055-1058. G. Vie, D. Thomas, and D. H. G. Crout, Enzyme Microb. Technol., 1997,20, 597-603. D. K. Watt, H. Ono, and K. Hayashi, Biochim. Biophys., 1998, 1385, 78-88. G. Vie and D. H. G. Crout, Tetrahedron: Asymmetry, 1994, 5, 2513--2516. M. Gelo-Pujic, E. Guibe-Jampel, A. Loupy, and A. Trincone, J. Chem. Soc., Perkin Trans. 1, 1997, 1001-1002. G. Ljunger, P. Adlercreutz, and B. Mattiasson, Enzyme Microb. Technol., 1994, 16, 751-755. N. Taubken and J. Thiem, Synthesis ( C ) , 1992, 517-518. H. Shinoyama and T. Yasui, Agric. Biol. Chem., 1988, 52, 2375--2377. H. Shinoyama, Y. Kamiyama, and T. Yasui, Agric. Biol. Chem., 1988, 52, 2197-2202. P. Drouet, M. Zhang, and M. D. Legoy, Biotechnol. Bioeng., 1994, 43, 1075-1080. J. Sulistyo, Y. Kamiyama, H. Ito, and T. Yasui, Biosci. Biotechnol. Biochem., 1994, 58, 13111313.
Carbohydrates in Chemistry and Biology Editor Beat Ernst, Gerald W. Hart, Pierre Sinay copyright OWILEY-VCH Verlag GmbH, 2000
30 Production of Heterologous Oligosaccharides by Recombinant Bacteria (Recombinant Oligosaccharides) Roberto A . Geremia and Eric Samain
30.1 Introduction During recent years it has been firmly established that oligosaccharides, either free or conjugated, are involved in many biological recognition processes, such as those that occur during fertilization, embryogenesis, inflammation, symbiotic nitrogen fixation, and host pathogen adhesion [l]. These advances have led to increased interest in potential applications of oligosaccharides and derivatives in therapeutics and in the agricultural industry. Both chemical and enzymatic methods have been developed for production of relevant amounts of biologically active oligosaccharides. Chemical methods still need many protection and deprotection steps; as the number of steps increases with the size of the oligosaccharide, longer oligosaccharides are produced in lower yields. Enzymatic methods use purified glycosylhydrolases or glycosyltransferases to assemble the oligosaccharide. Glycosyltransferases (GTs), are enzymes that transfer a sugar from a donor to an acceptor. With Leloir GTs the sugar donor is a sugar-nucleotide (Figure 1). The most specific enzymatic methods use glycosyltransferases, consequently these methods suffer from the relatively poor availability of glycosyltransferases and from the need to regenerate in situ sugar-nucleotides. Taking into account that enzymatic methods basically mimic in vitro the natural pathways of oligosaccharide biosynthesis, a reasonable short-cut is the production of the oligosaccharide in a living recombinant organism (e.g. Escherichia coli). Unfortunately this task is not simple to accomplish, because of the characteristics of the biosynthetic machinery involved in the synthesis of the desired products. Production in E. coli of active eukaryotic GTs in suitable amounts is not always possible or requires considerable effort for reasons that are not well understood. This problem can be overcome by use of homologous enzymes from prokaryotic organisms. A second possible difficulty consists in the availability of sugar-nucleotides. Bacteria recycle these compounds, because they are necessary for the synthesis of their cell wall carbohydrate polymers (murein, lipopolysaccharide, capsular poly-
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30 Production of Heterologous Oligosaccharides by Recombinant Bacteria
GT
Figure 1. Reaction catalyzed by 'Leloir' glycosyltransferases.
saccharides, etc. ) [2]. Occasionally, e. g. with GDP-fucose, the production of the sugar donor is probably coupled with the corresponding polysaccharide biosynthetic system, which is, in turn, induced by particular physiological or environmental conditions. For these reasons it is difficult to estimate the actual availability of the sugar donor. The solution consists either in adapting the culture conditions or genetically modifying the bacteria for the production of the desired sugarnucleotide. Finally, the most difficult problem to address is that of the acceptor. The physiological acceptor for glycosyltransferases involved in oligosaccharide synthesis is a mono- or oligosaccharide, which can be linked to a protein, a lipid, or another carrier molecule. The acceptor requirements are well studied for eukaryotic GTs, but little is known for the bacterial variety. Production of the oligosaccharide coupled to proteins or lipids requires engineering of the complete glycosylation pathway into the bacteria, which is an enormous task. The use of free sugars in vivo as acceptors is not feasible, because they are rapidly catabolized, thus their intracellular concentration is virtually zero. One possibility is to use lipid-linked intermediates as acceptors but they are present in small amounts [ 3 ] ,limiting the amount of oligosaccharide produced. The availability of an acceptor in sufficient amounts is, therefore, the limiting step for the production of oligosaccharides in bacteria. If the desired oligosaccharide is produced, it might be necessary to add nonglycosidic substituents, requiring the expression of the appropriate modifying enzyme and the presence of donors for the modification. Despite these drawbacks, this system remains attractive because once the genes are cloned and conditions established, the production is performed in vivo by the bacteria, without all the problems found in chemical or enzymatic synthesis. E. coli is commonly used for the production of heterologous DNA or proteins, and is a very well known organism. Its metabolism can be manipulated by varying the culture conditions. One of the most exciting features for our purposes is that it can be grown at high or very high cell density (OD optical density 100-200, in contrast with 2-5 under normal conditions). This means the amount of biomass obtained in a single culture can be more than 50 g L-' (dry weight). An exceptional opportunity for synthesizing oligosaccharides in E. coli, is provided by the rhizobial chitin oligosaccharide synthase NodC. The putative acceptor or primer for this enzyme has not yet been identified, but is present in E. coli, which enabled the production of chitin oligosaccharides in vivo. By use of the methodology developed by us and described below, E. coli strains expressing nodC produce 1-2 g L-' chitin pentasaccharide [4].Modification with glycosidic or non-glycosidic
30.2 Concept and Methodology of Heteroloyous Oligosuccharide Production
841
groups was also achieved by co-expression of the proper genes, with production yields between 0.2 and 1 g L-’ [4-61.
30.2 Concept and Methodology of Heterologous (‘Recombinant’) Oligosaccharide Production in E. cob Sugar-nucleotides and glycosyltransferases are already present in E. coli, because it produces a series of cell-wall polysaccharides. The enzymes for the biosynthesis of sugar-nucleotides are cytoplasmic, whereas most GTs involved in the biosynthesis of outer polysaccharides are peripheral membrane proteins whose catalytic domain is thought to face the cytoplasm [7, 81. Therefore, to use the pool of sugar-nucleotides, the recombinant GT should be targeted at the cytoplasm. An advantage of expressing recombinant bacterial glycosyltransferase in E. coli is that cytoplasm is their natural environment, and that having transmembrane domains, insertion into membrane in the E. coli host possibly occurs by similar mechanisms. Unfortunately, most eukaryotic GT are membrane-bound and present in the luminal side of endoplasmic reticulum or the Golgi apparatus, that is equivalent to the outside of cells. Because these organelles are not present in bacteria, it is necessary to remove the transmembrane domain (involved in targeting) to keep the protein in bacterial cytoplasm. It should be pointed out that the heterologous oligosaccharides are not secreted, and that they remain in the cytoplasm. The production of heterologous oligosaccharides uses recombinant DNA technology to produce recombinant GTs, and the E. coli cell machinery to produce the desired oligosaccharides. We have chosen to call the heterologous oligosaccharides obtained by this approach ‘recombinant oligosaccharides’.
30.2.1 Biosynthesis of Nod Factors One of the major sources of biological N2 fixation is the symbiosis relationship Rhizohium-leguminous. As part of this symbiosis a dedicated N2 fixation organ, called nodule, is formed. The nodule is a plant structure that is invaded by rhizobia, and its formation results from a ‘molecular dialog’ between the plant and the bacteria [9]. Briefly, the plant secretes specific flavonoids that induce the expression of nod genes, resulting in the synthesis of Nod factors. Upon secretion by specific proteins, the Nod factor will induce the deformation of hairy roots, and mitosis of plant cells. Eventually, the bacteria invade the root cell, migrate towards the cell division region, and are released into these cells. Finally, bacteria differentiate into a Nz-fixing form, the bacteroid. Nod factors consist of a chitin (tri-, tetra- or penta-) oligosaccharide, of which the non-reducing end N-acetyl group is substituted by a long-chain fatty acid. Several substitutions are found at the reducing- and non-reducing-end sugars, although substitutions on other residues are present in some species [lo, 111. The Nod factors
848
30 Production of Heteroloyous Oligosaccharides b-y Recombinant Bacteria
HO HO
0
*.
NodFE Figure 2. Structure of NodRmIV (Ac, S). Proteins involved in addition of each substituent are circled.
produced by R. meliloti are shown in Figure 2. The first committed step of Nod factor biosynthesis is the formation of the chitin oligomer by NodC using UDP-Nsatylglucosamine (UDP-GlcNAc) as the sugar donor [ 111. NodB specifically deacetylates the non-reducing terminus of the oligosaccharide. The NH2 group is finally acylated in a step that requires NodA. Other substituents are added before or after the acylation by specific enzymes (Figure 2). The final product, is secreted by a system that requires Nod1 and NodJ [ 1 I]. The nod genes were discovered as result of the identification of rhizobial nonnodulating mutants. These genes were isolated and sequenced [12, 131 but no function was assigned to them. Discovery of Nod factors [ 141 brought substance to the study of Nod protein function. The first hint of the function of NodC was the sequence similarity with cellulose and chitin synthases [ 151. The in vitro synthesis using rhizobial cell extracts or E. coli extracts containing recombinant NodC was then accomplished [ 161 using UDP-[ ''C]GlcNAc as sugar donor. The oligosaccharide formed was shown to bind to wheat germ agglutinin, and to be sensitive to chitinase, but the minute amounts of the chitin oligosaccharide available precluded unambiguous structural characterization. It was, nevertheless, discovered that the E. coli strain expressing NodC was able to produce, in vivo, small amounts of chitin oligosaccharides; these were characterized by mass spectrometry [ 171. The product consisted of a mixture of di-, tri-, tetra- and pentasaccharides, and purification involved several steps. The use of
30.2 Concept and Methodology of Heteroloyous Oligosucchuride Production
849
E. coli strains expressing several combinations of nod genes enabled the elucidation of the biosynthetic pathway and the function of these genes [17], by establishing the structure of the corresponding Nod metabolites. This was the first example of the production of a recombinant oligosaccharide and the starting point for the production of oligosaccharide in E. coli. Our further reasoning was that if a single batch culture (biomass representing OD = 2) can produce a few mg L-' Nod metabolites, in a culture of high density (OD = 100) it would produce a few hundred mg L-l. It was found by sequence analysis and tagging with reporter enzymes [ 181, that NodC is a membrane-bound protein with four transmembrane domains (one at the NH2 terminal end, and three at the COOH terminus) flanking a 300 amino acid cytoplasmic peptide. The cytoplasmic moiety of NodC shares sequence similarity with several glycosyltransferases and polysaccharide synthases [ 19-21] in two different domains (domain A towards the -NH2 end and domain B towards the -COOH end). Whereas both domains are conserved in polysaccharide synthases, only domain A is present in glycosyltransferases [21].This led to the hypothesis that domain B is involved in multiple sugar transfer. The suggested mechanism proposes the presence of one catalytic center in each domain. It was also proposed that the acceptor is formed by the enzyme [21].To date several authors have reported the in vitro formation of chitin oligosaccharides in the absence of the putative GlcNAc acceptor [22, 231.
30.2.2 Expression Systems and Cloning Strategy UDP-GlcNAc is a key metabolite of bacteria, because it is a precursor for the formation of murein and Lipid A, both necessaries for cell viability. UDP-GlcNAc is synthesized from fructose-6-phosphate in four steps. The two last reactions leading to UDP-GlcNAc from glucosamine-6-phosphate are catalyzed by a bifunctional enzyme encoded by glmU [24].The acetyltransferase activity of this enzyme is strongly inhibited by GlcNAc-1-P and by UDP-N-acetylmuramic acid, suggesting that this reaction is the main point of UDP-GlcNAc synthesis regulation. Because UDP-GlcNAc is substrate for NodC, the recombinant enzyme should be expressed at low levels, so as not to disturb cell wall synthesis. To regulate gene expression, the appropriate genes are cloned under the control of a given promoter. ~ ~ has the advantage that it Two promoters were used, P L and ~ ~PA^^. P L promoter allows a low-level expression in the absence of inducer, while PA^^ is a tight promoter [25], and can be used to tune the expression of the gene by use of an inducer (arabinose). For the production of a single oligosaccharide with glycosidic or nonglycosidic modifications, the expression of several genes is required. This can be achieved by cloning more than one gene into a given vector, or in two compatible vectors. With eukaryotic GT genes it is necessary to provide a bacterial ribosomal binding site, and to remove regions coding for transmembrane domains; one solution is to produce a fusion protein. Some of the plasmids used are shown in Figure 3; they are commonly available [4-61.
19 PLac@ PAra Neighbour non-translated DNA
Figure 3. Plasmids containing chitin oligosaccharide synthases and different modifying enzymes. Relevant genes are in black-filled arrows, promoters in hatched arrows. Because of the cloning strategy, DNA contiguous to the desired genes are present in the constructions (gray).
Vector DNA
-
9 a w.
00
30.2 Concept and Methodology of Heterologous Oligosaccharide Production
85 1
30.2.3 High Cell-Density Cultivation The cultivation of E. coli to high cell concentration is one way of maximizing the volumetric productivity of recombinant oligosaccharides. The main problem encountered in high cell-density culture (HCDC) is the risk of 0 2 limitation, which obliges the bacteria to shift to a fermentative metabolism, resulting in the production of growth-inhibiting metabolites such as acetic acid. Because the 0 2 delivery capacity of the fermenter is limited, the easiest way to prevent 0 2 shortage is to limit the bacterial growth by controlled feeding of the carbon source. Different feeding strategies have been proposed [26, 271. Although some strategies require sophisticated feedback control of substrate concentration to determine the feeding rate, reasonably high cell density can be also reached with simple fed-batch technique using pre-determined feeding rates to sustain carbon-limited growth [28]. This simple technique requires minimal fermentation equipment: a laboratory-scale fermenter with temperature, pH and 0 2 controls, and a peristaltic pump for the feeding. The protocol we routinely use to produce recombinant chitin oligosaccharides in high cell density cultures of E. coli includes the use of glycerol as carbon source because its catabolism does not provoke substantial accumulation of acetate, as does the use of glucose. The fed-batch strategy is outlined in Figure 4. During phase 1, cells grow exponentially until all the glycerol that was initially added in the starting media (17 g L-') had been consumed [4]. To prevent oxygen limitation,
phase 1
i phase 2
phase 3
100
80
-
60-
40
-
20 -
o+ 0
glycerol
I
5
10
15
20
25
30
35
Time (hours) Figure 4. High cell-density culture strategy. For details see Ref. [3].
40
45
50
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30 Production of Heterologous Oligosaccharidesby Recombinant Bacteria
growth is then carbon limited, and it is supported by the continuous feeding of a glycerol solution. Glycerol is first supplied at a high feeding rate in phase 2 to promote a rapid increase in biomass. Finally growth is conducted at a low rate (phase 3 ) to increase in the intracellular concentration of recombinant oligosaccharide.
30.2.4 Purification of Recombinant Oligosaccharides The recombinant chitin oligosaccharides are produced and accumulated in the cytoplasm and are not excreted in the culture medium. The first purification step consists of harvesting the cells by centrifugation to remove all culture supernatant. The cells are then suspended in distilled water and permeabilized either by a heat shock (30 min at 100 "C) or by a series of freeze-thaw cycles. The latter method is employed for compounds which are susceptible to damage at high temperature. These treatments enable the oligosaccharides to diffuse outside the permeabilized cell. After a second centrifugation the oligosaccharides are recovered in the supernatant and are then further purified by adsorption on charcoal-celite and selective elution with aqueous ethanol. Positively charged N-deacetylated chitin oligosaccharides and negatively charged sulfated compounds can be advantageously separated by ion-exchange chromatography. These purification procedures enable gram-scale preparation of oligosaccharides, which are found to be more than 90% pure by HPLC and NMR.
30.3 Examples of Recombinant Oligosaccharides 30.3.1 Production of Chitin Oligosaccharides in E. cofi Expressing NodC
The production of chitin oligosaccharides is achieved in E. coli DH1 cells containing ~ C Aor , PCR~ (Figure 3). R. meliloti produces Nod factors in which the oligosaccharide consists of a mixture of tetra- and pentasaccharide [29], whereas A. caulinodans NodC produces pentasaccharide as the major product [30]. The final degree of polymerization of the oligosaccharides is determined by NodC [5, 231 but the concentration of UDP-GlcNAc influences the size of the oligosaccharide [22, 231, perhaps by acting as a chain terminator. Because the concentration of the sugar donor can change during cell growth, and under different metabolic conditions, the size of the oligosaccharides was assessed at the end of phase 1 and at the end of phase 3 . On both occasions the major products were the pentasaccharide for ~ C A , , and a mixture of tetra- (70%) and pentasaccharide (30%) for p c ~ , [ 5 ] . Whereas these oligosaccharides were almost pure at the end of phase 3 , other products were found at the end of phase 1. These products comprise mainly derivatives of the chitin oligosaccharides whose reducing end was found to be substituted with a glycerol moiety, linked in the configuration to either the primary or secondary hydroxyl groups of glycerol [31].
30.3 Examples of Recombinant Oligosaccharides
853
Glycerol-substituted oligosaccharides are no longer detectable at the end of phase 3. During phase 1 the intracellular concentration of glycerol is high, because its uptake is mediated by the glycerol facilitator GlpF [32]. In contrast, during phases 2 and 3 the added glycerol is immediately used to sustain growth, therefore the intracellular concentration is low. These results strongly suggest that high intracellular glycerol concentration promotes its incorporation at the reducing end. The incubation in vitro of E. coli membranes containing NodC in the presence of glycerol and UDP[ 14C]GlcNAc, leads to the formation of chitinase-sensitive radioactive oligosaccharides, that migrate more slowly than the corresponding chitin oligosaccharides 1311.These results suggest that NodC adds the glycerol moiety to the oligosaccharide. 30.3.2 Production of Nod Factor Precursors
As mentioned above, much work has been devoted to understanding the function of the proteins involved in Nod factor biosynthesis, but their mode of action is not fully understood. Future trends are related to Nod factor recognition and signalling [33-361. Because chemical synthesis is long and tedious, we have proposed a new strategy involving the production of Nod factor precursors in E. coli expressing appropriate biosynthetic genes [ 371. These oligosaccharides are provided to chemists for further modifications. In the first instance, when E. coli DH1 strains harboring the plasmid ~ B C Awere , employed, chitin pentasaccharides N-deacetylated at the non-reducing terminus were obtained in yields close to 1 g L-’ of culture [4]. This oligosaccharide was acylated by chemical means [37]. and used for characterization of Nod factor protein binding sites 1381. Further chemical modification will enable the production of Nod factor derivatives useful for affinity chromatography, fluorescence and/or photoactivatable probes. These molecules will be useful tools for isolation and biochemical characterization of the Nod factor binding sites. The substantial amounts obtained will also facilitate the study of the signal transduction pathway as well as the regulation of plant modulation genes expression. The biological activity of R. meliloti Nod factors requires a tetrasaccharide and the presence of sulfate and 0-acetate. We have used several combinations of the nod genes (NodB, NodC, NodH, NodL); these have enabled us to produce as many as eight different molecules (see Figures 5 and 6) in the recombinant oligosaccharide system. In R. meliloti, three genes (nodPQH) are necessary for the sulfation of Nod factors 1391. The protein NodH catalyzes the transfer of sulfate from PAPS (3’phosphoadenosine 5’-phosphosulfate) to the reducing terminal 6-0 position of Nod factors [40]. The enzyme is also active on chitin oligosaccharides and their Ndeacetylated derivatives. Kinetic analysis indicates that the affinity of NodH for chitin oligosaccharides is an order of magnitude lower than for lipo-chitin oligosaccharides, suggesting that sulfation occurs in vivo after the acylation [41]. The K, value of ca 150-200 p~ found in vitro for chitin oligosaccharides should, however, be sufficient to enable the sulfation of recombinant chitin oligosaccharides which have been shown to accumulate in the cytoplasm of E. coli at a concentration higher than 10 mM.
854
30 Production of Heterologous Oligosaccharides by Recombinant Bacteria
Figure 5. Metabolic pathway leading to the synthesis of Nod factor precursors in E. coli. (NodL and NodH can both act on unsubstituted oligosaccharides.)
30.3 Examples of Recombinant Oligosaccharides
855
The proteins NodP (ATP sulfurylase) and NodQ (adenosine 5'-phosphosulfate (APS) kinase) are associated in a sulfate-activating complex that enables the synthesis of the sulfate donor (PAPS) for Nod factor sulfation [42]. PAPS is also the sulfate donor used for the synthesis of cysteine and it is produced in E. coli by the normal household proteins CysC, CysD, and CysN [43, 441. The genes cysCDN are part of the cysteine regulon which is repressed in the presence of cysteine and other reduced sulfur compounds [45]. This means that the E. coli machinery of PAPS biosynthesis can be used for chitin oligosaccharide sulfation if the bacteria are cultivated with sulfate as the only sulfur source. The total PAPS demand for the synthesis of cysteine and methionine (87 and 146 pmol g-' dried cells, respectively) [46] largely exceeds the maximum demand that can be estimated for chitin oligosaccharide sulfation (around 20 pmol g-' dried cells). High cell density cultivation of strain DH1 (~BCHR,)leads to the production of 330 mg L-' sulfated chitin oligosaccharides. This represented a sulfation yield of ca 50%. Attempts were made to improve this yield by expressing nodH in trans under the control of the PBADpromoter in a separate plasmid compatible with ~BCR,. The sulfation yields were similar to that obtained with nodH expressed in cis. Moreover, this approach has another advantage, because it limits the number of plasmids that have to be constructed to obtain strains expressing different possible combinations of genes. Strain DHI was co-transformed with plasmid pBADH and different plasmids carrying the gene nodC or nodBC. Cultivation of these strains enabled the production of sulfated N-peracetylated and non-reducing end Nmonodeacetylated chitin oligosaccharides. The choice between the gene nodC from A . caulinodans or from R. meliloti provides the additional possibility of producing these molecules as pentamers or as tetramers. The protein NodL is a transacetylase that utilizes acetyl-CoA as a substrate to 0acetylate lipo-chitin oligosaccharides and both N-peracetylated and non-reducing end N-monodeacetylated chitin oligosaccharides at the 6 position of the nonreducing terminal sugar [46-491. Because acetyl-CoA is a metabolite normally present in the bacterial cytoplasm, the coexpression of nodL with nodC can result in the production of mono 6-0-acetylated chitin oligosaccharide. The gene nodL was sub-cloned in pBBR plasmid which is compatible with both pUC and pBAD derivatives. Analysis of chitin oligosaccharides produced by strain DH 1 carrying nodC and nodL indicated that ca 700/0of the total oligosaccharide was 0-acetylated. Cultivation of strains expressing the four genes nodBCHL enables the production of the direct precursor for the preparation of NodRm-IV (Ac, S, CI62), i.e. the natural Nod factor produced by R. meliloti. 30.3.3 Production of Derivatives of N-Acetyllactosamine
The production of chitin oligosaccharides in E. coli opened the possibility of adding glycosyl substitutions to it. One interesting possibility is the addition of a p(1,4)linked galactosyl residue at the non-reducing end (Figure 6), to form a terminal LacNAc, the core of several biologically important oligosaccharides. This step is catalyzed by N-acetyllactosamine synthases (EC 2.4.1.90). For this purpose, we
856
30 Production
of
Heterologous Oligosaccharides by Recombinant Bacteria
choose LgtB, a bacterial p- 1,4 galactosyltransferase (Swiss-Prot Acc. Number Q51116) [50].LgtB is involved in the synthesis of the lacto-N-neotetraose moiety of the lipo-oligosaccharide produced by Neisseria meningitidis [ 501. Because E. coli produces a P-galactosidase, namely LacZ, we have used a lacZ- strain to prevent degradation of the terminal LacNAc residue. Co-expression of NodC and LgtB leads to the production in vivo of the expected compound [6]. The total recombinant oligosaccharide was produced at 0.8 g L-' of culture. On the basis of NMR data from the crude recombinant oligosaccharide fraction, the extent of galactosylation of the chitin oligosaccharide is close to 90%. Expression of the two genes in cis ( ~ C ALgtB) , slightly increased the galactosylation rate and total amount of recombinant oligosaccharides. Analysis of the recombinant oligosaccharides produced after phase 3 showed that the major product was the galactosyl chitin pentaose (Figure 6); other minor galactosylated and nongalactosylated oligosaccharides were also detected. Spectroscopic characterization of the galactosylated pentasaccharide confirmed the proposed structure [61. We have tested whether the Gal-(GlcNAc)5 is an acceptor for the bovine a-1,3 galactosyltransferase in vitro using UDP[ 14C] Gal. The incorporation rate was similar to that of the physiological acceptor (LacNAc) [ 5 ] .This result is promising in E. coli. for the production of aGall,3-~Gal-l,4(GlcNAc)~
30.4 Conclusions and Future Perspectives In the last three years, we have produced more than 50 g oligosaccharide using two 2 L and one 10 L fermenters. Typically, purification of gram amounts of an end Ndeacetylated chitin oligosaccharide takes one week. Nine different compounds have been produced (Figure 6), with production yields between 0.25 g L-' and 2 g L-' depending on the product (Table 1). The work presented here represents the first few steps towards the production of tailored oligosaccharides in E. coli. Obviously, there are several critical points that can be improved, as well as the production of other oligosaccharides and other applications that can be envisaged. 30.4.1 Production of Labeled Chitin Oligosaccharides to Study Their Interactions with Proteins
The recognition of different sugars by specific proteins (glycosyl hydrolases, GTs, lectins) is a key factor of biological processes. Molecular probes are needed to enable better understanding of the molecular basis of sugar recognition. Chitin oligosaccharides can be used to study the interaction with chitinase, and recognition by lectins or Nod factor binding proteins. To this end we have produced 13C-enriched chitin pentaose (20% enrichment) for NMR study of the mechanism of action of
x , XI
OH
NodC,, or Rm
v , VI
OH
HO
XI1
I, I1
L V I I , VIII
Figure 6. Recombinant oligosaccharides obtained.
HO
NodH
OH
HO
2-3
HO HO
2-3
HO
2-3
OH
XIV
OH
HO
L&tB
2-3
2-3
NodH
2-3
H
ul 4
00
cp
3F
9R
Q
P
L2
858
30 Production of Heterologous Oligosaccharides by Recombinant Bacteria
Table 1. The recombinant oligosaccharides obtained. Product
Plasmid used
Estimated production yield (g L-')
I I1 I11 IV V VI VII VIII IX X XI XI1 XI11 XIV
PcRm PCAc PBCRm PBCA, PCR, pBADH pCac pBADH PCR, PBBRLl PCA, + pBBRLl ~ C R , PBBRLl + pBADH PBCHR, pBCac pBADH PBCR, PBBRLl PBCHR, + PBBRLl PCA,LgtB
1.o 2.0 0.5 1.2 0.4 0.6 0.8 1.4 0.3 0.25 0.25 0.4 0.2 1.o
+ +
+ +
+ +
chitinases [ 5 11. The enrichment might be improved by using specific bacterial strains or specific growth conditions. 30.4.2 Improvement of Oligosaccharide Production, and Metabolic Engineering In the first instance, the volumetric yield of recombinant oligosaccharides can probably be increased by genetically engineering the bacteria, either adding extra copies of the genes coding for the synthesis of sugar donors (gmlU: UDP-GlcNAc, galE: UDP-Gal epimerase), or using stronger promoters. In the second instance, sugar-nucleotides are expressed either under certain conditions (GDP-fucose) or in certain strains (CMP-Neurominic acid). The constitutive production of these sugar-nucleotides could also be engineered to enable synthesis of more diverse oligosaccharides. 30.4.3 Production of More Complex Oligosaccharides
An important limitation to this method is the availability of appropriate GTs. Although the production in E. coli of eukaryotic G T active in vitro was reported, the activity in vivo was not assessed [52].Production of fully active GTs in E. coli will broaden the range of possible recombinant oligosaccharides. Research on the mode of action of GTs is also necessary for eventual engineering of available bacterial GTs to meet specificity requirements. Alternatively, new bacterial glycosyltransferases whose activity resembles those of eukaryotic GTs should be found. In this regard the results are encouraging, because the bacterial genes can be used for the production of oligosaccharides in E. coli as we did with LgtB.
References
859
Here we have presented an original strategy to produce derivatives of chitin oligosaccharides by a simple living organism. The products were obtained in amounts and purity that enable their use as substrates to perform chemical modifications or study biological properties. It is conceivable that the recombinant oligosaccharides can also be used as substrates for enzymatic methods. We think that the limits of this method have not yet been achieved, and it is possible that in the next few years the production of more complex oligosaccharides will become routine.
Acknowledgments The authors acknowledge Drs Serge Perez and Annemarie Lellouch (Cermav, Grenoble) for helpful discussions, and Dr Hughes Driguez for reading the manuscript. This work was supported by Xenotransplantation Project BI04CT972242 of the Biotech program from the European Union, and the program PCV 97 061 from the CNRS (France).
References 1. Varki, Glycobiology, 1993, 3, 97. 2. 0. Gabriel, H. E. Umbarger, Esherichia coli and Salmonella cellular and molecular biology. ASM Press, 1987, p. 504. 3. P. D. Rick, G. L. Hubbard, M. Kitaoka, H. Nagaki, T. Kinoshita, S. Dowd, V. Sirnplaceanu, C. Ho, Glycobiology, 1998, 8, 557. 4. E. Samain, S. Drouillard, A. Heyraud A, H. Driguez, R. A. Geremia, Carbohydv. Rex, 1997 302, 35. 5. E. Samain, V. Chazalet, R . A. Geremia, J. Biotechnol., submitted. 6. E. Bettler, E. Samain, V. Chazalet, C. Bosso, A. Heyraud, D. H. Joziasse, W. W. Wakarchuk, A. Imberty, R. A. Gerernia. Submitted. 7. Whitfield, M. A. Valvano, Advances in Microbial Physiology, 1993, 35, 135. 8. I. Roberts, Annu. Rev. Microbiol., 1996, 50, 285. 9. P. van Rhijn, J. Vanderleyden, Microbiol. Rev., 1995, 59, 124. 10. J. Denarie, F. Debelle, J. C. Prome, Annu Rev Biochem, 1996, 65, 503. 11. P. Mergaert, M. Van Montagu, M. Holsters, Mol. Microbiol., 1997,25, 81 1. 12. T. W. Jacobs, T. T. Egelhoff, S. R. Long, J. Bacteriol., 1985, 162, 469. 13. I. Torok, E. Kondorosi, T. Stepkowski, J. Posfai, A. Kondorosi, Nucleic Acids Rex, 1984, 12, 9509. 14. P. Lerouge, P. Roche, C. Faucher, F. Maillet, G. Truchet, J. C. Prome, J. Denarie, Nature, 1990, 344, 781. 15. E. Bulawa, Mol. Cell. Biol., 1992, 12, 1764. 16. R. A. Geremia, P. Mergaert, D. Geelen, M. Van Montagu, M. Holsters, Proc. Natl. Acad. Sciences USA, 1994, 91,2669. 17. P. Mergaert, W. D’Haeze, D. Geelen, D. Prome, M. van Montagu, R. A. Geremia, J. C. Prome, M. Holsters, J. Biol. Chem., 1995,270, 29217. 18. M. A. Barny, E. Schoonejans, A. Economou, A. W. Johnston, J. A. Downie, Mol. Microbiol., 1996, 19, 443. 19. M. Atkinson, S. R. Long, Mol. Plant-Microbe Interact., 1992, 5, 439. 20. Debelle, C. Rosenberg, J. Denarie, Mol. Plant-Microbe Interact., 1992, 5, 443. 21. I. M. Saxena, R. M. Brown Jr., M. Fevre, R. A. Gerernia, B. Henrissat, J. Bacteriol., 1995, 177, 1419.
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30 Production of Heterologous Oligosaccharides by Recombinant Bacteria
22. N. I. de Iannino, S. G. F’uepke, R. A. Ugalde, Mol. Plant-Microbe Interact., 1995, 8, 292. 23. E. Kamst, J. Pilling, L. M. Raamsdonk, B. J. Lugtenberg, H. P. Spaink, J. Bucteriol., 1997, 197, 2103. 24. D. Mengin-Lecreulx, J. J. van Heijenoort, J. Bacteriol., 1994, 176, 5788. 25. L. M. Guzman, D. Belin, M. J. Carson, J. Beckwith, JBacteriol, 1995, 177, 4121. 26. D. Riesenberg, Curr. Opin. Biotechnol., 1991, 2, 380. 27. S. Y. Lee, Trends Biotechnol., 1996, 14, 98. 28. D. J. Korz, U. Rinas, K. Hellmuth, E. A. Sanders, W. D. Deckwer. J. Biotechnol., 1995,39, 59. 29. M. Schultze, B. Quiclet-Sire, E. Kondorosi, H. Virelizer, J. N. Glushka, G. Endre, S. D. Gero, A. Kondorosi, Proc. Natl. Acad. Sci. USA, 1992, 89, 192. 30. P. Mergaert, M. van Montagu, J. C. Prome, M. Holsters, Proc. Natl. Acad. Sci. USA, 1993, 90, 1551. 3 1 . R. A. Geremia, personal communication. 32. C. Maurel, J. Reizer, J. I. Schroeder, M. J. Chrispeels, M. H. Saier Jr., J. Biol. Chem., 1994, 269, 11869. 33. J. J. Bono, J. Riond, K. C. Nicolaou, N. J. Bockovich, V. A. Estevez, J. V. Cullimore, R. Ranjeva, Plant J., 1995, 7, 253. 34. J. C. Prome, Curr Opin Struct Biol., 1996, 6, 671. 35. S. G. Puepke, Crit. Rev. Biotechnol., 1996, 16, 1. 36. K. van de Sande, T. Bisseling, Essays Biochem., 1997, 32, 127. 37. S. Drouillard, J. J. Bono, B. Henrissat, R. A. Geremia, F. Gressent, E. Samain, H. Driguez, 1997, 9th European Carbohydrate Symposium, 6-1 1 July, Utrecht, Nederlands. 38. J. J. Bono, F. Gressens, personal communication. See complete reference attached. 39. P. Roche, F. Maillet, C. Plazanet, F. Debelle, M. Ferro, G. Truche, J. C. Prome, J. Denarie, Proc. Natl. Acad. Sci. USA, 1996, 93, 15305. 40. D. W. Ehrhardt, E. M. Atkinson, K. F. Faull, D. I. Freedberg, D. P. Sutherlin, R. Armstrong, S. R. Long, J. Bacteriol., 1995, 177, 6237. 41. M. Schultze M, C. Staehelin, H. Rohrig, M. Joh, J. Schmidt, E. Kondorosi, J. Schell, A. Kondorosi, Proc. Natl. Acad. Sci, USA, 1995, 92, 2706. 42. J. S. Schwedock, C. Liu, T. S. Leyh, S. R. Long, J. Bucteriol., 1994, 176, 7055. 43. T. S. Leyh, J. C. Taylor, G. D. Markham, J. Biol. Chem., 1988,263, 2409. 44. T. S. Leyh, T. F. Vogt, Y. Suo, J. Biol. Chem., 1992, 267, 10405. 45. N. M. Kredich, Esherichia coli and Salmonella cellular and molecular biology. ASM press. Baltimore, 1996,pp. 514-527. 46. F. C. Neidhardt, H. E. Umbarger, Esherichia coli and Salmonella cellular and moleculur biology. ASM Press, 1996, p. 15. 47. P. Spaink, D. Sheeley, A. A. van Brussel, J. Glushka, W. S. York, T. Tak, 0 . Geiger, E. P. Kennedy, V. N. Reinhold, B. J. Lugtenberg, Nature, 1991, 354, 125. 48. V. Bloemberg, J. E. Thomas-Oates, B. J. Lugtenberg, H. P. Spaink, Mol. Microbiol., 1994, 11, 793. 49. G. V. Bloemberg, R. M. Lagas, S. van Leeuwen, G. A. Van der Marel, J. H. Van Boom, B. J. Lugtenberg, H. P. Spaink, Biochemistry, 1995, 34, 12712. 50. W. Wakarchuk, A. Martin, M. P. Jennings, E. R. Moxon, J. C. Richards, J. Biol.Chem., 1996, 271, 19166. 51. E. Samain, personal communication. 52. Fang, J. Li, X. Chen, Y. Zhang, J. Wang, Z. Guo, W. Zhang, L. Yu, K. Brew, P. G. Wang, J. Am. Chem. Soc., 1998, 120, 6635.
Part I Volume 2
IV Carbohydrate-Pr otein Interactions
Carbohydrates in Chemistry and Biology Editor Beat Ernst, Gerald W. Hart, Pierre Sinay copyright OWILEY-VCH Verlag GmbH, 2000
31 Protein-Carbohydrate Interaction: Fundamental Considerations Nikki F. Burkhalter, Sarah M. Dimick, and Eric J. Toone
31.1 Introduction A major-if not the major-rationale for the study of carbohydrate chemistry derives from the roles played by glycoconjugates in biology [l]. In almost all such roles a carbohydrate ligand must bind to a protein receptor. There exists, then, tremendous potential to modulate biological activity through the creation of high affinity mimics of native saccharide receptors; such compounds have potential therapeutic value in the treatment of viral, parasitic, mycoplasmal and bacterial infections, and the treatment of a range of human cancers [ 2 ] .The study of proteincarbohydrate interaction, then, forms a key focus of this discipline of carbohydrate chemistry and biology. The development of inhibitors of carbohydrate-mediated biological recognition is impeded by the exceptionally weak bindings that typify protein-carbohydrate interaction [ 3 ] . In this chapter we consider the energetic issues central to proteincarbohydrate interaction. We devote some space to a consideration of the general forces and interactions that provide both affinity and specificity during association in aqueous solution. A range of phenomena observed during protein-carbohydrate association appear to be dependent on the assay used to evaluate ‘binding’; we therefore consider the some of the more commonly utilized assays of protein carbohydrate interaction and delineate the microscopic events each assay is designed to evaluate. Finally, the last ten years has seen tremendous activity in the development of multivalent carbohydrate ligands; we thus explore the energetic issues surrounding multivalency in ligand binding. Throughout, we endeavor to relate energetic issues directly to protein-carbohydrate interaction. We stress, however, that a tremendous body of literature pertaining to the very issues central to protein-carbohydrate interaction exists in the context of other binding systems. Where appropriate, we draw on the lessons and experience of these adjacent disciplines in our consideration of the biophysical aspects of protein-carbohydrate interaction.
864
31 Protein-Carbohydrate Interaction: Fundumentul Considerations
31.2 Association in Aqueous Solution Two species combine to form a complex in water if the sum of the intermolecular forces between them more than offsets the sum of the loss of favorable interactions with solvent and any unfavorable interactions that develop between solutes during complex formation. Collectively the interactions between non-bonded species are referred to as cohesive forces, defined as those forces lost when the species are transferred to infinite separation in the gas phase. While it is common to classify chemical forces as covalent or non-covalent, the interactions are fundamentally the same; only the magnitude of the interactions varies. Cohesive, non-specific forces are weak compared to covalent interactions; typically we consider cohesive forces as those forces with strengths less than 10/0 of covalent bond strengths. We will see, however, that this definition is somewhat arbitrary and in fact a continuum of interaction energies exists. That non-covalent interactions between molecules are important has long been appreciated. By the middle of the 19th century failings in the ideal gas law were apparent, and by 1873 van der Waals had postulated his equation of state. Thus the functional dependence of intermolecular interactions on internuclear disvdnceattractive at long separations and repulsive at very short internuclear spacingswas clear long before there existed a complete understanding of the nature of the interactions involved. Association in aqueous solution is further complicated by the highly participatory nature of the solvent; as we shall see the contribution from solvation/desolvation processes to overall thermodynamic parameters is typically greater than that provided by interactions between the two associating solutes. We begin our discussion by considering the interactions that result during the approach of two species absent the effect of solvation. We later consider the effect of water on each of these terms.
31.2.1 Gas Phase Non Covalent Interactions
Dipole-Dipole Interactions Molecules in which the center of electron density differs from the center of mass possess a permanent dipole. The interaction of oppositely charged dipoles yields a coulombic attraction, while the analogous interaction of equivalently charged species provides a repulsive contribution to an intermolecular interaction energy..The product of the dipole separation and the partial charge of the dipoles yields the dipole moment p. Many species containing strongly electronegative elements show no dipole moments because of a symmetrical disposition of partial charge; examples include carbon dioxide and carbon tetrachloride. While this symmetry produces a net dipole moment of zero, the molecules have higher order moments. Thus carbon dioxide has a large quadrupole moment while carbon tetrachloride shows a large octopole moment. Dipole moments of groups commonly encountered in proteincarbohydrate interaction are listed in Table 1 [4].
31.2 Association in Aqueous Solution
865
Table 1. Dipole moments of representative small molecules [4].
H20 H2S CH3CH2F CHjCH2Cl CH3CH2Br CH3CH2I FCH2CH20H CH3CHzOH CH3CH20D CH3CH2SH HOCHzCHzOH HSCHzCH2SH CH30CH3 CH3SCH3 a
1.88 0.98 1.90 2.00 1.90 1.75 1.52 1.70 1.54 1.52 2.31 1.70 1.32 1.51
CH3CH20CH3 1.75 3.20 CH3CH2NO 3.50 CH3CH2CN 1.25 CH3CH2COOH 2.50 CH3CHzCHO CH3CH:!CONH2 3.60 CH3CH2NH2 1.30 NH2 CH2 CHI NH2 1.86 CH~CH~NOZ 2.25 3.95 CsH5NOz o - N O ~ C ~ H ~ N O ~5.90 M - N O ~ C ~ H ~ N O ~3.79 p-NO2C6HdN02 0.69
Debye
The contribution of interactions between permanent dipoles to the overall cohesive energy between two molecules depends on both the magnitude of the dipoles and their relative orientation. A stepwise consideration of each interactiondipole-dipole, dipole-quadrupole, quadrupole-quadrupole, etc.-provides the overall contribution of the interactions of partial charges to the total cohesive energy between two molecules. The strength of individual interactions drops off as a power function of the internuclear distance; the exact functional dependence varies with the interaction. In general, the interaction between multipoles of order n1 and 122 drops off as r - ( n l + n 2 + 1 ) .If we consider the interaction of two species each with both a dipole (p) and quadrupole (0)separated by an internuclear distance r and disposed relative to each other by the angles 0 ~ 0,, and c$ (Figure l), the intermolecular force is given by [ 5 ] :
866
31 Protein- Carbohydrate Interaction: Fundamental Considerations
where E , is the permitivity of free space. The dependence of each term on the respective dispositions of the multipoles is given by the expressions: f l ( 0 ~0, ~4), = sin 6, sin 0, cos $ - 2 cos 0~cos OB 3
f i ( e A , e B , $=) - [ C O S O ~ ( ~ C O S ~ O1)~ - 2sin0A sineBcosOBcos$] 2 3 f 3 ( e A , e B , $=) - [ I 4
-
5C0S2e~- 5 ~ 0 ~ ~~ ~ 0 ~c -o s ~ ~ ~ c o s ~ ~
+ 2(4 cos eAcos 0,
- sin eA sin 0,
cos $) 2 ]
The functional form of the relationship makes intuitive sense; f l reaches a favorable maximum when the dipoles are aligned antiparallel to each other, an unfavorable maximum when the dipoles are aligned in a parallel fashion, and zero when the dipoles are oriented perpendicular to each other. Averaged over all orientations, the terms sum to zero. Application of a Boltzman distribution provides an energy for dipole-dipole, dipole-quadrupole and quadrupole-quadrupole interactions of:
All interaction terms are negative (favorable) and inversely proportional to temperature. As the order of the multipole increases the distance dependence becomes increasingly severe, and interactions beyond quadrupole-quadrupole do not provide a significant contribution to overall interaction energies. Dipole-Induced Dipole A dipole is induced in a molecule with no net permanent dipole but with a polarizable electron cloud when it is brought into contact with a permanent dipole. The magnitude of the induced dipole and the strength of the resulting interaction with the permanent dipole depends on the magnitude of the permanent dipole and the polarizability of the second species: Pind = aE
where E is the electric field generated by the permanent dipole and a is the susceptibility, or polarizability, of the second species. The energy of the interaction is then given by:
31.2 Association in Aqueous Solution
867
The strength of the interaction again depends on the relative orientation of the two species; the effective field between a dipole and a polarizable species separated by a distance r and at an angle 8 to the normal is given by: E=pJ
1 + 3 cos2 8 47~5~3
and thus: U=
+
a p y 3 cos2 8 1) 2r6 ( 4 7 1 ~ ~ ) ~
As before, over all orientations the interaction sums to zero; application of a Boltzman correction yields an interaction energy given by:
Again, the magnitude of the term drops off as the sixth power of the internuclear distance, and is directional. Some values of polarizabilities of various groups commonly encountered in protein-carbohydrate interaction are shown in Table 2 [6]. Dispersive Interactions
The intuitive interactions described above--essentially Coulombic attractions between oppositely charged particles-cannot account for more than a fraction of the I Table 2. Group polarizabilities [6].
-H -F
-c1 -Br -I -0-H -S-H -NH2 -CN -NO2 -CH, -CH> -YO H I
-CI
a
10-25 cm3
a&!a
a?za
3.9 4.5 22.1 33.1 52.9 9.6 3.2 16.4 20.7 19.7 22.4 18.4 22.0
23.5
9.8
80.5
6.7 30.0 32.8 13.7 13.7 27.1 57.7 10.4 60.8 25.6
868
31 Protein-Carbohydrate Interaction: Fundamental Considerations
total cohesive energy between common materials in the condensed phase. By far the most important contributors to overall interaction energies are the dispersive forces first identified by Fritz London in 1930 [7, 81. Although it is impossible to properly consider dispersive interactions in a purely classical treatment, the concept is made intuitive by considering the structures of atoms and molecules on timescales shorter than electron reorganizations (i.e. the reciprocal of the dispersion frequencies). Frozen in time, all molecules possess instantaneous dipoles; interaction of these dipoles provides the London or dispersive interaction. The interaction energy between two species arising from London forces is given by the expression:
where VA and VB are the dispersion frequencies of A and B, and U A and U B the corresponding polarizabilities. Although individual dispersive interactions are weak, they have no orientational dependence. Summed over an entire molecule they are large, providing the bulk of the total cohesive energy. Specific Forces: Hydrogen Bonding and n-c Bonding
The forces described so far are weak, typically less than one percent the strength of a covalent bond. In many instances, interactions between non-covalently bonded species are significantly stronger than this limit. For example, the exothermic transfer of ethanol from the gas phase to triethylamine reduces the strength of the ethanol 0-H covalent bond by 13.5Y1: clearly the interactions formed between ethanol and triethylamine are significant on the scale of covalent bonds. These stronger interactions are referred to as specific forces, and result from the interaction of an electron lone pair with a permanent dipole (polarized o-bond). Such forces differ from the non-specific forces described above in several important ways: i) The interactions are directional and stoichiometric. Unlike other coulombic interactions that include a directional component to the force vector but that have no a priori defined stoichiometry, specific interactions have a discrete stoichiometry. Each lone pair of electrons forms a single interaction with an appropriately polarized n-G bond. It is thus correct to consider true equilibrium constants for the formation of two hydrogen bonds between water and acetone: H 0, H.
P-H ,H
Q O
K1 -
QoO'
K CH3
H3C
Scheme 1. Hydrogen bond formation.
K2
,H
P-H
Q0O'
KCH3
H3C
31.2 Association in Aqueous Solution
I
H 2.2
I Ca 1.o
cs
869
2.0 A1 1.6 sc
2.5 Si 1.9 Ge
3.0
P 2.2 As
3.5 S 2.6
4.0
Se
Br
c1 3.2
Ba
Figure 2. Representative Pauling electronegativities.
ii) The formation of spec$c interactions signiJicantly shortens the internuclear distance between the bound partners-typically to less than the sum of the van der Waal radii-while significantly lengthening the o-bond between the electropositive proton and its electronegative partner. This bond weakening is most readily observed as an increase in the IR stretch frequency. iii) The process is exothermic, despite the weakening of the polarized o-bond. The extent of polarization in an X-H bond that is required for effective activity as a hydrogen bond donor is unclear. A useful rule of thumb is that protons bonded to elements with Pauling electronegativities greater than three act as hydrogen bond donors. Most species with a lone pair of electrons act as hydrogen bond acceptors, including oxygen (alcohols and ethers, carbonyls, phosphine and amine oxides, sulfoxides), nitrogen (amines, pyridines, nitriles), and sulfur. Stable anions also form strong hydrogen bonds. The most reliable predictor of hydrogen bond strengths, at least in the gas phase, is the difference in proton affinity of the hydrogen bond donor and acceptor. By this criteria the FH-F- interaction is predicted as the strongest monomeric hydrogen bond; this prediction is borne out by a crystal phase heat of formation of -37 kcal mol-'. The value of hydrogen bond strengths in solvent, however, is greatly influenced by the interactions of both donor and acceptor with the solvent itself, and conclusions regarding the contributions of hydrogen bonds to overall net free energies of interaction in solution cannot be drawn from proton affinity data [9]. 31.2.2 The Effect of Water on Intermolecular Interactions Having considered intermolecular interaction in the absence of solvation, we now consider the consequence of addition of solvent to a system of interacting molecules. At the outset we note that the picture changes from one that considers the
870
31 Protein-Carbohydrate Interaction: Fundamental Considerations
Table 3. Proton affinities of representative small molecules [9]
a
PA"
PA"
226.8 222.3 222.2 218.0 214.9 194.1 198.6 189.3 203.5 189.8 165.4 187.4 196.4 183.7 180.3 165.2
168.5 166.4 173.2 165.7 163.3 209.4 142.5 209.4 195.0 188.7 185.6 179.6 209.1 194.9 190.5
kcal mol-'
interaction between solutes to one that considers the dzerence in interactions between solutes and solvent versus solute and solute. The transfer of all species from the gas phase to water is exothermic: by a variety of specific and non-specific mechanisms water interacts with all solutes such that the free energy of the solvated species is lower than that of the isolated gas-phase species. Binding two solutes thus necessarily involves the loss of favorable interactions with solvent, a loss that is ameliorated by the favorable interactions that arise during association in the absence of solvent. Below we consider the effects of solvation on each of the classes of interaction described above. Finally, we consider the 'hydrophobic effect' and its contribution to ligand binding. Coulombic Stabilization
We consider first the effect of aqueous solvation on all intermolecular stabilizations that derive from the interaction of charged or dipolar species. Because of its small size and significant dipole and quadrupole, water interacts strongly with all ionic and dipolar species. A binding event of charged or dipolar compounds thus proceeds with a significant loss of favorable cohesive interactions between solutes and water. The effect is most profound for ionic interactions; similar ameliorations of solute-solute interaction apply to multipole-multipole and dipole-induced dipole interactions. Gas phase ionic interaction energies are large, typically on the order of 50-200 kcal mol-'. The interaction of water with charged species is nearly as large; as a result solution-phase ionic interactions are weak, seldom more than five kcal mol-'
31.2 Associution in Aqueous Solution
871
and typically less than one kcal mol-'. Examples of this diminution, or leveling, of interaction energies are myriad. The binding constant for the association of the quaternary ammonium ion choline with acetyl cholinesterase differs from that of the uncharged analogue 3,3-dimethyl propanol by a factor of only 30. Likewise, a range of monovalent anions inhibit acetoacetate decarboxylase by interacting with the positively charged active site with binding constants ranging from 10 to lo5 M-', corresponding to binding free energies of only one to five kcal mol-'. The powerful effect of solvation is also apparent in the order of effectiveness of acetoacetate decarboxylase inhibition. The halogen anions bind with decreasing affinity in the order I- > Br- > C1- > F-, the opposite order to that predicted on the basis of electronegativities. Apparently the more effective solvation of smaller, harder anions leads to reduced binding to the protein: a decreasing favorable electrostatic interaction proceeding down the periodic table is more than compensated for by a more rapidly decreasing unfavorable desolvation energy. Similar arguments are readily apparent during the consideration of multipole-multipole and dipoleinduced dipole interactions. The effective strong favorable interaction of water with multipoles lost during binding precludes a significant energetic advantage from dipole-related interactions between solutes in the bound state relative to the solvated unbound state. It is important at this point to distinguish affinity from specificity, at least as it pertains to this discussion. Dipolar interactions are directional and, in an absolute sense, strong. Improperly satisfied dipolar interactions in a complex are strongly disfavored relative to the energy of the unbound solvated system. Thus dipolar and ionic interactions play important roles in determining the specificity of solute-solute interactions and the precise structure of the bound complex, even though they likely do not contribute strongly to the overall net negative free energy of complexation. Hydrogen Bonding The strongest known hydrogen bond is the FH-F- interaction. In the crystal phase this interaction is favorable by some 37 kcal mol-'. In aqueous solution the equilibrium constant for this hydrogen bond is 0.4 M-', corresponding to a free energy of 0.8 kcal mol-'. There is no evidence for the formation of any other monofunctional hydrogen bond in aqueous solution. The situation becomes somewhat more complex for intramolecular hydrogen bonds, which form without a loss of translational and rotational entropy. Most of the data collected in this regard involve pKa values of dibasic acids. Thus, for example, the two pKa values of fumaric acid, for which no intramolecular hydrogen bond is possible, are 3.03 and 4.54. From this reference, both pKa values are perturbed in the isomeric maleic acid, at 1.91 and 6.33. Presumably this larger 6pKa reflects the energetic contribution of an intramolecular hydrogen bond in the singly ionized species. Even with a loss of net favorable interaction energy, we stress again the important roles that hydrogen bonds play in determining both the specificity of solutesolute interactions and the structure of the bound complex. Because of the strong angular dependence of hydrogen bonds on the X-H+lone pair vector, hydrogen bonds provide an even more powerful orienting force than do dipolar interactions.
812
31 Protein-Carbohydrate Interaction: Fundamental Considerations
Dispersive Interactions Despite the fact that they are individually weak, dispersive forces contribute to the exothermicity of the interaction between solutes in aqueous solution. The magnitude of the dispersive component of the cohesive energy varies as the polarizability of the interacting solutes. Because oxygen polarizes very weakly, a binding cycle that replaces solute-water interactions with solute-solute interactions will be energetically favorable: the dispersive component of OH-OH self association is 470 kcal mol-’ A6, while that for CH2-CH2 interaction is 1160 kcal mol-’ A6 [ 5 ] . Additionally, the r6 dependence of the interaction is a powerful orienting force during ligand binding.
31.2.3 ‘Hydrophobic’ Interactions
It has long been appreciated that placement of most organic species into aqueous solution produces an energetically unfavorable perturbation to the structure of water. Ligand binding proceeds with the desolvation of some fraction of the interacting surfaces; to the extent that this desolvation relieves the perturbation of water structure the free energy of the bound system is lowered relative to the unbound state. This process is typically considered, at least philosophically, as a repulsion between water and ‘hydrophobic’ solutes. In reality, of course, the effect is the result of the strong attractive interactions between water molecues, providing a liquid with a boiling point of 100 “C and an enthalpy of vaporization of 540 cal g-’, despite a molecular weight of only 18 g mol-’ . The molecular origin of the hydrophobic effect remains obscure, despite nearly 100 years of intensive experimental and computational investigation. Water has a remarkably high cohesive energy density; a priori one might expect that the unfavorable free energy of transfer of ‘hydrophobic’ solutes from some non-aqueous condensed phase to water should come from the loss of water-water contacts as a cavity is formed to accommodate the solute. Such a process would lead to an unfavorable enthalpy of transfer; one would thus reasonably expect the unfavorable free energy of hydrophobic solvation to be dominated by an endothermic enthalpy of transfer. In fact, near room temperature the enthalpy of transfer of virtually all hydrophobic species is near zero or slightly exothermic. Rather, near room temperature the unfavorable free energy resulting from the transfer of ‘hydrophobic’ surface area to water derives from an unfavorable entropic term. Furthermore, both the enthalpy and entropy of transfer are strong functions of temperature: ‘hydrophobic’ dissolution proceeds with a large positive increment in the constant pressure heat capacity of the system, ACp. It is this term that most distinguishes water from all other solvents. In a seminal work Arnett and coworkers demonstrated that the heat capacity increment during aqueous dissolution was uniquely a property of water and is not found for any other solvent, regardless of polarity [ 101. During the past decades a variety of physical models designed to rationalize the observed thermodynamic behavior of nonpolar solutes in water have been proposed. It is beyond the scope of this work to describe each in detail; rather, the
31.2 Association in Aqueous Solution
813
reader is referred to any of several reviews and monographs on the topic [ 111. Here, we describe the most popular of the group-the ‘clathrate’ model of water-in enough detail to allow a qualitative, intuitive understanding of the energetic consequences of hydrophobic transfer to water. The transfer of a hydrophobic solute to water can be conceptually divided into two steps: the formation of a cavity of sufficient volume to accommodate the solute, and the transfer of the solute to that cavity followed by collapse of solvent onto the solute. The first of these steps must be endothermic; that the overall process is thennoneutral or exothermic near room temperature thus requires that the second conceptual step-placing the solute in the cavity and allowing the solvent to collapse on the solute-be exothermic. Water in bulk solution can form four hydrogen bonds in a tetrahedral orientation; in a solvation shell such bonding is impossible. The clathrate model of water asserts that water in a solvation shell orients to maximize favorable hydrogen bonds. In this structure a loss in the total number of hydrogen bonds is compensated for by an increase in the strength of the hydrogen bonds that can form. This increased strength comes at the price of reduced entropy, particularly the rotational entropy of water, in the clathrate solvation shell. The enthalpically enhanced hydrogen bonds of the clathrate shell also provide a mechanism for the positive increment in constant pressure molar heat capacity. The notion of clathrate water as a physical model to explain the unusual thermodynamics of hydrophobic hydration has been parameterized by both Muller and Ben-Naim to yield a semi-quantitative description of the event [12-141. In these constructs the enthalpy of transfer is given by the expression:
where f b and fhs is the fraction of hydrogen bonds in the bulk liquid and the hydration shell, respectively, and AHb and AHhs are the enthalpies of hydrogen bond formation in bulk solution and hydration shells, respectfully. While the clathrate model of water is intuitively accessible and explains both qualitatively and quantitatively many of the phenomena associated with hydrophobic hydration, many unanswered questions remain. Specifically, physical evidence for the existence of clathrates is lacking; indeed many studies specifically designed to observe such structures fail to note any increase in the order of water structure [ 15-20]. The notion of clathrate water is far from universally accepted and other models of water exist, especially a series of models based on scaled particle theory that explain the peculiar phenomenology of hydrophobic hydration based on the exceptionally small size of water [21-291. Putting aside issues of the molecular origin of ‘hydrophobic effect’, the more significant issue is the extent to which desolvation contributes to overall binding thermodynamics. A variety of experimental methodologies to evaluate the magnitude of this effect exist. The most commonly used tools are Hansch transfer parameters [30, 311. Briefly, a series of compounds are partitioned between water and watersaturated octanol. The value 7c is then derived for a substitutent according to the expression:
874
31 Protein-Carbohydrate Interaction: Fundamental Considerations
Table 4. Hansch parameters of representative functional groups [30, 311.
arom”
aliphb
0.15 0.70 1.02 1.26 -0.32 0.52 1.07 0.97 1.90 1.82 1.68 2.51 1.89 -0.61 -1.03 -0.04 0.62 - 1.23 0.18 0.24 -0.15 -0.72 -0.01
-0.73 -0.13 0.04
F CI Br I CN CH3 CF3 CH2CH3 n-Bu sec-Bu t-Bu cyclohexyl C6H5 OH CH20H OCH3 SCH3 NH2 N(CH3)2 NO2 COOH CH2COOH COOCH3 OCOCH3 COCH3 CONH2
-0.37 - 1.49
- 1.47
-1.80 -0.98 - 1.85 -0.95 - 1.26
-0.91 -0.91 - 1.26 -2.28
~~
“based on aromatic parent compound based on aliphatic parent compound
7t = log
P e l
where P and P, are the partition ratios of a substituted and parent compound, respectively. The value RTln7t is thus the incremental Gibbs free energy for the transfer of the substituent from water to water-saturated octanol, assuming at least rough group additivity. Hansch parameters for a range of commonly encountered substituents are shown in Table 4. Another approach to issues of solvation thermodynamics utilizes the differential hydrogen bonding properties of light and heavy water [32, 331. The use of thermodynamic solvent isotope effects, or the evaluation of the enthalpy of ligand binding in light and heavy water, allows evaluation of the extent to which desolvation aids
31.2 Association in Aqueous Solution
875
ligand binding. Again, our goal here is not to describe the technique exhaustively; the reader is referred to recent reviews of the subject for a more complete treatment. Briefly, the D-0 hydrogen bond is enthalpically favored relative to the H-0 analog by roughly 10%; an offsetting decrease in the entropy of bond formation leads to a free energy difference near zero between the two. Assuming a clatbrate model of water, the transfer of a solute from light to heavy water will be exothermic by an amount related to the enthalpy of clathrate formation. Straightforward extension of the Muller formalism above quantifies the enthalpy of transfer as [13, 341:
A schematic representation relating ligand binding in isotopic solvents can be created as:
AHt,"
AHt,b
\'
Figure 3. Cycle relating binding in isotopic waters. The subscripts represent light (1) and heavy (h) waters, and unbound (u) and bound (b)systems.
Thermodynamic parameters are state functions. Thus the difference in the two horizontal processes of Figure 3-binding in isotopic solvents-is equivalent to the difference in the two vertical processes-transfer of the free and bound systems from light to heavy water. From this formalism it is apparent that the difference in enthalpy of binding in isotopic waters is equivalent to the enthalpy of transfer of the jraction of the ligand and protein desolvated during binding. Because this enthalpy is related to the enthalpy of solvation, the thermodynamic solvent isotope effect is a measure of the enthalpy of binding that derives from removal of the binding site and ligand from solution. Thermodynamic solvent isotope effects have been utilized to evaluate the extent to which desolvation aids ligand binding [35,361. While the assumptions implicit in the calculations likely make precise determination of AHsolvimpossible, the relative role of desolvation for a series of bindings is clearly accessible (Table 5). This exercise facilitates two observations that are likely general. First, a significant fraction
876
31 Protein-Carbohydrate Interaction: Fundamental Considerations
Table 5. Solvation-associated enthalpies of ligand binding [ 351. Binding pair
AG
AH
Dioclea/3,6-di-(0-aMan)aManOMe Concanavalin A/aManOMe Concanavalin A/3,6-di-(0-aMan)aManOMe Vancomycin/diAcLys-D-Ala-D- Ala RNAse/CMP FK506/FKBP
-8.2 -5.0 -7.5 -7.2 -7.5 -12.3
-13.0 -7.1 -10.7
A& -4 -5
-5 -1
-11.5 -14.1
-14
-17.2
-18
of overall binding enthalpy derives from solvent reorganization; this trend is likely repeated in binding free energies. Secondly, the enthalpy derived from solvent reorganization is lower for protein-carbohydrate interaction than for any other interacting system studied. This observation hardly comes as a surprise, given the hydrophilic nature and high aqueous solubility of carbohydrates. On the other hand, the relatively small enthalpy of binding available from saccharide desolvation is the most probable cause of the low binding constants that typify protein-carbohydrate interaction. This limitation is fundamental, severe, and likely insurmountable. Together, the forgoing discussion leads one to a picture of binding that is largely driven by ‘hydrophobic’ effects. In this model, a range of polar interactions are largely responsible for the specificity of the interaction: if key hydrogen bonds, salt bridges and dipolar interactions are satisfied by solute-solvent interactions prior to association but lost during binding, an unfavorable contribution to the overall binding free energy results. If all interactions lost to solvent are adequately replaced in the complex, the net favorable free energy that derives from the desolvation of non-polar surface area drives formation of the complex. Lemieux described such notions two decades ago in the concept of a ‘polar gate’ [37]. More recently, several researchers have shown that a significant fraction of the total surface area of an oligosaccharide ligand can be replaced by non-carbohydrate surface [ 38-40],
31.3 The Evaluation of Protein-Carbohydrate Binding Discussion of the effect of ligand structure on protein-carbohydrate affinity requires an evaluation of complex stability constants. A number of biophysical techniques are appropriate for the study of protein-carbohydrate interaction; many of the more enlightening strategies are the topics of separate chapters elsewhere in this volume. We describe below three techniques used extensively in glycobiologyinhibition of hemagglutination, enzyme-linked lectin assay (ELLA), and isothermal titration microcalorimetry-and we consider the types of information provided by each technique in order to facilitate appropriate interpretation of the data.
31.3 The Evaluation of Protein-Carbohydrate Binding
811
31.3.1 Precipitin Assays
By far the most common of the techniques utilized to evaluate proteincarbohydrate interaction are a group of assays based on the hemagglutination process. Anticoagulated red blood cells spontaneously separate on standing in microtitre plate wells, as the denser erythrocytes settle to the bottom of the wells. Addition of a polyvalent lectin to a suspension of erythrocytes prevents this segregation and results in the formation of a gel-phase cross-linked lattice. This crosslinking process is referred to as hemagglutination, and provides a straightforward read-out for inhibition assays. Assays of protein-carbohydrate binding function by inhibiting this hemagglutination event. Addition of a soluble saccharide sets up a competition between glycoconjugates on erythrocyte surfaces. Typically serial dilutions are performed across a microtitre plate, and the minimum concentration of soluble ligand required to inhibit 50% agglutination is reported as an ZC50. Because the assay is run as a serial dilution errors are typically assumed at one well, or a factor of two. Absolute ZCso values are frequently not reproducible from lab to lab, although the order of inhibitory potency through a series of ligands is generally robust. Agglutination assays evaluate a Straightforward physical process, namely the ability of a soluble ligand to prevent a cross-linking aggregation process. The danger of agglutination assays lies in the assumption that ZC50 values are simply related to Keq for a reversible protein-carbohydrate binding event. At a minimum, the activity of a soluble ligand in an agglutination assay is the inhibition of an irreversible or quasi-reversible phase transition, from a soluble lectin to an ordered cross-linked gel matrix. Ligand binding is surely requisite for the initiation of this event, but all events that follow serve to produce a complicated set of coupled equilibria, each with a distinct microscopic set of kinetic and thermodynamic constants. The situation becomes significantly more complicated when the soluble ligands are themselves multivalent, since now there exists a competitive set of crosslinking/aggregation processes. In the limit, a protein-carbohydrate binding followed by an irreversible aggregation/precipitation will appear in this assay as an exceptionally high affinity association, since the binding equilibrium is coupled with the aggregation/precipitation event. Hemagglutination assays have been and will remain a mainstay of the study of protein-carbohydrate interaction. In many respects the assay is highly relevant to the study of biological processes; Nature doubtlessly uses the high valency of both carbohydrate ligands and binding proteins for a range of functions. On the other hand, ZC50 values from agglutination assays cannot be interpreted in terms of protein-carbohydrate affinity. In instances where protein-carbohydrate interactions have been evaluated by agglutination assay in addition to some other biophysical technique, hemagglutination ZC50 values do not correlate with K, values for the equivalent process. Recently we reported a study of the binding of multivalent saccharide ligands to concanavalin A [41]. While ZC50 values varied by a factor of 30 on a valence corrected basis, binding constants for the same ligands evaluated by titration microcalorimetry varied by less than a factor of two. Rather, hemaggluti-
+
818
31 Protein-Carbohydrate Interaction: Fundamental Considerations
nation IC50 values correlate with entropies of ligand binding, again demonstrating that hemagglutination evaluates an aggregation, rather than a binding, process. 31.3.2 Enzyme-Linked Lectin Assay (ELLA) Roy and coworkers have reported a modification of the precipitin assay [42-501. This assay evaluates competitive binding between soluble and immobilized ligands to a lectin-enzyme conjugate and is essentially a variant of quantitative ELISA. In the enzyme-linked lectin assay a high molecular weight polyvalent ligand is adsorbed to the surface of microtitre plates. The assay has been utilized most frequently for the evaluation of binding to mannose-specific proteins; in such cases yeast mannan serves as the immobilized ligand. Following blockage of non-specific binding with BSA, a lectin-horseradish peroxidase (HRP) conjugate is incubated in the microtitre plate wells with serial dilutions of a soluble ligand. Following an incubation period the microtitre plates are evacuated and the binding mixture replaced with hydrogen peroxide and a pro-dye substrate. Color formation, proportional to the amount of lectin bound to the microtitre plates, is read quantitatively with a UV plate reader. ELLA is designed to obviate the most significant complication of agglutination assays, namely the irreversible formation of aggregates. In theory, the competition between immobilized and soluble ligand should not involve the formation of kinetically trapped aggregates. On the other hand, data from ELLA assays do show evidence of kinetic contributions. Plots of inhibitor concentration versus fractional inhibition frequently asymptote to values less than 100% inhibition. Often different ligands asymptote to different fractional inhibitions, The most obvious explanation for this behavior is the formation of some irreversibly bound lectin species on the surface of the microtiter plate wells. The exact nature of this species is somewhat difficult to imagine, although the use of microheterogeneous polydisperse polysaccharide as an immobilized ligand likely presents a range of binding epitopes for the lectin-HRP conjugate. In summary, it is now clear that precipitin assays do not measure ligand binding. ELLA is a technique designed to overcome the issues that render agglutination unsuitable for evaluating ligand binding. However, until IC50 values obtained through this technique are verified by comparison to binding constants measured by other biophysical techniques, results from the assay should be regarded with some caution. 31.3.3 Isothermal Titration Microcalorimetry Isothermal titration calorimetry (ITC) has long been recognized as a useful tool for the evaluation of binding constants [51]. The field of calorimetry changed markedly during the early 1990s with the introduction of commercial titration microcalorimeters [52].These devices, available from several suppliers, operate with volumes near one to two milliliters. Virtually all the data reported to date on the thermody-
31.3 The Evaluation of Protein-Carbohydrate Binding
819
namics of protein-carbohydrate interaction has been obtained with one of several generations of the Microcal ITC; these instruments have sample cell volumes near 1.3 ml (Figure 4). The sensitivity requirements of the technique mandate a continuous power compensation design rather than the simpler and more traditional passive thermal conductivity experimental approaches. In this design, a sample and reference cell are heated at a constant rate, typically less than 100 microcalories per second. This heating rate alters the temperature of the cell contents by less than 0.1 "C during the course of a titration. The temperature between the two cells is evaluated and a second compensating voltage is applied to bring the two cells into precise thermal equilibrium. Addition of an aliquot of ligand through a syringe into the sample cell disrupts this equilibrium; in turn, the compensating voltage is adjusted to return the cells to equilibrium. The raw data is thus power as a function of time. Integration over time yields the more familiar plot of enthalpy per injection versus ligand concentration. The enthalpy evolved on each injection is a function of the concentrations of the two binding partners, the stoichiometry of the interaction, the binding constant (Keg)and the enthalpy of binding ( A H ) . A simple non-linear least squares fit to the data provides estimates of the binding stoichiometry, binding constant, and enthalpy of binding. The binding constant is related to the free energy of binding by the relationship:
AG
=
-RTInKeq
The entropy of binding is thus available by subtraction. The partial differential of enthalpy with respect to temperature defines the partial change in constant pressure heat capacity. Over short temperature ranges this derivative is approximated as
i3AH AC -AT
'-
AAH AT
-
The required concentrations of both binding partners are determined by the binding constant. The shape of the binding curve is determined by the unitless parameter c, equivalent to the product of the binding constant and the concentration of binding sites within the calorimeter cell. Figure 5 shows curves that result from values of c ranging over 1-1000. While in principle it is possible to fit curves anywhere within this range, values of c of 10-100 give optimal results. Practically, titration microcalorimetry operates to furnish estimates of both enthalpies and free energies of binding for systems with binding constants in the range of lo3 to lo7 M-'. At low affinity, protein solubility becomes limiting; a binding constant of lo3 M-' requires at least millimolar protein in solution. At high affinity, sensitivity becomes limiting. A binding constant of lo7 M-' limits the concentration of binding sites to 0.1 micromolar, which in turn limits the size of individual aliquot enthalpies. Across a complete titration, peak areas (i.e. enthalpies) should average no less than 100 microcalories. The range of lo3 to lo7 M-' thus assumes stable soluble protein and significant enthalpies of binding. The concentration of titrant in the syringe is typically 20-times the concentration of binding sites.
880
31 Protein- Carbohydrate Interaction: Fundamental Considerations
Sensor
?$
Lead Screw
Sensor Injector N
lie--
Plunger
Stirring
Syringe
Outer Shield Inner Shield
-
Reference Cell
Figure 4. The Microcal Omega titration microcalorimeter.
-
Sample Cell
31.3 The Evaluation of Protein-Carbohydrate Binding
-500
!
I
I
I
I
I
0
10
20
30
40
88 I
I
injection number Figure 5. The effect of c on calorimetric curve shape. Data calculated for AH = -6 kcal mol-' and 1 mM binding sites. Circles Keq = lo6; triangles Keq = lo5; diamonds Keq = lo4; squares Keq = 10'.
Even beyond the high affinity limit, calorimetry remains a powerful technique for the study of intermolecular interactions. With stoichiometric binding, enthalpies of binding are still accessible in a straightforward fashion. In this instance, the total enthalpy evolved on each injection is simply divided by the quantity of titrant added. As before, evaluation of the molar enthalpy of binding as a function of temperature provides ACp. Determination of the entropy of binding requires an independent determination of Keq;this protocol offers the additional advantage of avoiding correlated errors. That thermodynamic parameters are state functions provides another powerful methodology for determining both enthalpies and free energies of binding for very high affinity systems through a competitive cycle. If two species bind a single binding site with differing affinities, titration of the higher affinity ligand to binding sites loaded with a lower affinity ligand yields the difference in binding enthalpy and free energy between the two ligands. Thermodynamic parameters characterizing binding
882
31 Protein-Carbohydrate Interaction: Fundamental Considerations
of the lower affinity ligand can be determined independently and the corresponding values for the high affinity ligand are then equivalent to the sum of the two values. An elegant demonstration of this methodology was provided by Sigurskjold and coworkers in a series of papers describing the binding of acarbose to glucoamylase (vide infra) [53-561.
31.4 The Interpretation of Calorimetric Data While the measurement of thermodynamic parameters by ITC is straightforward, interpretation of the derived values is not. It is important to recall that thermodynamic properties represent the sum of all molecular processes that occur during a binding event. A range of intermolecular interactions must be considered carefully before interpreting thermodynamic properties in terms of solute-solute interaction. Below we consider some of the processes that contribute to net measured thermodynamic processes and, where possible, describe experimental techniques for the evaluation of the contribution of each set of microscopic events to overall ligand binding thermodynamics.
31.4.1 Solvation/Desolvation It has always been fashionable to reach conclusions regarding the role of solvation during association processes based on measured enthalpies and entropies of binding. Processes with favorable entropic components are frequently referred to as ‘entropy driven’-indicative of a ‘hydrophobically driven’ binding-while processes with large favorable enthalpies of binding are referred to as ‘enthalpy driven’; such a pattern is often interpreted in terms of favorable hydrogen bonding or salt bridge formation. There is no basis for this distinction. The predicted magnitude and sign of enthalpies and entropies arising from desolvation of both ligand and binding site are unclear. Indeed, the thermodynamic parameter most uniquely associated with solvation processes in aqueous solution is ACp. The compensating dependence of both enthalpy and entropy on this term requires that over a sufficient temperature range both the magnitude and sign of a measured enthalpy and entropy will change. Surely, however, the same physical processes aid and oppose binding over this range. Two methodologies allow independent evaluation of the role of solvation in ligand binding processes. The first requires measurement of thermodynamic solvent isotope effects, the differential enthalpy of binding in light and heavy water [33]. The fraction of the enthalpy of binding arising from solvent reorganization is directly related to this quantity. In principle, an absolute solvation-associated enthalpy is obtainable through this approach; in practice, derivation of such a value requires the use of a proportionality constant not known with either precision or accuracy. Thus the absolute value of solvation-associated enthalpies derived in this
31.4 The Interpretation of Calorimetric Data
883
way likely have substantial error associated with them. On the other hand, the error is a constant, deriving from a poor understanding of the precise difference in enthalpies between the H-0 and D-0 interaction. Comparative thermodynamic solvent isotope effects are thus free of this error. TSIE is thus most useful for evaluating the effect of ligand modification on binding thermodynamics [36]. The fraction of an overall enthalpy of binding attributable to solvation/desolvation for two ligands is readily compared by comparing solvent isotope effects. In this way assignment of changes in the enthalpy of binding in response to a change in ligand structure to either changes in solute-solute interaction or to changes in ligand solvation is straightforward and unambiguous. We have previously shown that thermodynamic solvent isotope effects correlate linearly with the change in molar heat capacity that accompanies binding [35].This correlation demonstrates that ACp is exclusively a measure of solvation, and comparison of AC, values for related ligands allows a similar interpretation of differences in binding enthalpies in terms of protein-carbohydrate interaction versus differences in solute-solvent interactions prior to association [ 571. The relative random errors of the two methodologies are unclear; in the one instance small values must be measured accurately while in the other errors in individual determinations are propagated during curve fitting. In any event, a meaningful discussion regarding the molecular origin of measured thermodynamic parameters is impossible without some evaluation of the contribution of solvation to overall measured enthalpies of binding. The entropy of binding arising from solvation is also available with knowledge of the change in molar heat capacity accompanying binding. Overall entropies of binding can be conceptually dissected to describe the contributions of changes in translational and rotational entropy, solvation-associated entropy, and changes in entropy from restriction of conformational degrees of freedom during binding:
Separation of overall entropies of binding into component segments in this fashion again allows relatively sophisticated conclusions to be drawn regarding the molecular origin of measured thermodynamic parameters. In this formalism, entropies of binding are divided into three contributions and knowledge of any two of the three provides the third by subtraction. Evaluation of ASsolv and AS,,, are both feasible. Solvation Entropy It has long been recognized from efforts to understand protein folding that solvation associated entropies are related to changes in molar heat capacity accompanying binding. Thus,
where T is the temperature at which the entropy of binding was measured and T * is the so-called entropy convergence temperature, or the temperature at which all
884
31 Protein- Carbohydrate Interaction: Fundamental Considerations
solvation associated entropies approach zero [ 58, 591. AS*solv includes contributions to entropy from proton transfer and electrostatic effects; for most proteincarbohydrate interactions this term is negligable. Evaluation of ACp thus allows extraction of the fraction of the total entropy of binding attributable to solvent reorganization. Note that this term is temperature dependent; again, this dependence makes intuitive sense. As the temperature of the system is raised the residual order of water slowly dissipates, and at 385.15 K disappears entirely. Translational/RotationalEntropy
A major fraction of the entropy of a particle derives from its ability to translocate in three dimensions and rotate on three axes. The magnitude of this entropy varies as the logarithm of the particle mass and the principle moment arms describing the distribution of mass. Because of the logarithmic relationship ligand binding proceeds with a loss of entropy roughly equivalent to the translational and rotational entropy of the smaller particle. The magnitude of this term has long been a contentious issue. Evaluation of A&+R in the gas phase is straightforward, and follows from the Sakur-Tetrode equation. Transfer to condensed phase, however, greatly complicates the matter. All solutions show greatly reduced molecular motion relative to the gas phase. Water is especially severe with regard to the restriction of molecular mobility: as a highly associated liquid with a tremendous cohesive energy density, water interacts strongly with both adjacent water molecules and with dissolved solutes. A wide range of values of the contribution of AST+R to complexation entropies has been suggested; these are values of 7-50 eu (Table 6 ) . The entropy values reported in Table 6 correspond to the best estimates of the transla-
Table 6. Reported values of AST+R. Investigators
AST+R~
TAS~
Doty & Myers Dunitz Erickson Horton & Lewis Janin Janin & Chothia Jencks Murphy et al. Page & Jencks Searle & Williams Searle et al. Spolar & Record Tidor & Karplus Williams et al.
122 7 (0-7) 23-37 21 100 (50) 57-74 45 (35) 8 40-50 (25-35) 40 (0-40) 40 40-60 100 (77) 39
36.4 2.1 7-1 1 6.2 30 (15) 17-22 13.4 (10.4) 2.4 12-15 (7.5-10.4) 12 12 11.9-17.9 30 (23) 11.5
ae.u. bkcal mol-' at 298K
xnta
0-7
50 10
15 0-40
23
Ref. 60 61 62 63 64 65-67 68 58 69 70 71 72 73 74
31.4 The Interpretation of Calorimetric Data
885
tional and rotational entropy loss during association. Many of these values refer to total immobilization, i.e. total loss of translational and rotational entropy with no account for residual internal entropy. In those cases for which an estimate of the residual internal entropy was made, the corrected translational and rotational entropy loss has been placed in parentheses after the value for total immobilization, and the compensating internal energy (Sint)has been noted. In order to interpret changes in solution thermodynamics in response to changes in ligand structure, we require ASsolv and ASconf.With a methodology for evaluation of ASsolv in hand knowledge of AST+R would provide ASconf by subtraction. Although accurate knowledge of AST+R remains elusive, it is important to recall that values of AST+R are relatively insensitive to molecular size and shape. Accordingly, an arbitrary value of AST+R can be selected for the purpose of this exercise; this value will remain constant across a set of ligands. Changes in entropies of binding arising from ligand modification can thus confidently be ascribed to changes in solvation-associated entropies, or interactions of solvent and solute prior to association, or to changes in conformational degrees of freedom in ligand and protein during binding. Again, the range of values in Table 6 precludes extraction of absolute contributions to overall measured entropies arising from the loss of conformational degrees of freedom. 31.4.2 Other Contributions to Thermodynamics of Association Proton Transfer Many ligand binding events proceed with the transfer of a proton, either from or to buffer; in such instances the enthalpy of buffer ionization will be included in measured values of AHB. Enthalpies of buffer protonation range from near zero to greater than 10 kcal mol-' (Table 7) [51]. Evaluation of enthalpies of binding as a function of buffer ionization enthalpy is a straightforward method of determining the extent of protonation contributions to overall binding enthalpies. A plot of measured enthalpies of binding versus buffer ionization enthalpy provides a plot with a slope equal to the number of protons transferred during binding, and a yintercept equal to the enthalpy of binding in the absence of buffer ionization. Salt Effects/Binding Site Reorganization In some instances, a binding site undergoes reorganization during association. An equilibrium can be written describing the enthalpy, entropy and free energy difference between the two states. Ligand binding thus involves a coupled equilibrium, and the reported values of thermodynamic properties are sums of those values for binding site reorganization and ligand binding to the preformed site. If environmental conditions affect the position of this equilibrium, the precise conditions under which binding is measured will affect the measured enthalpy of binding. An example of such an effect is observed in the binding of mannosides by concanvalin A [36]. Here, a salt bridge between Asp16 and Arg288 in the unbound form of the protein is ruptured during ligand binding, a process that proceeds with an unfa-
886
31 Protein-Carbohydrate Interaction: Fundumentul Considerations
Table 7. Enathalpy of ionization of common buffers [51]. Trivial Name
Formula
AHB"
MES
O/ N L +J H C H ~ C H ~ S O ~ -
12.68
bis-tris ACES ADA
(HOCH2)3CN+H(CH2CH2OH)2 H2NCOCHzNfH2CH2CH2SO3H2NCOCH2N+H(CH2COO-)l
29.25 30.12 11.51
MOPS
O~+HCH~CH~CH~SO~-
19.0
PIPES
Na03SCH2CH2NnN+HCH2CH2S03LJ
8.70
BES
(HOCH2CH2)2N+HCH2CH2SO3-
23.10
HEPES
HOCH2CH2HN+r\NCH2CH2SOju
16.40
TES Ethyl glycinate Glycinamide
(HOCH2)3CNtH2CH2CH2S03H3NfCH2COOCH2CH3 H3N+CH*CONH2
29.25 46.32 44.77
HEPPS
HOCH2CH2 HtNC\NCH2 CHzCH2SO3
17.95
Tricine THAM Glycylglycine Bicine TAPS N,N-Dimethylglycine
(HOCH2)3CN+H2CH2COO(HOCH2)3CN'H3 H3N+CH2CONHCH2COO(HOCH2CH2)2N+HCH2COO(CH3)2N+HCHzCOO-
30.50 47.28 43.72 26.23 40.12 31.51
CAPS
cNtH2CH2CH:CH2S03-
48.53
(HOCH2)3CNtH2CH2CH2CH2S03-
"kcal mol-'; enthalpy of removing the H from the quaternary nitrogen atom
vorable enthalpy. Increasing ionic strength cleaves this intramolecular salt bridge prior to ligand binding, diminishing the unfavorable contribution to the overall enthalpy of binding. As a consequence, enthalpies of mannoside binding vary by a factor of two over a salt range from 5 mM to 1 M.
31.4.3 van't Hoff versus Calorimetric Enthalpies A measured difference between van't Hoff and calorimetric enthalpies of binding has been offered as a possible test of contributions other than ligand binding to calorimetric enthalpies of binding [75-771. van't Hoff enthalpies of binding are determined from the temperature dependence of the binding free energy according to the expression: AHVH
1nK = 1nA - RT where A is a pre-exponential term related to the entropy of binding. A plot of 1nK
31.5 The Thermodynamics
of'Protein
Carbohydrate Interaction
887
versus 1/T thus gives a slope equivalent to AHU"/R, This relationship has been utilized extensively by the protein folding community to observe coupled equilibria unrelated to folding. There is, however, a range of problems associated with its use in ligand binding. First, associations in aqueous solution universally proceed with a change in molar heat capacity, i.e. ACp # 0. The expression above then is predicted to be nonlinear, since A H is itself a function of temperature. The fit required to extract A H Uis~thus a non-linear regression for two variables using values that have themselves been extracted from a non-linear regression for three variables. Appropriate permutation of errors almost certainly precludes observation of all but extraordinarily large differences between the calorimetric and van? Hoff enthalpies with any degree of confidence. Secondly, it is somewhat difficult to devise a physical model that corresponds to a measured difference. van't Hoff enthalpies will differ from calorimetric enthalpies only to the extent that calorimetric enthalpies contain contributions from processes that do not contribute to the free energy of binding. Because thermodynamic processes are state functions, this condition can hold only for situations where perfect enthalpy-entropy compensation exists, and even then only if the compensating terms have equivalent temperature dependencies. Because ACp affects both A H and A S , this condition can be true only if A S for the compensating process is large compared to ACp. Thus while in principle differences between van't Hoff and calorimetric enthalpies contain important information regarding the precise molecular processes that occur during binding, such differences should be regarded cautiously.
31.5 The Thermodynamics of Protein-Carbohydrate Interaction Having considered those intermolecular interactions that contribute to overall net measured thermodynamic parameters, we turn now to a review of thermodynamic measurements of protein-carbohydrate binding reported during the last five years. Values reported prior to this time can be found in earlier reviews. Calorimetrically derived changes in enthalpies, entropies, free energies and molar heat capacity that occur during protein-carbohydrate binding are shown in Table 8. Extension of the database of known protein-carbohydrate thermodynamic parameters continues to reinforce the basic concepts identified some time ago. Virtually all of the examples in Table 8 show binding free energies of 4-8 kcal mol-', corresponding to association constants of 103-105 M-'. Typically, enthalpies of binding are more negative than the corresponding free energies of binding and the entropic contribution to AG is unfavorable. The database of changes in molar heat capacity accompanying protein-carbohydrate binding remains small, but continues to grow. Again, in almost all cases exceptionally small values of ACp are recorded, almost always less than 200 cal mol-' deg-', and usually less than 100 cal mol-' deg-'. A small number of values in Table 8 are positive; this observation is unusual. In most instances the errors in individual values of both A H and the A H versus T fits are large and the validity of these data is unclear. There is at least some correlation between ACp and AG. Thus, for example, the binding of acarbose to glu-
fragment free concanavalin A concanavalin A
Succinyl concanavalin A
CeNumonasJimi CBD
Heparin Heparin Heparin Heparin Heparin
Antithrombin 111 K'2'-A'34 Antithrombin 111 K"'AA Antithrombin I11 K'25AA Antithrombin 111 R'29AQ Antithrombin I11 K'33AA CBDNl
-AHb
34.5 14.9 36.0 48.6 57.5 9.7 PG~c(~-~~)[PG~c(~-+~)]~G~cOH 12.7 PGk( 1--+4)[PGl~(l-+4)]3Gl~OH 13.0 PGlc(l-+4)[PGl~( I+4)]4GlcOH Barley a-glucan 13.7 14.4 Oat P-glucan 13.4 Hydroxyethyl Cellulose Acid Swollen Cellulose 7.7 Cellulose I 1.2 Cellulose I1 0.2 14.5 1 2 13.8 5.0 3 14.3 Mana(l+6)[Mana(l-3)]-Man aMeGlc 5.3 6.6 6.7 6.8 aMeMan 8.2 8.4 aMe2dMan 7.2 5.9 C-allylglucose 5.9 C-allylmannose 1 8.5 2 11.1 3 6.2
Ligand"
Protein
Table 8. Thermodynamics of protein-carbohydrate binding.
-28.0 -7.6 -31.2 -45.8 -53.9 -4.1 -6.6 -6.9 -7.4 -8.2 -7.3 -1.8 9.7 8.2 -9.7 -8.7 0.2 6.7 -0.7 -2.1 -2.1 -1.5 -2.9 -2.8 -1.9 -0.2 -0.8 -3.5 -5.0 -1.0
TASb 10.1 4.5 4.3 4.1 4.8 5.0 6.1 6.1 6.3 6.2 6.1 5.9 10.8 8.4 4.8 5.1 5.2 31.6 4.6 4.5 4.6 5.3 5.3 5.6 5.2 5.0 5.1 5.0 6.1 5.2
-AG~
-103
-50.0 -61.9
ACpc
81
78 78 78 78 78 79 79 79 79 79 79 79 80 80 81 81 81 82 81 82 83 81 82 84 83 81 81 81 81
Ref.
aGlcOPh 1dGlc aGlcF 2dGlcOH
3,4dMana(1+3)[Mana(1+6)]2,4dManOMe
Mana( 1+3)[Mana(l-6)]ManOMe 2dMana(l+3)[Mana( lk6)lManOMe
3dMana(l+3)[Mana(l+6)]4dManOMe 3dMana(l+3)[Mana(l+6)]2,4dManOMe
3dMana( 1+3)[Mana( 1+6)]2dManOMe
6dMana(l+6)[Mana(l-+3)]ManOMe Mana(l+3)[Mana(l+6)]2dManOMe Mana(l+3)[Mana(1+6)]4dManOMe
2dMana(1+6)[Mana( 1+3)]ManOMe 4dMana(l+6)[Mana(1+3)]ManOMe
2dMana(l+6)[Mana(l+3)]-ManOMe
4dMana( 1+3)[Mana( 1+6)]-ManOMe
aGlc( 116)GlcOH aMe2dGlc Mana( 1i 2 ) M a n Mana( 1j 2 ) M a n O M e Mana( 1+2)Mana( l i 2 ) M a n Mana( 1i 3 ) M a n Mana( 1+3)ManOMe Mana( 1-6)Man Mana( lh6)ManOMe GlcNAcP(1-2)Man Mana(1+6)[Mana(l+S)]-Man 4
aMeGlcNAc aGlc( 114)GlcOH 6.2 6.2 6.2 6.7 7.3 9.9 10.5 10.7 10.2 10.7 9.4 8.4 5.3 14.1 10.6 10.6 12.3 14.9 14.0 11.2 11.7 11.6 13.4 12.1 10.6 9.7 8.7 14.7 14.1 8.9 3.5 4.2 7.1 4.8 -2.1 -1.9 -1.9 -1.6 -2.6 -3.6 -3.5 -3.1 -4.5 -4.5 -3.8 -3.1 -0.1 -6.6 -2.2 -2.2 -5.5 -7.1 -6.2 -4.9 -5.7 -5.5 -6.5 -5.7 -4.4 -3.8 -3.2 -7.1 -6.9 -3.3 1.8 0.7 -4.2 -1.4 4.1 4.3 4.3 5.1 4.7 6.3 7.0 7.6 5.7 6.2 5.6 5.3 5.2 7.5 8.4 8.4 6.8 7.8 7.1 6.3 6.0 6. I 6.9 6.4 6.2 5.9 5.5 7.6 7.2 5.6 5.3 3.5 2.9 3.4 -19.4 -6.7 41.1 1.8
82 82 83 83 82 82 82 82 82 82 82 82 82 82 82 84 85 85 84 85 85 85 85 85 85 85 85 84 84 84 76 76 76 76 W
00
00
z
Y
Ni-concanavalin A
Co-concanavalin A
Protein
Table 8 (continued)
2-OMe aManOMe 2-OEt aManOMe 2-OPr aManOMe 2-OBn aManOMe 3-OMe aManOMe 2,3-diOMe aManOMe 3-OMe aMan(l-3)-2-OBn aManOMe aGlcOMe 5 aMan (lk2)aManOMe aMan(1-3)aManOMe aMan(144)aManOMe aMan(1-6)aManOMe aGlc(1-4)GlcOH aGlc(1+6)GlcOH aMan(l+3)[aMana(l+6)]-aManOMe aManOMe ManOH aGlcOMe GlcOH aManOMe ManOH aGlcOMe GlcOH
2FGlcOH 3FGlcOH aManOMe
Ligand" 6.5 3.8 6.8 6.8 4.8 2.8 4.2 6.2 5.3 6.8 7.5 5.3 5.9 7.0 7.4 7.4 6.9 3.7 4.6 10.2 6.4 5.3 4.8 3.6 6.2 4.0 4.5 3.4
AH^ -3.0 -0.7 -1.5 -1.5 0.4 3.4 1.2 -0.2 -0.3 -2.0 -1.7 -0.7 -0.7 -0.2 -1.4 -2.3 -1.6 0.7 -0.2 -2.8 -0.9 -1.0 -0.1 0.1 -0.6 0.6 0.0 0.3
TAS~
3.5 3.1 5.3 5.3 5.2 6.2 5.4 6.0 5.0 4.8 5.8 4.6 5.2 6.8 6.0 5.0 5.3 4.4 4.4 7.4 5.5 4.4 4.6 3.1 5.6 4.6 4.5 3.6
-AGb
-93.0
-44.0
-110
40.8 -9.6 -50.0
ACpc
76 76 36 86 86 86 86 86 86 86 86 36 36 36 36 36 36 36 36 36 87 87 87 87 87 87 87 87
Ref.
10
co
g'
'
5
$
9
& $
'$
%'
Z
2
.1"
$
1
3 g.
2
0
Dioclea grandi3ora
concanavalin A (pH 5.2)
Cd-concanavalin A
a M a n ( l i 3 ) [ a M a n (lh6)laManOMe
aMan(1-4)ManOMe aMan(1+6)ManOMe
aMan(143)ManOMe
aGlcCally1 aManCal1yl a2dGlcOMe aMan (1+2)ManOMe
aManOMe
aManOMe ManOH aGlcOMe GlcOH ManOH aMan(1-3)ManOH aMan (146)ManOH aMan(l+3)[aMan (1+6)]aManOMe aGlcOMe
7.5 5.5 4.6 3.9 5.0 8.4 7.2 14.2 4.4 5.0 4.4 7.8 8.2 7.8 2.4 6.3 7.5 9.9 6.4 10.1 11.4 8.4 8.4 8.6 16.2 13.0 15.2 11.0 14.6 15.1 12.8 10.0 10.4 9.8
-2.2 -1.0 -0.1 -0.1 -0.5 -2.7 -1.6 -6.8 -0.2 -0.3 -0. I -1.5 -3.3 -2.9 2.0 -1.7 -2.9 -3.8 -0.5 -4.3 -5.1 -3.5 -3.3 -3.6 -7.9 -4.8 -7.2 -4.5 -6.8 -7.1 -5.5 -4.0 -4.5 -4.0
5.3 4.5 4.5 3.8 4.5 5.6 5.7 7.4 4.2 4.7 4.2 4.8 4.9 4.8 4.4 4.6 4.6 6.1 5.9 5.8 5.5 5.0 5.1 5.1 8.3 8.2 8.0 6.5 7.8 8.0 7.3 6.0 5.9 5.8 -96.0
-22.0
-40.0
-56.0
87 87 87 87 88 88 88 88 81 84 36 81 84 36 81 81 84 84 36 84 36 36 84 36 84 36 84 84 84 84 84 84 84 84
Erythrina corallodendron
Protein
Table 8 (continued)
7.3 6.8 5.7 6.4 4.7 4.7 5.2 4.3 4.2 3.6 3.9 3.9 4.2 4.4 4.3 4.4 4.4 4.2 4.3 4.3 4.4 4.7 4.5 5.0 5.8 5.5 5.4 5.0 5.0 4.9
-7.5 -6.0 -4.2 1.8 -1.4 -1.6 -1.7 -0.1 -0.2 -0.8 -1.0 -0.5 -2.9 -1.2 -1.2 0.9 1.1 -1.1 -0.9 -2.5 -1.9 -5.7 -5.4 -5.9 -6.8 -5.5 -6.0 -6.3 1.1 0.6
14.8 12.8 9.9 4.6 6.1 6.3 6.9 4.4 4.4 4.4 4.9 4.4 7.1 5.6 5.5 3.6 3.3 5.3 5.2 6.8 6.3 10.4 9.9 10.9 12.6 11.0 11.3 11.3 3.9 4.3
aMan (1 -+3)[aMan(1+6)]-2daManOMe aMan (1+3)[aMan (1+6)]-4daManOMe 3,4daMan (1+3)[aMan(l+6)]2,4daManOMe
pGal(1+4)PGlcNAcOMe aFuc( 1+2)PGal( 144)GlcOH
PGa1(1-+4)GlcNAcOH
PGalNAcOMe PGa1(1-+4)GlcOH
aGalOMe
GalOH
GalNAcOH
aGlc ( I -4)GlcOH aGlc (1 +6)GlcOH 5 aGlc (144)GlcOH aGlc( 1+6)GlcOH PGalOMe
4
-AGb
TAP
-AHb
Ligand"
93.2
ACp'
84 84 84 84 83 83 36 36 36 89 90 90 89 90 YO 90 90 90 90 89 89 90 90 89 89 90 90 89 90 90
Ref.
5'
f
9 $ $
2
$ & 2
o
3
'2.
3
2
2
$
$
1
g.
2
2
\D h,
co
Gal- 1 (dimer)
FGF-1 cyclic mimic FGF-1 Native Binding Site FGF- 1: D-Pro 136 Gal-1 (Bovine Spleen)
Erythrina indica
Erythrina cristagalli
bGal(1+4)PGlcNAcOMe Heparan Sulfate Heparan Sulfate Heparan Sulfate PGdl (1-4)Gk pGal(1-4)pGlcOMe pGa1(1-+4)FrucOH pGd( 144)ManOH pGal(1t3)AraOH pGal( 1-3)GlcNAcOH 2-OMe pGal (lh4)GlcOH Galp( 144)GlcNAcOH Thio Galp(l+l)PGal OGal(1+4)GlcNAcOH Dithiogalactoside
PGal(1+4)PGlcNAcOMe PGalOMe GalNAcOH PGalNAcOMe pGa1(114)GlcOH
PGalOMe GalNAcOH PGalNAcOMe pGa1(1+4)GlcOH pGal(1+4)GlcNAcOH
FucOH
aMe-N-dansylcalaminide
1.1 4.7 7.3 6.8 6.0 10.9 12.8 10.3 5.9 4.9 5.2 10.4 7.4 13.7 10.9 10.4 24.4 23.2 22.9 5.0 7.1 8.2 8.4 8.8 7.1 7.3 7.8 11.1 6.6 3.8
1.o
4.4 5.5
2.9 2.1 2.6 2.5 -0.7 -2.9 -2.5 -1.1 -5.4 -6.5 -4.8 -2.1 -0.7 -0.8 -5.6 -2.7 -7.8 -5.1 -4.8 -16.9 -15.9 -15.9 0.2 -2.0 -2.9 -3.2 -3.7 -1.3 -1.5 -2.3 -5.6 -1.4 0.9
7.3 7.6 3.6 3.6 4.1 4.4 4.3 4.9 5.6 6.3 5.5 3.8 4.2 4.4 4.8 4.7 5.9 5.9 5.7 7.5 7.3 7.1 5.2 5.0 5.4 5.5 5.1 5.7 5.8 5.5 5.5 5.2 4.8 66.3 6.7 12.5 -48.6 -20.0 -35.1 -29.1 -57.1 -82.4 -88.4
-244
-200
105
89 89 89 89 89 91 91 91 75 75 75 75 75 75 75 75 75 89 89
90 90 90 90 89 89 89 89 89 89 89 89 89 89
w
\o
co
=. 2
f:
3
%
9
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8.A
2
q
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rp
3 3
$
GI : Gly 174Cys
G1: Thr173Gly
G 1: Gln 172Asn
GI: Asnl71Ser
GI: Trpl20Phe
GI: Arg54Lys GI: Arg54Leu GI: Serll9Tyr
GI: Trp52Phe
GI: Tyr5OPhe
Gal-] triple mutant Glucoamylase G1
Gal-I C2S mutant
Protein
Table 8 (continued)
~~
~
Acarbosed 1-Deoxynojirimycin Acarbosed 1-Deoxynojirimycin Acarbosed Acarbosed Acarbosed 1-Deoxynojirimycin Acarbosed 1 -Deoxynojirimycin Acarbosed 1-Deoxynojirimycin Acarbosed 1-Deoxynojirimycin Acarbosed 1-Deoxynojirimycin Acarbosed 1-Deoxynojirimycin
9
pGal(1-+4)GlcNAcOH Dithiogalactoside PGal(1+4)GlcNAcOH 1-Deoxynojirimycin Acarbosed Methyl a,&acarviosinide P-cyclodextrin 6 7 8
Ligand" 2.8 2.6 0.6 1.7 7.9 7.4 12.9 20.2 21.4 22.5 16.5 9.5 2.4 12.7 4.5 9.9 8.6 7.2 2.2 6.9 6.9 12.3 3.6 11.3 3.3 12.8 4.1 8.4 2.4
-AHb 1.9 2.1 4.8 4.8 8.6 2.1 -6.5 -10.2 -11.2 -11.8 -5.6 7.3 3.9 2.9 0.6 -0.4 0.4 9.3 4.5 2.7 -0.7 4.3 2.9 5.8 2.8 4.0 2.4 8.8 4.0
TASb 4.8 4.7 5.4 6.4 16.5 9.5 6.4 10.0 10.2 10.7 11.0 16.8 6.2 15.6 5.1 9.5 9.0 16.4 6.7 9.6 6.2 16.7 6.5 17.0 6.1 16.8 6.5 17.2 6.4
-AG~ ACPC
~
89 89 89 53 53 53 55 55 55 55 55 56 56 56 56 56 56 56 56 56 56 56 56 56 56 56 56 56 56
Ref.
5.
@
2
,"
-
2 g.
&
6'
'2
&
2
3
2
$
g
Q
1
2 2 g.
2
P
v,
m
Lentil Lectin
Hevein
Gyrase B
G2: Tyrl75Phe
Glucoamylase C 2
G1: Trp317Phe GI Starch Binding Domain
G1: Arg305Lys G1: Asp309Glu
G1: Serl85His
G1: Glul80Gln
G 1: Asp 176Asn
8 9 Acarbose' 1-Deoxynojirimycin Clorobiocin Novobiocin PGlcNAc( 1-4)GlcNAcOH PGlcNAc( 1+4)PGlcNAc( 1-+4)GlcNAcOH aGluOPh ldGlu aGluF
7
6
Acarbosed 1-Deoxynojirimycin Acarbosed 1-Deoxynojirimycin Acarbosed 1-Deoxynojirimycin Acarbosed Acarbosed 1-Deoxynojirimycin Acarbosed GlcSGlc2 GlcSGlc3 GlcSGlCd P-cyclodextrin 1-Deoxynojirimycin Acarbose' D- Gluco-dihydroacarbose L-Zdo-dihydroacarbose aSGlc( 1+4)aGlcOMe cLSGlc(1+4)BGlcOMe aS,SGlc( lh4)aGlcOMe
9.4 3.2 4.7 3.4 13.0 5.4 7.3 1.4 2.8 8.5 9.1 17.7 11.1 13.1 2.7 9.7 7.1 2.3 1.4 3.3 1.4 8.0 7.6 6.5 9.9 8.9 2.9 8.8 9.3 4.9 6.0 2.6 3.6 5.9
6.0 2.8 1.4 2.4 2.2 0.4 -0.5 6.1 3.7 2.4 3.4 13.2 6.4 6.6 3.4 6.6 3.2 5.0 4.8 1.9 3.4 >3.0 >3.4 >4.3 >1.1 9.2 2.5 1.o 0.8 -1.2 -0.2 0.7 -1.1 -3.4
15.4 6.1 6.1 5.8 15.2 5.0 6.8 14.2 6.5 10.9 4.3 4.5 4.7 6.5 6.2 16.3 10.3 7.3 6.2 5.2 4.8 >11.0 >11.0 >I03 >11.0 18.0 5.3 9.8 10.1 3.8 5.8 3.3 2.4 2.5 -23.4
-64.5 -83.7 - 12.1 -9.0 -44.7
-200
56 56 56 56 56 56 56 56 56 56 54 54 54 54 53 53 53 53 53 53 53 55 55 55 55 56 56 92 92 93 93 76 76 76 s
4 g.
3
?
e
$
Y
Ricin cornrnunis Agglutinin
Peptide: YYWIGIR-NH2
Pea Lectin
MBP (E. coli)
MBP-C (rat liver)'
MBP-A (rat serum).
Protein
Table 8 (continued)
ThiodiGal
PGal(1+4)GlcOH
OMeGlcNAc NAcYD(G-(CH&-Man)2 aGlc( 1+4)GlcOH 0-cyclodextrin aGlcOPh 1dGlu aGluF 2dGluOH 2FGluOH 3FGluOH 30MeGluOH sLeX LeX aNeuAc(2-.3)PGal( 1-t4)GlcNAcOH pGa1(I +4)GlcNAcOH
2dGluOH 2FGluOH 3FGluOH 30MeGluOH aManOMe OMeGlcNAc NAcYD(G-(CH2)6-Man)2 aManOMe
Ligand" 4.3 3.7 3.5 2.9 4.7 5.2 5.2 5.1 0.0 4.1 7.4 2.8 3.0 3.0 4.3 4.3 5.0 3.7 3.0 3.4 73.7 18.6 3.2 6.9 6.6 7.6 8.1 7.5 12.2 12.0
AH^ -1.7 -0.6 -1.3 1.6 -0.9 -1.4 -1.4 -1.3 0.4 -0.9 -1.5 10.9 4.4 0.9 -1.4 -1.4 -1.9 0.0 0.0 1.2 -67.3 -12.4 0.9 -0.89 -0.6 -1.7 -1.6 -2.1 -6.8 -6.5
TAS~
2.6 3.1 2.2 4.5 3.8 3.8 3.8 3.8 0.4 3.8 5.9 8.1 1.4 3.9 2.9 2.9 3.0 3.6 2.8 4.6 6.4 6.3 4.1 6.0 6.1 6.0 6.0 6.0 5.4 5.5
-AGb
-207 162 -4.5 11.0 -5.0 -40.3 -46.3 8.7 -1.8
-4.8 -33.4 -17.4 -53.5
ACpc
76 76 76 76 94 94 94 94 94 94 94 95 95 76 16 76 76 76 76 76 96 96 96 97 97 91 97 91 97 91
Ref.
Soybean Agglutinin
Snowdrop Lectin
Sheep Spleen Lectin (L-14)
Se 155.4
PGalOMe
aMan(l+3)[aMan(l+6)]uManOMe
GalOH aGalOMe PGalOMe aMan(1 i 3 ) u M a n O M e aMan(1+6)aManOMe
MumaGal
MumSLac
pGal(144)GlcNAcOH
ThiodiGal
14
12 13
11
MumbPGalNAc GalOH 10
SGalOMe
uGalOMe
MumbPGal
MumbaGal
5.8 5.2 9.5 10.4 5.3 5.3 5.4 5.5 7.2 5.8 6.8 8.8 8.2 8.6 7.0 12.9 11.5 10.0 10.5 8.6 10.3 8.8 8.6 2.4 2.2 0.6 0.7 0.5 3.1 2.1 3.9 10.6 8.9
-1.4 -0.8 -3.6 -4.6 -0.9 -1.2 -0.3 -0.8 -1.7 -1.4 0.3 -1.8 -1.2 -1.5 0.7 -1.4 -5.5 -3.8 -4.3 -2.9 -4.5 -3.6 -3.4 2.4 2.9 -1.7 -1.7 -1.7 1.7 2.1 0.9 -6.9 -4.9 4.4 4.5 5.9 5.8 4.3 4.4 5.0 4.8 5.5 4.3 7.1 1.0 7.0 7.0 7.6 5.5 6.0 6.2 6.2 5.7 5.7 5.3 5.3 4.8 5.0 2.4 2.4 2.2 4.8 4.2 4.8 3.1 4.0 -93.2
99 99 99 99 99 36 36 36 89 89
99
98 99 99 99 99 99
91 97 97 97 97 97 97 97 97 97 98 98 98 98
W 4
00
'3
$
is9 %
g.
2
q
$,2
3
'2
R
9
9.5 13.9 12.1 5.5 8.2 1.5 4.7 6.3 5.1 5.1 6.2 5.9 3.9 5.0 6.5 5.8 6.1 7.0
GalNAcOH PGalNAcOMe
PGal( 1-4)GlcOH PGal( 1-4)GlcNAcOH pGal( 1-14) PGlcNAcOMe PGlcNAc( 1+4)GlcNAcOH PGlcNAc( I -+4)PGlcNAc( 1+4)GlcNAcOH PGlcNAc( 1+4)[ PGlcNAc( 1+4)]2GlcNAcOH PGICNAC(1+4)[ PGlcNAc( 1 +4)]3GlcNAcOH GalOH 1 HGal 2HGal FucOH alFGal 2FGalOH 6FGalOH 20MeGalOH
-AHb
Ligand" -4.1 -8.0 -5.9 -2.4 -4.3 -3.8 -0.8 -1.2 0.5 0.8 -1.8 -1.6 -0.7 -1.5 -2.1 -1.6 -1.9 -1.8
TASb
3.9 5.1 5.6 5.9 4.4 4.3 3.2 3.5 4.4 4.1 4.3 5.2
3.1
5.4 6.0 6.2 3.2 3.9
-AGb
-36.4 8.4 -23.2
-13.1 -3.0 -6.0 24.9
- 100
ACp'
100 100 101 101 101 101 101 101 101 101
100 100
89 89 89 89 89 89
Ref.
"structures of ligands 1-14 are shown in Attachment to Table 8; d = deoxy; reducing sugars are assumed to be equilibrating mixtures of anomers bkcal mol-' cal mol-' deg-' by displacement data obtained by fixing n
Winged Bean Agglutinin I
Urtica dioica Agglutinin'
Protein
Table 8 (continued)
zri
6' 3
5-9
$.
0
n
E ??
2
g.
2
6'
2
g
2
B
.$
g
1
2 g.
2
w \o w
899
31.6 The Role of Multivalency in Protein-Carbohydrate Interaction Attachment to Table 8: Ligands 1-14. OH
0
0
"3 0
0
HO&OEH
HO
OH
H z + H o o p ~ H
0
0
p
OH HO OH
1
2
p1-2
GlcNAc -M a n q l - 6 ManPI-2 / GlcNAc -Man a?-3
OH
5
4 -OH
HyA:
HO
3
900
31 Protein-Carbohydrate Interaction: Fundamental Considerations
Table 9. Acarbose binding by displacement method [53]. Enzyme
Ligand
K"
G2
1-deoxynojirimycin acarbose (apparent)" acarbose 1-deoxynojirimycin acarbose (apparent)" acarbose methyl a$-acarviosinide acarbose (apparent)d acarbose
3.30 2.70 x 8.80 x 4.70 x 2.00 x 9.40 x 7.80 x 3.80 2.90 x
G1
G1
104 107 10" 104 107 10"
lo6 104 10"
-AGb
-AHh
TAS~
6.2 10.1 16.3 6.4 10.0 16.5 9.5 6.3 15.8
2.7 7.0 9.7 1.7 6.2 7.8 7.4 1.7 9.1
3.5 3.1 6.6 4.8 3.9 8.6 2.1 4.6 6.6
aM-1
kcal mol-' Inhibited by 1-deoxynojirimycin Inhibited by methyl a$-acarviosinide
coamylase G2 proceeds with a free energy of binding of 16.3 kcal mol-' and a ACp of -200 cal mol-' deg-' [53].Similarly, the binding of maltose to maltose binding protein shows a free energy of binding of 8.14 kcal mol-' and an accompanying change in molar heat capacity of -207 cal mol-' deg-' [95]. It seems likely that the source of enhanced affinity is highly efficient removal of solvent from poorly solvated surfaces. Sigurskjold and coworkers have utilized a displacement strategy to evaluate the binding thermodynamics of the highest affinity protein-carbohydrate complexes known, that of acarbose and Aspergillus glucoamylase (Table 9) [53]. The high affinity of this complex precludes direct calorimetric titration of the protein with ligand. Instead, this group takes advantage of the additivity of binding thermodynamic parameters and displaces the moderately binding ligand 1-deoxynojirimycin with the high affinity ligand acarbose. In an appropriate concentration domain, the enthalpy of acarbose binding is roughly equivalent to the sum of the enthalpy of 1-deoxynojirimycin binding and the enthalpy of 1-deoxynojirimycin displacement by acarbose. Similarly, the free energies of binding are additive, requiring that the complex stability constants are mulitiplicative. The effect of metal ion identity on the thermodynamics of concanavalin Aoligomannoside binding has been investigated [87].The role of metal ion in legume lectin binding has long been questioned; although metal ions are required for binding they do not appear to contact saccharide ligands. Through the series cobalt nickel and cadmium only minor alterations in enthalpies and free energies of binding were observed, suggesting a primary role for metal ion in the maintenance of protein structure rather than in ligand binding. Previously, we and others have commented on the similarities and differences between patterns of saccharide association for lectins and antibodies [3]. Unfortunately, the database of thermodynamic parameters for antibody-carbohydrate complexation remains small. While important structural and energetic differences may exist that provide clues to the origin of both affinity and association in aqueous solution, it is impossible to reach any conclusions at this time.
31.6 The Role of Multivalency in Protein-Carbohydrate Interaction
901
31.6 The Role of Multivalency in Protein-Carbohydrate Interaction The tremendous potential of saccharide ligands for the treatment of a wide range of human diseases continues to drive much of contemporary glycoscience. The major impediment to the use of carbohydrate or carbohydrate mimetics continues to be the low affinity of saccharide ligands for their lectin receptors. As we note above, the inherently low enthalpy and free energy available from desolvation during protein-carbohydrate complexation likely places fundamental limitations on the affinity that might be achieved through modification of monomeric ligands. Lectins are seldom found in vivo as monomeric species; rather, they are aggregated into higher order oligomeric structures. A reasonable conclusion drawn from this observation is that nature takes advantage of the remarkable diversity of carbohydrate structures to achieve high selectivity, while overcoming the low inherent affinity through the use of multivalency. From this concept an enormous array of multivalent carbohydrate ligands has been synthesized and evaluated in a range of binding assays. Many such ligands show remarkable enhancements in activity compared to an equivalent concentration of the corresponding monovalent ligand; indeed some show affinity enhancements as large as lo9 on a valence corrected, or per mole of saccharide, basis. In most instances the physical basis of the observed effects is unclear. Here, we review briefly the phenomonology of multivalency effects in protein-carbohydrate interaction, then consider the thermodynamic consequences of tethering recognition domains. The goal of this exercise is to provide a molecular basis for the ‘cluster glycoside effect’ and evaluate its potential for therapeutic application.
3 1.6.1 Phenomenology The ‘cluster glycoside effect’, as defined by Y. C. Lee, was first observed using the hemagglutination assay [ 11. Evaluating several hepatic lectins against several monosaccharide types tethered by amino acids, Lee noted the ZCso of these multivalent neoglycoconjugates was lower than expected based on the saccharide content of the ligand (Figure 6). Since this time an enormous variety of multivalent ligands have been prepared and bound (Table 10) [ 102, 1031. The magnitude of the cluster glycoside effect varies over nine orders of magnitude across the range of neoglycoconjugate structures that have been evaluated. Some of the first multivalent ligands reported are a group of glycosylated polyacrylamides. These ligands are prepared by copolymerization of acrylamide and an acrylic acid ester [ 1041. Two conceptual routes have been reported, varying in the nature of the acrylic acid ester. In one instance the ester contains the saccharide recognition domain; in the other the acryloyl ester incorporates an Nhydroxysuccinamide ester that is later displaced by a saccharide recognition domain tethered through an amine spacer (Figure 6). The molecular weight range achieved with these methods spans 10,000-450,000 Da, with larger polymers typically formed by incorporating the saccharides during
precipitation
4.7 x 10' 2.6 x 103 5.1 x 10' 1.8 x lo2 2.6 x 10' 6.6 x 10' 1.4 103 1.0 107 5.6 107 6.0 x 10' 1-2 x 100 8.3 x lo2 4.9 x 100 1.4 107 3.3 103 1.7 103 2.6 105 2.0 x 10' 1.2 x 102
2 2 3 12 4 16 16 6 6 4 2 2 2 270 7 7 3 105 25 4
Chicken hepatic lectin Chicken hepatic lectin LimaxJiavus lectin Linzax Jiauus lectin Concanavalin A Pea lectin Human mannose receptor Human mannose receptor Strepococcus suis Viral hemagglutinin Intact influenza virus Immobilized E-selectin Viral hemagglutinin Cholera toxin B subunit Heat labile enterotoxin Concanavalin A P-selectin Concanavalin A
"calculated by dividing K, for multivalent ligand by K, for monovalent refrence compound Hemagglutination Assay Enzyme Linked Immunosorbent Assay Enzyme Linked Lectin Assay
precipitation precipitation ELLAd ELLA ELLA ELLA Displacement Displacement HA HA HA ELLA Fluorescence ELLA variation ELLA variation HA Inhibition HA
47 41 42 42 106 106 107 108 108 109 110 111 111 112 113 41
1 1
1
104 104 104 104 105
HA^ HA ELISA" ELISA precipitation
5.0 x lo6 5.0 x 105 1.0 x 106 1.6 x 10' 1-2 x 100
~
200-2000 200 2000 200-2000 200- 2000 2-3
Viral hemagglutinin Viral hemagglutinin Viral hemagglutinin Viral hemagglutinin Serum-type mannose binding protein rat hepatic lectin
Sialylated glycopolymer; pre-P Sialylated glycopolymer; co-P Sialylated glycopolymer; pre-P Sialylated glycopolymer; co-P Bivalent and trivalent mannosylated glycoclusters Bivalent glycopeptide based NAcYD(GG-ah-GlyC) Bivalent glycopeptide based YEE (ah-GlyC) Tris-GlcNAc glycocluster Sialylated PAMAM glycodendrimer Sialylated PAMAM glycodendrimer Mannosylated PAM AM glycodendrimer Mannosylated PAMAM glycodendrimer Lysine-based mannosylated glycoclusters Lysine-based mannosylated glycoclusters Galabioside glycoclusters Bivalent sialosides Bivalent sialosides sLeXdimer Lyso-GM3-PGA glycopolymer oligo-GM1 propylene-imine glycodendrimer oligo-GM1 propylene-imine glycodendrimer C-mannosyl. Glycopolymer 3', 6' sulfated galactosyl glycopolymer Mannosylated glycodendrimer
Ref.
Assay Method
p"
Ligand valency
Lectin
Ligand
Table 10. Representative binding enhancements through multivalency.
31.6 The Role of’A4ultiualency in Protein-Carbohydrate Interaction
903
Figure 6. Representative multivalent ligands. From top left: mannosylated ROMP polymers; acrylamide/acryloyl ester copolymers; peptide-based dendritic ligands. See text for descriptions.
the copolymerization, rather than adding the saccharides to an activated backbone. Several other groups have reported a similar strategy [ 114-1 191. The performance of glycopolymers in agglutination assays is remarkable; indeed, the largest cluster glycoside effects observed to date are for the acrylamide polymers. Spaltenstein and Whitesides observed a reduction of 1O5 in IC50 for polyacrylamide glycopolymers bearing a-sialic acid residues compared to monosaccharide assayed against influenza viral hemagglutinin, placing minimum inhibitory concentrations in the nanomolar range [ 1201. Remarkably the efficacy of polymeric inhibitors varied both as the content of ligand and as the method by which it was incorporated: IC50 values for glycopolymers prepared by the activated backbone method were 75 times lower than equivalent sized glycopolymers prepared by copolymerization of sialic acid acrylamide monomers [ 1211. Apparently the mechanism by which these polyvalent ligands inhibit hemagglutinin-mediated agglutination is dependent on both the content and spatial orientation of saccharide epitopes. Polyacrylamides bearing N-acetyllactosamine, prepared by Tsuchida and co-workers, showed ZC50 values enhanced by roughly lo3 relative to unmodified monovalent ligand in agglutination assays against plant lectins [ 1181.
904
31 Protein-Carbohydrate Interaction: Fundamental Considerations
Kiessling and coworkers have prepared a range of glycopolymers using a ringopening metathesis polymerization (ROMP) strategy (Figure 6) [ 1121. Again, enhancements in affinity near lo3 on a valence-corrected basis are observed in agglutination assays. Significantly, greatly reduced enhancements in apparent affinity are observed when the ligands are assayed by other methodologies. A surface plasmon resonance study of the binding of mannosylated ROMP ligands to concanavalin A showed enhancements of five to 40-fold [122]. Kiessling and coworkers have also demonstrated a size dependence during glycopolymer inhibition [ 1231. Mannosylated glycopolymers showed enhancements in the range of 50-3,000 compared to monosaccharide, increasing exponentially with polymer chain lengths up to 143 units in agglutination assays against concanavalin A. Increasing chain lengths beyond this value failed to provide a continued valence corrected enhancement, although overall ligand affinities apparently continue to increase. With very large polymers (MW lo6) enhancements near lo5 were observed. The cell surface presents a multivalent display of carbohydrate exquisitely suited to maximize protein-carbohydrate binding while minimizing entropic penalties. On the one hand, carbohydrate epitopes are free to orient themselves in two dimensions in a configuration that optimizes favorable contacts with the lectin. On the other hand, the bulk of the translational entropy and all of the rotational entropy has already been lost during placement of the ligand in the lipid bilayer. This situation is mimicked by a variety of synthetic glycosylated liposomes and, like glycopolymers, glycosylated liposomes show remarkably enhanced performance in agglutination assays compared to an equivalent concentration of the corresponding monovalent ligand. The general method for synthesis of glycosylated liposomes is: i) conjugation of the glycosyl moiety to a lipid chain; ii) incorporation of some amount of this glycolipid into a mixture of phosphatidyl choline and cholesterol; and iii) sonication or extrusion through a membrane to form the liposome [124]. Kingery-Wood and co-workers produced a series of sialic acid containing liposomes with mole fractions of sialic acid (compared to phosphatidyl choline and cholesterol) of 2-25%. By hemagglutination assays, a maximum inhibition was observed at 5% sialic acid incorporation, with ZC50 values in the nanomolar range. DeFrees and co-workers repeated this procedure with the sialyl Lewis' epitope [ 1251. Ketis et al. incorporated the glycoprotein glycophorin into liposomes as a 33% component for binding assays with wheat germ agglutinin [126]. Charych and co-workers have developed an innovative methodology for glycoliposome production [ 127- 1291. Incorporation of diacetylenic lipids followed by UV (254 nm) irradiation yielded a highly colored (blue) polymerized liposome species. The incorporated chromophore provides a unique binding read-out: upon lectin binding the liposome turns red, presumably as the lipid bilayer adopts a conformation optimal for ligand binding. A much larger group of multivalent ligands include the so-called glycoclusters and glycodendrimers. These ligands are typically smaller in size than the polymers, and require significantly greater synthetic effort. On the other hand, the resulting species are homogenous in nature and more easily characterized than polymeric
31.6 The Role of Multivalency in Protein-Carbohydrate Interaction
905
ligands; additionally, greater control is achieved over the spacing and orientation of glycosyl moieties. A complete description of this group of ligands is beyond the scope of this document; rather, the reader is directed to several recent reviews of the field [102, 1031. In contrast to the remarkable performance of glycopolymers, glycoclusters and glycosylated dendrimers provide much more modest enhancements in affinity. With valences ranging to 36, typical valence-corrected enhancements in affinity are on the order of 10-103, although some exceptional enhancements have been reported. Hansen and co-workers reported increases of 102-103 in ZCso values for several low-valency galabiose inhibitors relative to unmodified galabiose against the Grampositive bacterium Streptococcus suis [ 1071. Nonetheless, this enhancement raised the approximate range of binding efficacy from micromolar to nanomolar, approaching ranges required for therapeutic utility. Knowles and coworkers observed a 100-fold increase in the relative potency for a series of dimeric sialosides compared to unmodified sialic acid when evaluated against the influenza virus in agglutination assays [ 1081. A peptide-based mannosylated glycocluster reported by Biessen and coworkers showed a cluster glycoside effect of lo6 relative to mannose when evaluated against human mannose receptor; this dramatic increase in relative inhibitory potential is one of the largest seen with non-polymeric systems [ 1061. Again, the effect is also a function of the analytical methodology utilized, and apparent enhancements in binding measured by ELLA are much smaller than those seen in hemagglutination assays 142-501. For example, many of the glycosylated PAMAM dendrimers, with valencies ranging over 2-1 6, show enhancements in apparent affinity relative to the unmodified saccharides ranging from 2- to 15-fold when considered on a per saccharide basis 147, 1301. Similarly, Ashton and coworkers report only modest enhancements in affinity during evaluation of glycodendrimers based on a combination aliphatic/aromatic core [ 1311. 31.6.2 The Energetic Consequence of Ligand Linkage
Having considered the reported phenomonology of multivalency effects in proteincarbohydrate interaction, we now aim to understand the origin of the cluster glycoside effect at a molecular level. To the extent that a set of design principles relevant to high affinity can be extracted, multivalency is among the most promising strategies towards the development of therapeutically useful carbohydrates. We consider here only a brief treatment of multivalency effects in aqueous association; a more complete treatment of the topic has recently appeared [ 1031. A meaningful discussion of multivalency in ligand binding first requires a precise definition of the terms involved. Consider the binding of N monovalent ligands versus a ligand of valency N, LN, to an N-valent lectin. Assuming the binding sites are equivalent and non-interacting, thermodynamic parameters describing the association of each monovalent ligand can be represented as AJN. The corresponding terms for association of the multivalent ligand are related to those for binding of the monovalent ligand by the expression:
A J N = NAJN + AJi
906
31 Protein- Carbohydrate Interaction: Fundamental Considerations
where AJi is an interaction term, describing the energetic consequences of physical linkage [68]. A discussion of multivalency effects next requires distinction be drawn between an overall affinity for the multivalent ligand and affinity on a per saccharide basis. These concepts have been variously termed functional afinity and intrinsic afinity, or avidity and ufinity, respectively [103, 1321. Whitesides and coworkers provided a more precise rendering of these concepts by defining the quantities a and b as:
or, in free energy terms:
Defined in this way, c1 is a measure of what is traditionally referred to as positive cooperativity; in the analysis of Jencks, values of a greater than unity correspond to favorable (i.e. negative) interaction free energies. Determination of a values require knowledge of the number of ligands bound. Since in most instances this information is not known, the quantity fl describes simple phenomenology:
At its simplest level enhanced affinity from multivalency requires only that jAGNI > IAGI, where AGN represents the change in Gibbs’ free energy upon binding of the N-valent ligand and A G represents the change in Gibbs’ free energy upon binding of each of the constituent monomeric ligands to an individual lectin binding site. Note that in this construct, the contribution of each monovalent recognition epitope to the overall binding free energy need not be greater than-or even equivalent to-that of monovalent ligand. The only requirement for the observation of enhanced affinity of the ligund is that IAJil be less than I(N - l)A&I; in such instances p will be greater than or equal to one. At a second level, a multivalency effect requires that the interaction free energy be favorable. This case describes what is normally referred to as positive cooperativity; here lAGNI > INAGNI. The key value describing the effect of ligand multivalency on binding thermodynamics is the interaction energy, AGi. A range of physical processes contribute to these interaction free energies; the concepts are intuitively more accessible if considered as separate enthalpic and entropic terms. Enthalpic Contributions to AGi The addition of a linker region has enthalpic consequences to overall binding free energies. First, a linker must be long enough to facilitate optimal placement of ligands within binding sites. Linkers too short or too rigid to facilitate optimal placement of recognition epitopes within binding sites have the effect of contributing unfavorably to interaction enthalpies. Assuming the recognition epitopes
31.6 The Role of Multivalency in Protein-Carbohydrate Interaction
901
can in fact bind successfully, the linker will almost certainly contact the surface of the protein at the periphery of the binding site, on the surface of the protein beyond the binding sites, or both. These contacts can be either favorable or unfavorable, contributing postively or negatively to interaction energies. The a priori prediction of favorable or unfavorable contributions to AHi is virtually impossible. Combinatorial approaches using, for example, peptide spacers may be a useful method of sampling wide ranges of linker-peptide interaction. Finally, the interaction of the linker region with solvent prior to binding leads to an energetic consequence during binding if the linker region is desolvated: this desolvation could make a favorable or unfavorable contribution to AHi, depending on the precise molecular details of the linker surface. Again, association in solution requires consideration both of the interactions between solutes and the interactions of both solutes with solvent prior to association. Entropic Contributions to AGi
Energetic consequences of ligand tethering are typically considered primarily in entropic terms; there is little doubt that there will be a significant contribution to binding free energies attributable to an entropic interaction term. Predicting the magnitude-or even sign-of the entropic consequence of tethering ligands, however, is not straightforward. As described above, an overall entropy of binding can be conceptually separated into a translational and rotational entropy, a conformational entropy, and a solvation-associated entropy. A similar exercise facilitates consideration of entropic contributions to ASi. In this construct, A& is considered as the sum of the differences in each entropic term for binding of the N-valent ligand and N monovalent ligands; that is:
where MNLNrepresents multivalent and monovalent complexes, respectively. The first term describes the entropic ‘savings’ that derives from minimizing the loss of translational and rotational entropy-the ability of a molecule to translocate in three dimensions and rotate on three axes-by converting N-ligands into a single species. Because translational and rotational entropies scale as the logarithm of the molecular size, AST+R for a multivalent ligand will be approximately equivalent to that of the corresponding monovalent species. As previously noted, the magnitude of this term is largely unknown. Table 6 above shows the wide range of values currently assigned to this term. The second term of the entropy decomposition describes the loss in conformational degrees of freedom during binding, typically the restriction of flexible dihedrals. In the context of interaction entropies, this term primarily arises from losses in degrees of freedom in a flexible linker region during binding. Although the magnitude of this term is somewhat unclear, the situation is considerably less opaque than for A S T + R . Some agreement seems to have emerged for a value near 0.5
908
31 Protein-Carboh ydrate Interaction: Fundamental Considerations
kcal mol-' near room temperature for complete restriction of a bond previously free to equilibrate among three energetically equivalent staggered conformers [ 133, 1341. Obviously not all bonds restricted during binding possess such freedom prior to complexation, and not all are completely constrained following binding. Linkers designed to span binding sites separated by 20-70 8, possess considerable conformational flexibility; the loss of this flexibility will unquestionably lead to a significant unfavorable contribution to ASl. Based on an entropic analysis, the prescription for the design of linker regions is clear. Linkers must be long enough to facilitate optimal placement of the saccharide recognition domains within the binding site, but with enough rigidity to minimize a conformational entropy penalty. An alternative strategy might be to make linkers of sufficient length that residual flexibility following binding minimizes the unfavorable contribution of ASconf to ASl. A number of studies on the role of flexibility in linker regions have been reported. Glick et al. evaluated a series of bivalent sialic acid ligands for the viral hemagglutinin, varying in length and linker flexibility [ 1081. A linker of intermediate flexibility provided the tightest binding as evaluated by hemagglutination assay. Unfortunately, without the required thermodynamic data, molecular interpretation of the result is impossible. Bundle and coworkers also explored the role of ligand flexibility, albeit in a somewhat different context [98, 1351. Two decades of intense study on small molecule recognition in organic solvent clearly demonstrated that preorganization and complementarity are key requirements of ligand structure for the observation of high affinity. From that observation, ligands with diminished conformational entropy, or preorganized ligands, should show enhanced affinity relative to ligands with freely rotating bonds. Monoclonal antibody Sel55.4 binds the trisaccharide methyl 2-(a-~-ga~actopyranosyl)-3-(a-~-3,6-dideoxyxy~opyranosy~)-~-~ mannopyranoside. This binding buries the dideoxyxylopyranose (abequose) moiety completely, and consequently freezes rotation about the AbeMan glycosidic bond. A series of ligands were prepared that preorganize the ligand by freezing this rotation (Figure 7). Binding of each restricted ligand proceeded with thermodynamic parameters remarkably unchanged from those of the native-presumably less ordered-ligand. The origin of this effect is unclear. On the one hand, the inherent flexibility of the localized bond is low; the exo-anomeric effect likely limits the entropic gain available. On the other hand, the tether used to preorganize the ligand may interact unfavorably in the complex. The role of ligand flexibility/preorganization in determining affinities has also been probed through the use of C-glycosides [ 136, 1371. Absent the anomeric oxygen and consequent exo-anomeric effect, C-glycoside ligands should show enhanced flexibility around the glycosidic bond relative to the corresponding 0-glycoside. This additional flexibility in the unbound ligand should enhance the conformational entropy loss upon binding. Again, the results to date fail to unambiguously demonstrate general design principles with regard to anomeric flexibility. A series of ligands that sequentially replace both the anomeric and pyranosidic oxygen with a methylene and thioether failed to yield a clear picture of structure-activity relationships. As with Bundle's work, however, the alteration in flexibility predicted to arise from such modification is small. Assuming an exo-anomeric effect of roughly
31.6 The Role of Multivalency in Protein-Curbolzydrute Interaction
909
R1=Oor S R2=(CH2), or CH2C6H4CH2
HO
Figure 7. Ligands designed to assess the role of conformational entropy in ligand binding. See text for description.
1-2 kcal mol-' and that the gg-configuration about the glycosidic linkage is precluded in both the C- and 0-glycoside, deletion of the anomeric oxygen alters the predicted rotomeric distribution about the anomeric linkage by roughly a factor of two. Furthermore, the uncertain role of changes in ligand solvation renders a molecular basis for changes in affinity opaque; further calorimetric study of such ligands may illuminate the relevant issues. Beyond concepts of linker flexibility, unpredictable values of ASsOlv and AHi
9 10
31 Protein-Carbohydrate Interaction: Fundamental Considerations
produce intractable design problems. The large potential energetic consequences of even small ligand modifications, coupled with the relatively weak interaction free energies that typify protein-carbohydrate interaction require a careful and stepwise exploration of the relationship between ligand structure and binding energetics. There likely do exist a simple set of rules to govern intelligent construction of multivalent ligands. Finally, it is imperative that the analytical methodology used to observe cluster glycoside effects be carefully considered before a molecular explanation of phenomenology is offered. 31.6.3 A Molecular Basis for the Cluster Glycoside Effect
Having considered the phenomenology of the cluster glycoside effect and the thermodynamic considerations surrounding aqueous association of multivalent ligands, we now turn briefly to possible molecular explanations for the affinity that apparently derives from multivalency. At this time, there are no recorded definitive examples of cooperativity in protein-carbohydrate binding deriving from favorable interaction energies. It seems most likely, then, that most cluster glycoside effects reported to date are in fact measures of the ability of multivalent saccharide ligands to drive the formation of some form of higher order aggregate. While formation of this aggregate might be favorable, providing a coupled equilibrium that would, in turn, enhance protein-carbohydrate affinities, there is no evidence to suggest that this is in fact true. Alternatively, Mammen, Choi, and Whitesides postulated a theory for the remarkable performance of glycopolymers in agglutination assays [ 1031. After binding several sialic acids to the viral hemagglutinin, these large, sterically demanding molecules may form a water-swollen, gel-like layer between the lectin and the erythrocyte surface. This barrier prevents additional binding from proceeding, but does not require thermodynamically enhanced saccharide-lectin binding. In some respects, protein-carbohydrate interaction is a poor system with which to study multivalency effects in aqueous association. The low monovalent affinities, poor analytical methodology for evaluation of binding, and the propensity of lectins to aggregate in the presence of multivalent saccharide ligands provides a daunting series of complications to the interpretation of binding data. More recently the study of protein-carbohdyrate interaction using bacterial two-component toxins suggests that some enhancement in affinity may be possible through multivalency [ 1381. These proteins, which do not aggregate polyvalent saccharides and show relatively small distances between binding sites, are more amenable to study. The enhancements in affinity available in these systems is not yet clear. Finally, we note that the appropriate analytical methodology and model of binding is dependent on the question asked. It may well be the case that in the context of biological systems, ligands especially capable of driving aggregation/ precipitation processes might act as useful agents. The concern, rather, is a precise definition of the issue at hand. With regard to ligand binding, in the commonly accepted definition, it appears unlikely that multivalency can afford high affinity through positive cooperativity.
Rejerences
9 11
Acknowledgments
The authors gratefully acknowledge the assistance of Mr. J. Lundquist and Ms. Aimee Butler for preparation of figures. Financial support for this work was provided by the National Institutes of Health (GM57179). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.
Lee, Y.C.; Lee, R.T. Acc. Chem. Res. 1995,28, 321. Dwek, R.A. Chem. Rev. 1996,96,683-720. Toone, E.J. Current Opinion in Structural Biology 1994, 4, 719-728. McClellan, A.L. Tables of Experimental Dipole Moments; Rahara Enterprises: El Cerrito, 1974. Rigby, M.; Smith, E.B.; Wakeham, W.A.; Maitland, G.C. The Forces Between Molecules; Oxford: New York, 1986. Zeegers-Huyskens, T.; Huyskens, P. Intevmolecular Forces; Huyskens, P. L.; Luck, W. A. P.; Zeegers-Huyskens, T., Ed.; Springer-Verlag: New York, 1991, pp 1-30. London, F. Z. Physik 1930, 63, 245. London, F. Z. Physik Chem. Leipzig 1930, 11, 222. Hunter, E.P.L.; Lias, S.G. J. Phys. Chem. Ref: Data 1998. 27, 413-656. Mirejovsky, D.; Arnett, E.M. J. Am. Chem. Soc. 1983, 105, 1112. Water: A Comprehensive Treatise, Vol. 1-7; Franks, F., Ed.; Plenum Press: New York, 1974. Muller, N. J. Solution Chem. 1988, 17, 669. Muller, N. Acc. Chem. Res. 1990,23, 23-28. Ben-Naim, A.M., R. J. Phys. Chem. B 1997, 101, 11221-11225. Soper, A.K.; Luzar, A.J. J. Phys. Chem. 1996, ZOO, 1357. Turner, J.; Soper, A.K. J. Chem. Phys. 1994, 101, 6116. Turner, J.Z.; Soper, A.K.; Finney, J.L. J. Chem. Phys. 1995, 102, 5438. Turner, J.; Soper, A.K.; Finney, J.L. Molecular Phys. 1990, 70, 679. Finney, J.L.; Soper, A.K.; Turner, J.Z. Pure Appl. Chem. 1993, 65, 2521. Leberman, R.; Soper, A.K. Nature 1995,378, 364. Reiss, H.; Frisch, H.L.; Lebowitz, J.L. J. Chem. Phys. 1959, 31, 369. Reiss, H.; Tully-Smith, D.M. J. Chem. Phys. 1971, 55. 1674. Reiss, H. Adv. Chem. Phys. 1966, 9, 1. Frisch, H L. Adv. Chem. Phys. 1963, 6, 229. Pierotti, R.A. A. Chem. Rev. 1976, 76, 711. Stillinger, F.H. J. Solution Chem. 1973, 2, 141. Ben-Naim, A,; Tenne, R. J. Chem. Phys. 1977, 67, 627. Lee, B. Biopolymers 1991, 31, 993. Lee, B. Methods Enzymol. 1995,259, 555. Fujita, T.; Iwasa, J.; Hansch, C. J. Am. Chem. Soc. 1964, 86, 5175. Iwasa, J.; Fujita. T.; Hansch, C. J. Med. Chem. 1965, 8, 150. Arnett, E.M.; McKelvey, D.R. in Solute-Solvent Interactions Coetzee, J.F.; Ritchie, C.D., Ed.; Marcel Dekker: New York, 1969, pp. 342. Oas, T.G.; Toone, E.J. Adv. Biophys. Chem. 1997, 6, 1-52. Muller, N. J. Solution Chem. 1991,20, 669. Chervenak, M.C.; Toone, E.J. J. Am. Chem. Soc. 1994, 116, 10533-10539. Chervenak, M.C.; Toone, E.J. Biochemistry 1995,34, 5685-5695. Lemieux, R.U. Chem. Soc. Rev. 1989, 18, 347-374. Simanek, E.E.; McGarvey, G.J.; Jablonowski, J.A.; Wong, C.H. Chem. Rev. 1998, 98, 833862. Huwe, C.M.; Woltering, T.J.; Jiricek, J.; Weitz-Schmidt, G.; Wong, C.H. Bioorg. Med. Chem. 1999, 7, 773-788.
9 12
31 Protein-Carbohydrate Interaction: Fundamental Considerations
40. Du, M.; Hindsgaul, 0. Synlett 1997, 395-397. 41. Dimick, S.M.; Powell, S.C.; McMahon, S.A.; Moothoo, D.N.; Naismith, J.H.; Toone, E.J. J. Am. Chem. Soc. 1999, in press. 42. PagC, D.; Zanini, D.; Roy, R. Bioorganic & Medicinal Chemistry 1996, 4, 1949-1961. 43. PagC, D.; Roy, R. Bioconjugate Chem. 1997, 8, 714-723. 44. PagC, D.; Roy, R. Glycoconjugate J. 1997, 14, 345-356. 45. Roy, R.; Tropper, F.D.; Romanowska, A. Bioconjugate Chem. 1992,3,256-261. 46. Roy, R. PagC, D.; Perez, S.F.; Bencomo, V.V. Glycoconjugate J. 1998, 15, 251-263. 47. Zanini, D.; Roy, R. J. Am. Chem. SOC.1997,119,2088-2095. 48. Tropper, F.D.; Romanowska, A.; Roy, R. Methods in Enzymology 1994,242,257-271. 49. Arya, P.; Kutterer, K.M.K.; Qin, H.; Roby, J.; Barnes, M.L.; Kim, J.M.; Roy, R. Bioorg. Med. Chem. Lett. 1998,8>1127-1132. 50. Garcia-Lopez, J.J.; Hernandez-Mateo, F.; Isac-Garcia, J.; Kim, J.M.; Roy, R.; SantoyoGonzalez, F.; Vargas-Berenguel, A. J. Org. Chem. 1999, 64, 522-531. 51. Grime, J.K. Analytical Solution Chemistry; John Wiley & Sons: New York, 1985; Vol. 79. 52. Wiseman, T.; Williston, S.; Brandts, J.F.; Lin, L.-N. Anal. Biochem. 1989, 179, 131. 53. Sigurskjold, B.W.; Berland, C.R.; Svensson, B. Biochemistry 1994, 33, 10191-10199. 54. Sigurskjold, B.W.; Svensson, B.; Williamson, G.; Driguez, H. Eur. J. Biochem. 1994, 225, 133-141. 55. Sigurskjold, B.W.; Christensen, T.; Payre, N.; Cottaz, S.; Driguez, H.; Svensson, B. Biochemistry 1998, 37, 10446--10452. 56. Berland, C.R.; Sigurskjold, B.W.; Stoffer, B.; Frnadsen, T.P.; Svensson, B. Biochemistry 1995, 34, 10153-10161. 57. Sturtevant, J.M. Proc. Natl. Acad. Sci. USA 1977, 74, 2236-2240. 58. Murphy, K.P.; Privalov, P.L.; Gill, S.J. Science 1990, 247, 559-561. 59. Murphy, K.P. Biophys. Chem. 1994,51, 311-326. 60. Doty, P.; Meyers, G.E. Discussions Faraday Soc. 1953,13, 51. 61. Dunitz, J.D. Science 1994, 264, 670. 62. Erickson, H.P. J. Mol. Biol. 1989, 206, 465-474. 63. Horton, N.; Lewis, M. Protein Science 1992, 1, 169-181. 64. Janin, J. Proteins Struct. Funct. Genet. 1995,21, 30-39. 65. Janin, J.; Chothia, C. J. Mol. Biol. 1976, 100, 197-211. 66. Janin, J.; Chothia, C. Biochemistry 1978, 17, 2943-2948. 67. Janin, J.; Chothia, C. J. Biol. Chem. 1990, 265, 16027-~16030. 68. Jencks, W.P. Adu. Enzymol. 1975, 43, 219-410. 69. Page, M.I.; Jencks, W.P. Proc. Natl. Acad. Sci. 1971, 68, 1678-1683. 70. Searle, M.S.;Williams, D.H. J. Am. Chem. Soc. 1992, 114, 10690-10697. 71. Searle, M.S.;Williams, D.H.; Gerhard, U. J. Am. Chem. Soc. 1992,114, 10697-10704. 72. Spolar, R.S.; Record, M.T., Jr. Science 1994,263, 777-784. 73. Tidor, B.; Karplus, M. J. Mol. Biol. 1994, 238, 405-414. 74. Williams, D.H.; Searle, M.S.; Mackay, J.P.; Gerhard, U.; Maplestone, R.A. Proc. Natl. Acad. Sci. USA 1993, 90, 1172-1 178. 75. Schwarz, F.P.; Ahmed, H.; Bianchet, M.A.; Amzel, L.M.; Vasta, G.R. Biochemistry 1998, 37, 586775877, 16. Schwarz, F.P.; Misquith, S.; Surolia, A. Biochem J. 1996, 316, 123-129. 77. Schwarz, F.P. J. Solution Chem. 1996,25,471-484. 78. Tyler-Cross, R.; Sobel, M.; McAdory, L.E.; Harris, R.B. Arch. Biochem. Biophys. 1996,334. 206-21 3. 79. Tomme, P.; Creagh, A.L.; Kilburn, D.G.; Haynes, C.A. Biochemistry 1996,35, 13885-13894. 80. Creagh, A.L.; Ong, E.; Jervis, E.; Kilburn, D.G.; Haynes, C.A. Proc. Natl. Acad. Sci. USA 1996, 93, 12229-12234. 81. Weatherman, R.V.; Mortell, K.H.; Chervenak, M.; Kiessling, L.L.; Toone, E.J. Biochemistry 1996,35, 3619-3624. 82. Mandal, D.; Kishore, N.; Brewer, C.F. Biochemistry 1994, 33, 1149-1 156. 83. Dam, T.K.; Oscarson, S.; Sacchettini, J.C.; Brewer, C.F. J. Biol. Chem. 1998, 273, 3282632832.
References
9 13
Dam, T.K.; Oscarson, S.; Brewer, C.F. J. Biol. Chem. 1998,273, 32812-32817. Gupta, D.; Dam, T.K.; Oscarson, S.; Brewer, C.F. J. Biol. Chem. 1997, 272, 6388-6392. Chervenak, M.C.; Toone, E.J. Bioorg. Med. Chem. 1996, 4, 1963-1977. Sanders, J.N.; Chenoweth, S.A.; Schwarz, F.P. J. Znory. Biochem. 1998, 70, 71-82. Swaminathan, C.P.; Surolia, N.; Surolia, A. J. Am. Chem. Soc. 1998,120, 5153-5159. Gupta, D.; Cho, M.; Cummings, R.D.; Brewer, C.F. Biochemistry 1996, 35, 15236-15243. Surolia, A,; Sharon, N.; Schwarz, F.P. J. Biol. Chem. 1996,271, 17697-17703. Fromm, J.R.; Hileman, R.E.; Weiler, J.M.; Linhardt, R.J. Arch. Biochem. Biophys. 1997,346, 252-262. 92. Tsai, F.T.F.; Singh, O.M.P.; Skarzynski, T.; Wonacott, A.J.; Weston, S.; Tucker, A,; Pauptit, R.A.; Breeze, A.L.; Poyser, J.P.; O’Brien, R.; Ladbury, J.E.; Wigley, D.B. Proteins Struct. Funct. Genet. 1997, 28, 41-52. 93. Garcia-Hernandez, E.; Zubillaga, R.A.; Rojo-Dominguez, A,; Rodriguez-Romero, A,; Hernandez-Arana, A. Proteins: Struct., Funct., Genet. 1997,29, 467-477. 94. Quesenberry, M.S.; Lee, R.T.; Lee, Y.C. Biochemistry 1997,36, 2724-2732. 95. Thomson, J.; Liu, Y.; Sturtevant, J.M.; Quiocho, F.A. Biophys. Chem. 1998, 70, 101-108. 96. Briggs, J.B.; Larsen, R.A.; Harris, R.B.; Sekar, K.V.S.; Macher, B.A. Glycohiology 1996, 6, 831-836. 97. Sharma, S.; Bhardwaj, S.; Surolia, A,; Podder, S.K. Biochem. J. 1998, 333, 539-542. 98. Bundle, D.R.; Alibes, R.; Nilar, S.; Otter, A,; Warwas, M.; Zhang, P. J. Am. Chem. Soc. 1998,120, 5317-5318. 99. Ramkumar, R.; Surolia, A,; Podder, S.K. Biochem J. 1995, 308, 237-241. 100. Lee: R.T.; Gabius, H.J.; Lee, Y.C. Glycoconjugate J. 1998, IS, 649-655. 101. Swaminathan, C.P.; Gupta, D.; Sharma, V.; Surolia, A. Biochemistry 1997, 36, 13428-1 3434. 102. Roy, R. Curr. Opin. Struct. Biol. 1996, 6 , 692-702. 103. Mammen, M.; Choi, S.K.; Whitesides, G.M. Angew. Chem. Znt. Ed. 1998, 37, 2754-2794. 104. Sigal, G.B.; Mammen, M.; Dahmann, G.; Whitesides, G.M. J. Am. Chem. SOC.1996, 118, 3789-3800. 105. Lee, R.T.; Ichikawa, Y.; Kawasaki, T.; Drickamer, K.; Lee, Y.C. Arch. Biochem. Biophys. 1992,299, 129-136. 106. Biessen, E.A.L.; Noorman, F.; vanTeijlingen, M.E.; Kuiper, J.; BarrettBergshoeff, M.; Bijsterbosch, M.K.; Rijken, D.C.; vanBerkel, T.J.C. J. Biol. Chem. 1996,271,28024-23669. 107. Hansen, H.C.; Haataja, S.; Finne, J.; Magnusson, G. J. Am. Chem. SOC.1997, 119, 69746979. 108. Glick, G.D.; Toogood, P.L.; Wiley, D.C.; Skehel, J.J.; Knowles, J.R. J. Biol. Chem. 1991, 266,23660-23669. 109. Wittman, V.; Takayama, S.; Gong, K.W.; Weitz-Schmidt, G.; Wong, C.H. J. Ory. Chem. 1998,63, 513775143, 110. Kamitakahara, H.; Suzuki, T.; Nishigori, N.; Suzuki, Y.; Kanie, 0.;Wong, C.H. Angew. Chem. Int. Ed. Engl. 1998, 37, 1524-1528. 111. Thompson, J.P.; Schengrund, C.L. Glycoconj. J. 1997, 14, 837-845. 112. Mortell, K.H.; Weatherman, R.V.; Kiessling, L.L. J. Am. Chem. Soc. 1996, 118, 2297-2298. 113. Manning, D.D.; Hu, X.; Beck, P.; Kiessling, L.L. J. Am. Chem. Soc. 1997, 119, 3161-3162. 114. Nishida, Y.; Dohi, H.; Uzawa, H.; Kobayashi, K. Tet. Let. 1998, 39, 8681. 115. Cao, S.; Roy, R. Tet. Let. 1996, 37, 3421. 116. Bovin, N.V.; Gabius, H.J. Chem. Soc. Rev. 1995, 413. 117. Bovin, N.V. Glycoconj. J. 1998, IS, 431. 118 Tsuchida, A,; Akimoto, S.; Usui, T.; Kobayashi, K. J. Biochem. 1998,123, 715. 119 Park, K.H.; Takei, R.; Goto, M.; Maruyama, A,; Kobayashi, A,; Kobayashi, K.; Akaike, T. J. Biochem. 1997, 121, 997. 120. Spaltenstein, A.; Whitesides, G.M. J. Am. Chem. Soc. 1991, 113, 686. 121. Lees, W.J.; Spaltenstein, A,; Kingery-Wood, J. E.; Whitesides, G. M. J. Med. Chem. 1994, 37, 3419. 122. Mann, D.A.; Kanai, M.; Maly, D.J.; Kiessling, L.L. J. Am. Chem. Soc. 1998, 120, 1057510582. 123. Kanai, M.; Mortell, K.H.; Kiessling, L.L. J. Am. Chem. Soc. 1997, 119, 9931. 84. 85. 86. 87. 88. 89. 90. 91.
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31 Protein-Carbohydrate Interaction: Fundamental Considerations
124. Kingery-Wood, J.E.; Williams, K.W.; Sigal, G.B.; Whitesides, G.M. J. Am. Chem. SOC.1992, 114, 7303. 125. DeFrees, S.A.; Phillips, L.; Guo, L.; Zalipsky, S. J. Am. Chem. SOC. 1996, IZ8,6101. 126. Ketis, N.V.; Girdlestone, J.; Grant, C.W. M. Proc. Natl. Acad. Sci. USA 1980, 77, 3788. 127. Charych, D.; Cheng, Q.; Reichart, A.; Kuziemko, G.; Stroh, M.; Nagy, J.O.; Spevak, W.; Stevens, R.C. Chern. & Biol. 1996,3, 113. 128. Spevak, W.; Foxall, C.; Charych, D.H.; Dasgupta, F.; Nagy, J.O. J. Med. Chern. 1996, 39, 1018.
129. Pan, J.J.; Charych, D. Langmuir 1997,.13, 1365. 130. Zanini, D.; Roy, R. J. Org. Chem. 1998, 63, 3486. 131. Ashton, P.R.; Hounsell, E.F.; Jayaraman, N.; Nilsen, T.M.; Spencer, N.; Stoddart, J.F.; Young, M. J. Org. Chem. 1998, 63, 3429. 132. Hornick, C.L.; Karush, F. Immunochem. 1972, 9, 341. 133. Mammen, M.; Shaknovich, E.I.; Deutch, J.M.; Whitesides, G.M. J. Org. Chem. 1998, 63, 3821-3830. 134. Mammen, M.; Shaknovich, E.I.; Whitesides, G.M. J. Org. Chern. 1998, 63, 3168-3175. 135. Alibes, R.; Bundle, D.R. J. Org. Chern. 1998,63, 6288-6301. 136. For example: Espinosa, J.F.; Bruix, M.; Jarreton, 0.;Skrydstrup, T.; Beau, J.M.; JimenezBarbero, J. Chem. Eur. J. 1999, 5, 442-448, and references therein. 137. Aguilera, B.; Jimenez-Barbero, J.; Fernandez-Mayoralas, A. Carbohydr. Res. 1998, 308, 1927. 138. Toone et al., Unpublished Data.
Carbohydrates in Chemistry and Biology Editor Beat Ernst, Gerald W. Hart, Pierre Sinay copyright OWILEY-VCH Verlag GmbH, 2000
32 Structural Analysis of Oligosaccharides: FAB-MS, ES-MS and MALDI-MS Anne Dell, Howard R. Morris, Richard Easton, Stuart Haslam, Maria Panico, Mark Sutton-Smith, Andrew J. Reason, and Kay-Hooi Khoo
32.1 Introduction In the glycobiology field no structural technique can match mass spectrometry for the breadth of problems that can be addressed, the complexity of samples that can be successfully analyzed, and for the amount of structural information that can be obtained from sub-nanomolar amounts of material. The introduction of fast atom bombardment-mass spectrometry (FAB-MS) at the beginning of the 1980s [ l ] revolutionized the structure determination of a very wide range of carbohydratecontaining biopolymers [2, 31 and this revolution has continued with the newer techniques of electrospray ionization (ES-MS) [4] and matrix assisted laser desorption ionization (MALDI-MS) [ 5 ] . A summary of each of these techniques is given below in order to facilitate understanding of their problem-solving capabilities. All three technologies permit the direct ionization/desorption of non-volatile substances and are applicable to intact glycoconjugates as well as fragments.
32.2 Fast Atom Bombardment-Mass Spectrometry (FAB-MS) Double focusing sector mass spectrometers are employed in the majority of biopolymer FAB-MS studies. These consist of a source where the ions are generated, an analyzer comprising an electric sector for energy focusing and a magnetic sector for separating ions of different mass to charge ratios, and a detector. In the FAB experiment, an accelerated beam of atoms (usually xenon) or ions (usually cesium) is fired from an atom or ion gun towards a small metal target attached to the end of a probe (see Figure la). Prior to insertion of the probe into the FAB source, it is loaded with a viscous liquid called the matrix in which is dissolved the sample to be analyzed. When the atom or ion beam collides with the matrix, kinetic energy is
9 16
32 Structural Analysis of Oligosacclzarides
Source Slit
+To
Analyser
Laser attenuator Variable-voltage
Flight
detector
source chamber Ground Grid
\
Collision
Sampling Quadrupole Analyser
Sample in Solution
+
Drying Gas Atmospheric Pressure
1
1
-1 'mbar
-
10-4 10-5 m bar
Figure 1. (A) Fast Atom Bombardment (FAB): the sample is dissolved in a liquid matrix and ionization/desorption is effected by a high energy beam of particles fired from an atom or ion gun. @) Matrix Assisted Laser Desorption Ionisation (MALDI): the sample is dried on a metal target in the presence of a chromophoric matrix and sample ions are produced by energy transfer from matrix molecules that have absorbed energy from the laser pulse. (C) Electrospray (ES): a stream of liquid containing the sample of interest enters the source through a capillary interface, where the sample molecules are stripped of solvent, leaving them as multiply charged species.
32.3 Matrix Assisted Laser Desorption Ionization-Time
9 17
transferred to the surface molecules, many of which are sputtered out of the liquid into the high vacuum of the ion source. A significant number of these molecules are ionized during the sputtering process. Thus gas-phase ions are generated without prior volatilization of the sample allowing the analysis of polar, involatile, and thermally labile compounds. Both positive and negative ions are produced during the sputtering process, and either can be recorded by an appropriate choice of instrumental parameters. Molecules are ionized by protonation or by the addition of a cation such as sodium, potassium, ammonium (positive ion formation), or by the loss of a proton, or addition of an anion such as chloride, thiocyanate (negative ion formation). During ionization, some internal energy is imparted to the molecule resulting in fragmentation of labile bonds. The sputtered molecular and fragment ions are accelerated to about 8,000 eV prior to passage through the analyzer sectors to a detector where the mass to charge ratios are recorded by a computer to give the mass spectrum. Magnetic sector analyzers separate ions on the principle that charged molecules are deflected by strong magnetic fields. Ions of larger mass are deflected by the magnetic field less than ions of smaller mass according to the equation m / z = B2r2/2V where m / z is the mass to charge ratio of the ion, B is the strength of the magnetic field, r is the radius of the circular path through which the ion is travelling through the magnet and V is the accelerating voltage. Ions of different m / z values are brought into focus at the detector by scanning B with V and r being kept constant. Modern high field magnetic sector mass spectrometers are capable of focusing ions up to 15,000 Da at full sensitivity, but the working mass range of FAB-MS for carbohydrate polymers is limited to about 6,000 Da. Higher molecular weight samples are either too difficult to desorb from the matrix or do not produce a sufficient abundance of molecular ions to allow detection above background. The FAB experiment often yields structurally informative fragment ions as well as molecular ions but the quantity and quality of fragmentation can be very variable depending on factors such as the purity of the sample and whether it has been derivatized. To ensure that sufficient fragment ions are produced to allow structural assignments it may be necessary to employ tandem MS techniques (MS/MS) in which ions produced in the FAB source pass through the mass analyzer into a chamber containing an inert gas. Collisions with the gas provide sufficient energy for bond cleavages and the resulting fragment ions (called daughter ions) are detected after passing through a second (tandem) analyzer. Tandem MS is especially powerful when mixtures are being analyzed because it allows unambiguous attribution of fragment ions to individual components of the mixture.
32.3 Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry (MALDI-TOF-MS) In the MALDI experiment (Figure lb) the sample is embedded in a low molecular weight, UV absorbing matrix which enhances sample ionization. When the matrix
9 18
32 Structural Analysis of Oligosucchurides
absorbs the laser pulse enough energy is transferred to the sample, via mechanisms that are not well understood, to enable the formation of molecular ions. The matrix is present in a vast excess over the sample and therefore isolates individual sample molecules. This results in the volatilization of predominantly monomeric molecular ions, although dimers, trimers etc are observed in some MALDI spectra. Ions produced in MALDI-MS are usually analyzed by Time of Flight (TOF) instrumentation. TOF analyzers work on the principle that when ions are accelerated with the same potential from a fixed point and at a fixed initial time and are allowed to drift, the ions will separate according to their mass/charge ratios. Lighter ions drift more quickly to the detector and heavier ions drift more slowly. The time required for ions to reach the detector can therefore be related to their mass. Following acceleration, ions exhibit a broad energy distribution. This energy spread can be minimized using Delayed Extraction (DE) which involves applying a high voltage pulse at a pre-determined time following ion generation in a weak electrical field leading to enhanced resolution and mass accuracy. Most modern MALDI instruments allow both linear and reflector mass analysis. In the linear mode, ions travel without interference down the flight tube to the detector which records the m / z ratio and signal intensity. A reflector is a single-stage gridded mirror that focuses energy. In reflector mass analysis mode, a uniform electric field is applied to the mirror to reflect ions. This filters out neutral molecules, corrects dispersion of the ions occurring in the flight tube and thus provides greater mass accuracy and resolution. In contrast to the magnetic sectors used for analysis in FAB-MS, the TOF analyzer has an almost unlimited mass range. MALDI is also considerably more efficient than FAB in ionizing large biopolymers and is the method of choice for defining the molecular weights of carbohydrate polymers, particularly at high mass.
32.4 Electrospray-Mass Spectrometry (ES-MS) ES-MS is a method by which a stream of liquid containing the sample of interest is introduced into the atmospheric pressure ion source of a mass spectrometer (Figure lc). This can be achieved by direct injection into buffer which is pumped at the rate of a few microliters per minute through a metal-tipped glass capillary. Alternatively the eluent of a microbore LC, after stream splitting, can be passed via the capillary into the ES source. Many newer instruments have high sensitivity nanospray sources capable of operating at flow rates of as low as a few nanoliters per minute. With this type of source, samples are introduced using a probe with an attached nanospray needle, the latter being pre-loaded with about a microliter of a solution of the sample (see 3.9.2, Protocol 2). Irrespective of the introduction method, an aerosol of microdroplets is generated in the source which then traverses a series of skimmers, encountering a drying gas, the net effect of which is the creation of charged molecular species, devoid of solvent. These gaseous ions, whose charge depends on the number of ionizable groups
32.5 Appearance o j Mass Spectra Obtained in FAB-MS
919
in the molecule, are passed into the mass analyzer which is commonly a quadrupole mass filter. This type of analyzer is compact and relatively inexpensive. It comprises four parallel rods which act as mass filters when rf and dc fields are applied to diagonally opposed rods. By sweeping the rf and dc voltages in a fixed ratio, ions of successive mass to charge ratios follow a stable path to the detector. Ions with m / z values above about 4,000 cannot be detected with a quadrupole analyzer. However this is not too problematical for a significant portion of biopolymer ES-MS applications because of the presence of multiple functional groups capable of carrying charge. For example proteins and glycoproteins in excess of 100 kDa can be amenable to ES-MS analysis. Uncharged or poorly charged polysaccharides are not, however, amenable to ES-MS on a quadrupole instrument. The ionization process in ES-MS is very gentle thus resulting in very few fragment ions and little or no sequence information. To overcome this problem many ES instruments have triple quadrupole analyzers for collisional activation MS/MS experiments. The first quadrupole is used to select the parent ion, the second is the collision chamber and the third separates the resulting fragment ions. Whilst triple quadrupole instruments are very powerful, their sensitivity is limited by a number of factors including scanning ion detection and poor fragment ion resolution. These problems have been overcome in a novel mass spectrometer, called the Q-TOF, which was introduced in the mid-1990s [6, 71. The Q-TOF has both quadrupole and TOF analyzers which are arranged orthogonally with a collision cell between them (Figure 2). For normal mass spectra, the quadrupole is used in the rf-only mode as a wide-bandpass filter to transmit a wide mass range. The collision cell is not pressurized, and ions are transmitted to the TOF for mass analysis. In the MS/MS mode the quadrupole operates in the normal resolving mode and is able to select precursor ions up to m / z 4,000 for collisional activation in the hexapole gas cell. The fragment ions are transmitted to the TOF for mass analysis. The orthogonal geometry, and parallel rather than sequential detection of the ions, leads to a significant improvement in sensitivity over scanning instruments when used to acquire full spectra. The Q-TOF is an immensely powerful instrument. The good signal/noise ratios in MS/MS on the Q-TOF correspond to low femtomole/attomole sample consumption. Definitive unambiguous sequence assignment is facilitated by daughter ion resolutions of greater than 3,000 (which allows easy assignment of the z value of m / z ) and daughter ion mass accuracies of 0.05 Da.
32.5 Appearance of Mass Spectra Obtained in FAB-MS, MALDI-MS and ES-MS Experiments FAB spectra are characterized by a high level of chemical “noise”, arising from the matrix and sample, giving a peak at every mass upon which are superimposed the signals for molecular and fragment ions of the sample and matrix. The chemical noise is much lower in MALDI-MS which results in better signal to noise for sam-
920
32 Structural Analysis of Oligosaccharides
SAMPLING CONE QUADRUFQLE IN NARROW BANDPASS MODE
HDV\POLE GAS COLLISION CELL
-
Q TOF MSIMS MODE
REFLECTRON
7
Figure 2. Schematic illustration of the Q-TOF in the MS-MS mode of operation. The quadrupole is set to transmit the parent ion of interest and the hexapole collision chamber contains the collision gas. The orthogonal TOF separates the daughter ions.
ple ions. Most of the molecular and fragment ions observed in FAB and MALDI spectra are singly charged, e.g. [M+H]+, [M-HI-, [M+Na]+, [M-H+2Na]+ etc. In contrast to FAB-MS and MALDI-MS, electrospray mass spectra are characterized by multiply charged ions. The raw data obtained following ES-MS analyses is complex because ions can carry a range of charges due to the different ionisable sites in the molecule. Multiple m / z signals are therefore recorded for each mass value. Fortunately these data can be processed using a simple computer algorithm to produce a mass spectrum of comparable appearance to a FAB or MALDI spectrum. Ions are observed as clusters in all types of mass spectra because of the existence of isotopes. The contribution of I3C (1.1% natural abundance) means that for every carbon atom in the molecule there is a 1.1% chance that it will be a 13Catom and so have an atomic mass of 13 instead of 12. The relative intensity of the different peaks in the cluster reflects this probability distribution, i.e. the height of the signal at m / z X + 1 (where X corresponds to the molecular ion that contains only 12C)reflects the probability of finding one I3C atom in the molecule. The individual signals in a
32.7 Derivatisation
92 1
cluster will be observed when the instrument resolution is set to resolve nominal masses. At lower resolutions the clusters are present as an unresolved envelope. For very high molecular weight ions these envelopes may embrace more than one cluster of molecular ions, e.g. [M+H]+ and [M+Na]+, resulting in a very broad peak which cannot be accurately mass-assigned.
32.6 Assignment of Mass Values There are four principal ways of denoting the mass of an ion in the mass spectrum: i) Nominal mass-this is the sum of the integer atomic weights of the isotopes comprising the ion, giving the specific peak whose mass is being assigned. ii) Accurate mass-this is the sum of the accurate atomic weights of the isotopes comprising the ion giving the specific peak whose mass is being assigned. Accurate masses are obtained when spectra are computer assigned by comparison with a calibration spectrum containing ions whose accurate masses are known (the calibration standard is often an alkali halide such as CsI). iii) Average mass (chemical mass)-this is the average of the accurate isotope mass of each element, weighted by the relative abundance of the isotopes, i.e. it corresponds to the sum of the chemical atomic weights. Average masses are assigned when an isotopic cluster is recorded at such low resolution that the cluster is completely unresolved. The average mass is the centre of gravity of the unresolved cluster. iv) Peak top mass-this is the accurate mass of the top of an unresolved cluster. At high masses, above about m l z 5,000, the peak top mass is very close to the average mass because the isotopic distribution results in a Gaussian shape for the cluster. ES-MS assignments are normally based on peak top masses.
32.7 Derivatisation Although native samples are amenable to FAB-MS, MALDI-MS and ES-MS, it is often desirable to prepare derivatives prior to analysis. As a general rule glycans whose hydroxyl groups are protected by functional groups, such as methyl or acetyl, fragment more reliably than their native counterparts. Also derivatives are easier to obtain free from salt impurities which may prejudice the MS experiment and sensitivity is significantly improved when hydrophobic moieties are present. Derivatization methods can be broadly divided into two categories: (i) “tagging” of reducing ends, and (ii) protection of most or all of the functional groups. Commonly used tagging reagents include p-aminobenzoic acid ethyl ester (ABEE) and 2-aminopyridine (2-AP). This type of derivatization facilitates chromatographic
922
32 Structural Analysis of Oligosaccharides
purification and enhances reducing-end fragment ions in MS and MS/MS experiments. Protection of functional groups by permethylation or per(deuter0)acetylation is by far the most important type of derivatization employed in carbohydrate MS. These derivatives fragment reliably to yield abundant A-type fragment ions (see below) which are extremely useful for sequencing.
32.8 Fragmentation Pathways The following general rules apply to the fragmentation behavior of oligosaccharides and glycoconjugates [3]: i) The most abundant fragment ions are formed by cleavages at glycosidic linkages. Glycosidic cleavage is accompanied by a hydrogen transfer to the glycosidic oxygen if the charge on the fragment ion is not specifically located at the point of cleavage. ii) Ring cleavage, when it occurs, is best rationalized as arising from the sequential movement of electron pairs around the ring resulting in the breakage of single bonds and the formation of double bonds (see Figure 3). iii) Fragment ions can sometimes be formed by two or more cleavage events occurring in different parts of the molecule. This phenomenon is more frequently observed in native samples than in derivatives. Unambiguous sequencing of native samples is often not possible if “double cleavage” ions are formed in abundance. iv) The fragment ions produced by glycopeptides are derived predominantly from cleavage of the glycosidic linkages. Fragment ions resulting from cleavage of the peptide bonds are often of low abundance. v) If a permanent charge is present in the molecule (e.g. a sulfate moiety) then fragment ions produced by cleavage of labile bonds in the vicinity of the charge dominate the spectrum. The major fragmentation pathways relevant to carbohydrate MS are summarized below: i) A-type cleavage-Glycosidic cleavage (Figure 3) yields an oxonium ion thereby locating the charge at the point of cleavage on the non-reducing fragment. The term A-type cleavage is normally applied to this type of fragmentation based on nomenclature used in electron impact mass spectrometry. A-type cleavage is the major mode of fragmentation of permethylated and per(deuter0)acetylated oligosaccharides. If HexNAc residues are present in the sequence, cleavage occurs predominantly (and sometimes exclusively) at the amino sugar residues. A secondary fragmentation associated with A-type cleavage is elimination of the substituent at the 3-position of the HexNAc oxonium ion thus defining whether the 3-position is occupied by a sugar such as fucose.
32.8 Fragmentation Pathways
923
0
A-type ion
B-cleavaae and 6-elimination
J.
J.
p-cleavage ion
0-elimination ion
0-elimination ion
b-cleavage ion
(non-reducing)
(reducing)
(non-reducing)
(reducing)
Rina cleavaae
CH20R
I
bR bR
bR
bR
Figure 3. Key fragmentation pathways in glycopolymer mass spectrometry are shown in this figure. The terms A-type cleavage, P-cleavage, ring cleavage etc are useful descriptors of these pathways. The reader should consult [ 191 for information on how to systematically name fragment ions arising from each of these pathways.
924
32 Structural Analysis of Oligosacchavides
ii) P-cleavage-and p-elimination-This involves cleavage on either side of the glycosidic linkage with hydrogen transfer to the glycosidic oxygen resulting in one fragment having a hydroxyl group at the position of glycosidic linkage and the other having a double bond (Figure 3 ) . These are referred to as P-cleavage and 0-elimination products respectively. Note that, unlike A-type cleavage, a charged moiety is not produced as a result of cleavage. Depending on where the charge is located in the fragmenting molecule, either or both reducing and nonreducing P-cleavage and/or P-elimination ions will be observed. This type of cleavage is favored by native oligosaccharides and glycopeptides and is especially prominent in samples modified with a reducing end “tag”. iii) Ring cleavages-These are frequently observed in MS/MS experiments especially when cationised molecular ions are subjected to collisional activation. They can provide useful information on attachment sites of functional groups and glycosidic linkages. Examples of ring cleavages are shown in Figure 3.
32.9 Protocols for MS Analysis In this section we describe some of the MS protocols employed in our laboratories. These are given for illustrative purposes in order to facilitate understanding of the case studies presented in later sections of this Chapter. The protocols exemplify general strategies underlying each technique and are not intended to be exclusive. 32.9.1 Protocol 1-Sample
Loading for FAB-MS Analysis
i) Dissolve the sample in either 50/0 acetic acid (for underivatized samples) or methanol (for derivatives) to such a concentration that 1 pl aliquot of the sample contains the desired amount of sample for FAB-MS analysis. ii) Smear about 1 pl of the matrix onto the metal target which is attached to the end of the FAB probe. iii) Load about 1 pl of the sample on the surface of the matrix using a micropipette or syringe. iv) Introduce the probe into the FAB source and collect data immediately. v) When a wide mass range of scanning is required, e.g. mlz 4,000 to 200, it may be necessary depending upon instrument type to acquire scans covering the high and low mass range separately with two separate sample loadings. vi) Use different matrices and matrix additives to improve the quality of the data or alter the molecular and/or fragment ion patterns: use monothioglycerol as the matrix for general analysis - use rn-nitrobenzyl alcohol matrix for salty samples. add 1 pl of dilute aqueous (100-200 mM) HC1 to the matrix to minimize cationization - other matrix additives, e.g. sodium acetate and ammonium thiocyanate can also be used in specific cases [3]. ~
~
32.9 Protocols for M S Anulysis
925
32.9.2 Protocol 2-Sample Loading for NanoES-MS and MS-MS Analysis on the Q-TOF
i) Samples should be solubilized in an ES-MS compatible solvent such as acetonitrile/O.l% aqueous TFA (1 : 1, v:v). It should be noted that when using nanospray, it is observed that increased beam stability is achieved sometimes when using methanol. 1-2 pl of sample can be loaded to the end of the metal coated capillary using either plastic gel loader tips or a microlitre syringe. Care must be taken in handling the capillary because the 1-2 pm tip through which the sample is sprayed is very fragile and easily broken. ii) Place the metal coated capillary (square end first) into its holder. This consists of a knurled nut, a 5 mm length of conductive elastomer and a Swagelok union. Once secured, screw the capillary/holder onto the probe and place into the source. iii) Having inspected the MS spectrum, and tuned the quadrupole to, for example, a double or triply charged ion of interest, the argon collision gas pressure is adjusted to approximately lop4 (this will vary for optimum collision-induced fragmentation) and the collision energy is set between 10 and 60 eV relative to the size of the molecule under study, and its charge state. iv) Collect data until summation of scans produces a good quality spectrum. Collection times may vary depending on sample concentration, its propensity for ionization and state of capillary tips. However, for a 100 femtomole per yl solution, 0.5 min should be adequate for a high quality spectrum. v) Manipulation of the capillary tips by fracturing may be needed during ionization to maintain the spray without air bubbles and particulate blockages. 32.9.3 Protocol 3-Sample Q-TOF
Loading for LC-ES-MS and LC-ES-MS-MS on the
i) Dissolve the sample in an appropriate volume by using starting condition buffer and load into a 20 pl sample loop. Elute using 0.1% aqueous trifluoroacetic acid (buffer A) and a gradient up to 100% acetonitrile in 0.1% aqueous trifluoroacetic acid (buffer B) at a flow rate of 50 pl/min. The elution is monitored by UV absorbance at 214 nm in a nanoflow cell. ii) After the column a methoxyethanol :isopropanol (1 : 1, v :v) solvent mixture is added at a flow rate of 50 pl/min; this helps to counteract the signal suppressive effects of trifluoroacetic acid. The flow is then stream-split to allow collection of approximately 85 pl fractions from the column at 1 min intervals, with the remaining 15 pl/min directed on-line to the electrospray source of the Q-TOF instrument. 32.9.4 Protocol 4-Sample
Loading for MALDI-MS Analysis
i) Dissolve the sample to be analyzed in pure water (for native carbohydrates) or 80 :20 methanol :pure water (for derivatized carbohydrates) to produce a sample concentration of around 1 10 pmoles/yl. ~
926
32 Structural Analysis of Oligosaccharides
ii) Prepare the appropriate matrix: - 2,5-Dihydroxybenzoic acid (DHB) is used as general matrix; 10 mg/ml in 90 : 10 water :ethanol (for native carbohydrates) or 80 :20 methanol :water (for derivatized carbohydrates) - 2-(4-hydroxyphenylazo)-benzoicacid (HABA) is used for enhancement of polar carbohydrates; 1.3 mg/ml in 50 : 50 water :acetonitrile (for native carbohydrates) or 80 :20 methanol :water (for derivatized carbohydrates) iii) Pipette 1 p1 of the sample solution (using a Gilson P2 pipette or similar) to the metal target followed by 1 pl of the matrix solution. iv) Dry under vacuum. v) Introduce into the MALDI source and operate the instrument - DE-MALDI-TOF-MS analysis in the reflector mode is the method of choice for carbohydrate samples in the mass range 100-10,000 Da. - DE-MALDI-TOF-MS analysis in the linear mode is the method of choice for carbohydrate samples in the mass range >10,000 Da.
32.10 Applications of FAB-MS, MALDI-MS and ES-MS in Glycobiology Broadly speaking FAB-MS, MALDI-MS and ES-MS can be exploited in two general ways in the glycobiology field: i) detailed characterization of purified individual glycopolymers or mixtures of glycopolymers which usually requires acquisition of a considerable body of structural information, and ii) rapid screening of cell-, tissue- and whole animal-extracts where a limited number of MS experiments on relatively crude extracts are often sufficient to answer the questions addressed. Below we have selected examples from recent glycopolymer research which exemplify the versatility of problems amenable to current technology. 32.10.1 Case Study l-Molecular MALDI-MS
Weight Profiling of Polysaccharides by
The following study of laminarin polysaccharides illustrates the potential of MALDI-MS for rapidly and sensitively defining the degree of polymerization (d.p.) of carbohydrate polymers and for revealing structural modifications which result in altered molecular weight. Laminarins are a class of low-molecular-weight storage fi-glucans of brown algae consisting of (1-3)-linked P-D-glucopyranose residues in which some 6-O-branching in the main chain and some fi(1-6)-intrachain links are present. The majority of
32.10 Applications of FAB-MS, MALDI-MS and ES-MS in Glycobiology
927
laminarins contain polymeric chains of two types, polymeric glucopyranose only (G-chains) and polymeric glucopyranose terminated with 1-@substituted Dmannitol residues (M-chains). Both native and permethylated laminarins give excellent MALDI data [8]. The permethylated derivatives were examined in order to facilitate discrimination between M- and G-laminarins which differ by only 2 mass units in their native form but by 16 mass units after permethylation. Typical spectra are shown in Figure 4a for native laminarin from Laminaviu cichorioides (a mannitol-containing laminarin) and in Figure 4b for permethylated laminarin from Chorda crinitu (a mannitol-free laminarin). The L. cichorioides sample shows a Gaussian-like d.p. profile peaking at about d.p. 27 and ranging over about d.p. 9-40. The C. crinitu sample has a bimodal d.p. profile with abundant oligomers at both low and high mass. Interestingly, close examination of Figure 4b reveals two regions of the spectrum where clusters of ions suggest additional components, namely d.p. 5-10 and d.p. 16-17. The latter show abundant signals 46 mass units lower than the corresponding Goligomer which is consistent with cyclic components of d.p. 16 and 17. The former exhibit satellite peaks 41 mass units higher than the corresponding G-oligomer. This mass interval is consistent with the presence of a HexNAc residue replacing a hexose residue in a minority of the sample. This was an unexpected discovery because aminosugars had not previously been found in laminarins and illustrates the power of the MALDI technique for revealing the presence of novel minor constituents in complex mixtures. 32.10.2 Case Study 2-Analysis of Glycoproteins by LC-ES-MS and FAB-MS
The following study of glycodelin A illustrates how the strengths of LC-ES-MS and FAB-MS can be exploited in a complementary manner to obtain structural information on low micromolar quantities of novel glycoproteins [9]. Glycodelin-A (GdA), is a human amniotic fluid-derived glycoprotein that has potent contraceptive and immunosuppressive activities. GdA has 162 amino acids and there are three potential N-linked glycosylation sites at Asn-28, Asn-63 and Asn-85. Together LC-ES-MS and FAB-MS gave sufficient data to define the site occupancy and the sequences of the N-glycans present on GdA. The strategy for analyzing GdA is shown in Figure 5. The key experimental steps were as follows: i) LC-ES-MS and LC-ES-MS-MS analyses of tryptic and cyanogen bromide digests of GdA confirmed the protein sequence, established the non-occupancy of Asn-85 and showed that Asn-28 and Asn-63 were glycosylated. ii) Glycans were released from LC fractions containing the glycopeptides identified by ES-MS analysis. After permethylation these were analyzed by FAB-MS yielding abundant molecular ions and A-type fragment ions. The latter define the non-reducing sequences in the antennae of complex-type and hybrid Nglycans whilst the former define the overall compositions of each type of glycan including high mannose structures. FAB-MS is the method of choice for this
928
32 Structural Analysis of Oligosuccharides
d
8 In
d
8 P
$ %
:: siuno3
d
d
Counts
N
8 0
E a
0
I
a
3 v
L C
930
32 Structural Analysis of Oligosaccharides
PNGase F
PNGase F
$1SeP-Pak -:
4
Void permethylation
$1-
Void exoglycosidase
4
Hydrolysis
4 4 Acetylation 4 Reduction
$1c=( Permethylation
Void Permethylation
$1-
$1-
Sep-Pak
Sep-Pak
$1-
$1-
Figure 5. The experimental strategy used to characterize glycodelin A.
type of analysis because it reliably produces A-type ions without the need for MS/MS experiments. iii) The products of various exoglycosidase digests were analyzed by FAB-MS after permethylation. iv) Linkage analysis by GC-MS was used to define sites of glycosyl attachment in the N-glycan sequences. From these experiments the structures of the majority of the oligosaccharides present in GdA were defined as shown in Figure 6. The Asn-28 site was shown to carry high mannose, hybrid and complex-type structures, whereas Asn-63 is exclusively occupied by complex-type glycans. 32.10.3 Case Study 3-Characterization of a Novel N-Glycan by FAB-MS and FAB-MS-MS The following study of Haemonchus contortus N-glycans demonstrates how novel glycans present in complex mixtures can be rigorously identified by strategies based on FAB-MS and FAB-MS-MS analyses [lo].
32.10 Applications of FAB-MS, MALDI-MS and ES-MS in Glycobiology
931
Key
V
= Fucose
0
= Mannose = Galactose
0 = Sialic acid 0 = N-acetylglucosamine = N-acetylgalactosamine
Figure 6. This cartoon structure shows the major glycoforms of glycodelin A. Further information can be found in [9].
932
32 Structural Analysis of Oligosaccharides FUC a14
I
M a n p1-4GlcNAc p14GlcNAc
I FUCal-3
FUc aI ~ - 3
Figure 7. Structure of the novel highly fucosylated core of some H. contortus N-glycans.
Adult Haernonchus
contortus
+ + +
Reduction I Carbnxymethylation Trypsin digest
Glycopeptides I peptides PNGase F digest I Sep-pak pnrifreation
J FAB-MS
PNGase A digest I Sep-pak puritiultion
1..
Exoglycosidye digestion /
J
FAB~MS
Linkage analysis
,
+
rdasedigestion
Linkage analysis FAB-MS
+ +
MS-MS Mild methmolysis
4 .
Linkage analysis
f
Linkage analysis
Figure 8. The experimental strategy used to characterize the H. contortus N-glycan shown in Figure 7.
H. contortus is an economically important nematode that parasitizes domestic ruminants. In a programme of work aimed at identifying carbohydrate antigens that could be targets for vaccine development, the structures of N-glycans present in H. contortus extracts have been investigated. Novel N-linked glycans with trifucosylated cores (Figure 7) have been identified in adult animals using the strategy outlined in Figure 8. The key experimental steps were as follows:
i) FAB-MS analysis of permethylated glycans released from H. contortus glycopeptides by peptide N-glycosidase (PNGase F) and PNGase A (the latter releases glycans with fucose attached to the 3-position of the proximal GlcNAc of the core which are resistant to PNGase F) established the presence of high mannose structures, minor amounts of complex structures and unusual truncated glycans substituted with up to three fucose residues. ii) Digestion of released glycans with a- and (3-mannosidase greatly reduced
32.10 Applications of FAB-MS, MALDI-MS and ES-MS in Glycobioloyy
933
the complexity of the mixture of glycans facilitating subsequent MS-MS experiments. iii) Selected molecular ions were subjected to FAB-MS-MS which provides sequence and linkage informative daughter fragment ions. iv) Linkage analysis by GC-MS was used to confirm sites of glycosyl attachment. An especially important element of the above structural strategy is the MS-MS component because the novel glycans could not be isolated in sufficient quantities for individual structural studies. Some of the MS-MS data which helped to establish the sites of fucosyl attachment are shown in Figure 9.
32.10.4 Case Study 4-High Sensitivity Sequencing of a Novel Glycopeptide by Q-TOF ES-MS-MS and MALDI-MS This study of a novel glycopeptide derived from Dictyostelium sp. SKPl exemplifies the power of the Q-TOF nano-electrospray mass spectrometer for high sensitivity sequencing [ 1 I]. The efficacy of MALDI-MS for screening enzyme digests is also demonstrated. SKPl is a cytoplasmic protein which has been identified in a variety of eukaryotes including yeast, mouse and man. It is found as part of a multiprotein complex which is involved in the ubiquitination of certain cell cycle and nutritional regulatory proteins thus condemning them to proteasomal degradation. Prior to the Q-TOF study described below, metabolic labelling and other experiments had indicated that, unusually for a cytoplasmic protein, SKPl from Dictyostelium is modified by an oligosaccharide containing Fuc and Gal. To investigate the glycosylation of SKPl , cells were metabolically labelled with '[HIFuc, radioactive SKPl was purified, reduced and alkylated and finally digested with endo-Lys-C. Components of the digest were separated by gel filtration and reverse phase HPLC yielding a single radioactive peak which was likely to be the expected fucoglycopeptide. Parallel experiments with non-radioactive material were performed and the fraction eluting at the position of the radioactive peak was subjected to nanospray-ES-MS-MS on the Q-TOF. Analysis of a few picomoles in the MS-only mode gave a major [M+3H]+++ signal at m / z 829.42 which was subjected to collisional activation yielding a spectrum which was remarkably rich in doubly and singly charged daughter ions (Figure 10). A portion of the spectrum is shown at high magnification (Figure 11) to illustrate the excellent signal to noise and resolution of even very minor fragment ions. This is an important strength of Q-TOF instrumentation especially since information such as sugar attachment sites in glycopeptides is often carried by low abundance fragment ions. Detailed interpretation of the MS-MS data allowed assignment of the carbohydrate sequence, Hex-Hex-deoxyHex-Hex-HexNAc, and the complete peptide sequence, N-D-F-T-P(0H)-E-E-E-E-Q-I-R-K and also the site of attachment of the carbohydrate, which was unexpectedly found to be hydroxyproline. Some of the key fragment ions used in the structure assignment are shown in Figure 12. MALDI-MS of the glycopeptide gave an [M+H]+ signal at m / z 2487 thus providing corroborative evidence for the structure proposed from the Q-TOF data. To
934
32 Structural Analysis of Oligosaccharides
a)
Figure 9. Data from a FAB-MS-MS experiment which provided evidence for the trifucosylated structure shown in Figure 7. The [M+H]+ and [M+Na]+ ions at rn/z 1263 (A) and 1285 (B) respectively were selected for collisional activation. Note that because of the different internal energies of these two molecular ions, the fragmentation pathways are different. Structurally useful fragment ions, produced via fragmentation pathways outlined in Figure 3, are shown on the inset. These ions provide important information on the attachment sites of the fucosyl residues.
assign linkages, and to rigorously establish the types of sugar present, the glycopeptide was treated with a variety of exoglycosidases with the products being analysed by MALDI-TOF-MS. Representative data are shown in Figure 13 which shows the evidence for the non-reducing sequence being Gala 1-6Galal -?Fucal -2. Similar experiments showed that the proximal disaccharide in Figure 12 is GalP13GlcNAc.
935
32.10 Applications of FAB-MS, MALDI-MS and ES-MS in Glycobiology
1
-+-
I
259 c-) 1049
CHzOMe
CHzOMe
0-
4 *- :*M a'
OMe
AcMeN
676
0 OMe
NMeA!?
1. 3
OMc
li
845
OMe
1095
'I' 25
259
I
426
b)
Figure 9 (continued)
32.10.5 Case Study 5-FAB-MS Content
i
Screening of Biological Samples for Glycan
Although detailed structural analysis is fundamental to a full understanding of structure/function relationships, there are many situations where the rapid acquisition of partial structural information is of greater value to the program of research than a time-consuming strategy leading to complete structure assignments. For example, many months of painstaking research was required to characterise the novel N-glycan described in Case Study 3 . Once its structure was known it was of interest to determine whether its synthesis was developmentally regulated and whether it was found in other parasites occupying a similar niche to H. contortus. These issues can be addressed by screening experiments [ 121 in which tissue extracts are analysed for their glycan content using FAB-MS or MALDI-MS to screen for molecular ions
?I0
2
[M+2H]2’ 9: 32
927.4
826.41 825.90
[M+ZH]
26.92
1
HexNAc, Hex
I
Peptide
& 10
,1008.46 ,1009.43
[M+2H]
[M+2HI2+
HexNAc
HexNAc, Hex, Fuc
I
Peptide
’+28.42,1008.94’+
Peptide
I
Peptide HexNAc
1651.79 1650.78
[M+H]
Peptide
HexNAc
I
Peptide
Figure 10. Q-TOF analysis of a glycopeptide from SKPI: MS-MS collision activated decomposition spectrum of m / ; 829.42’++
I
163.08
+
m
W
W
.
382.91
175.63
174.54
1274.64
y" I0
y" 0 I
HexNAc 1376.61
y"11
Figure 11. Part of the MS-MS spectrum shown in Figure 10 expanded to show fragment ions more clearly.
1081.4
D82.41
[M+2H] 2+ 1c 98
HexNAc, Hex, Fuc
I
Peptlde
57 y" 9
I I
HexNAc, Hex
y-9
y" 10
mlz 0
938
32 Structural Analysis of Oligosaccharides
N-D-F-
PEEEEQ IRK
I?-
l - t n -
-
-
-
HexNAc
tA-
!Hex
-
-- -
+ 1173.57 1376.61
+ 1538 84
deoxyHex Hex Hex
Figure 12. Structure of the SKPl glycopeptide deduced from the Q-TOF data showing some of the diagnostic fragment ions used to assign the position of attachment of the sugar.
and FAB-MS to screen for non-reducing structures via A-type fragment ions. This type of experiment can be completed in a few weeks in contrast to the months or years required to fully characterize individual glycans in complex mixtures. Screening methods which are applicable to a wide range of biological material, including organs, cell lines and whole parasites, are exemplified by the following profiling study of a variety of mouse organs in which the FAB-MS data are interpreted in the context of previous knowledge of murine glycosylation. This work is part of a study using knockout mice to address fundamental issues of glycan function in which FAB-MS is being used to identify changes in glycosylation occurring when particular glycosyltransferase and glycosidase genes are ablated. The experimental strategy employs the following steps:
i) Detergent extraction of the tissue/organ followed by detergent removal ii) Reduction/carboxymethylation, tryptic digestion and Sep-pak separation of the peptide/glycopeptide pool from salts, free sugars etc iii) Release of N-glycans by peptide N-glycosidase F digestion iv) Sep-pak separation of N-glycans from the peptidelo-linked glycopeptide pool v) Permethylation of N-glycans and FAB-MS analysis vi) Reductive elimination of 0-glycans from peptidelo-glycopeptide pool and Dowex purification vii) Permethylation of 0-glycans and FAB-MS analysis These experiments provide compositional information (via the molecular ions) on the majority of neutral and sialic acid containing glycans in the tissueslorgans together with information on the types of non-reducing structures present (via the A-type ions). This is illustrated by the data in Figures 14 and 15. Figure 14 shows the molecular ion region of the N-glycan pools from mouse brain, liver and lung. A unique composition can be ascribed to each molecular ion as shown. Figure 15 shows the A-type fragment ions formed from these molecular ions. The A-type ions are assigned using a variety of information including their masses, the presence or absence of associated fragment ions and prior knowledge of glycosylation in the mouse. The data show that all organs are rich in both high mannose and complextype glycans. The brain and kidney have complex-type glycans that are rich in Lewis x antennae (note the A-type ion at m / z 638 in Figures 15A and B) whilst
32.10 Applications of FAB-MS, MALDI-MS and ES-MS in Glycobiology
939
A. Hex- Hex- deoxyHex- Hex- HexNAc- OHPro 2487
B. [Galal,6) Hex- deoxyHex- Hex- HexNAc- OHPro 2325
-b C. [Galal,6- Gala1 )deoxyHex- Hex- HexNAc- OHPro
0. [Galal,6-Galal ,-Fuccrl,2).Hex-HexNAc-WPro
201 7
4 Figure 13. MALDI-TOF analysis of the SKPl glycopeptide: (A) spectrum from the native glycopeptide, (B) spectrum after treatment with X. manihotis a 1-3/6 galactosidase, (C) spectrum after treatment with green coffee bean a-galactosidase, (D) spectrum after treatment with green coffee bean a-galactosidase and X. manihotis a 1-2 fucosidase.
those of the lung are characterised by sialylation (note the A-type ions at m/z 825 and 855 in Figure 15C) or alpha-Gal capping (note the A-type ion at m / z 668 in Figure 15C). The above screening strategy is remarkably reproducible with very little variation in relative molecular ion abundance occurring between data from different experiments on the same organ or on organs from different animals. Hence
940
32 Structural Analysis
of
Oligosaccharides
A Fuc,HeqHexNAc.
.--
NeuAc,Fuc,He~Hm~NAc.
03 2dOO 2200 2200 23100 24'00 .2+00 2dOO 27'00 2dOO 2dOO 30100 31100 32'00 33'00 34'00
'm/z
Figure 14. Molecular ion region of FAB spectra obtained from screening experiments (see text) on murine (A) brain, (B) liver and (C) lung. Compositions of major signals are shown.
32.10 Applications of FAB-MS, MALDI-MS and ES-MS in Glycobiology 1
941
A
+
HexNAc
/
+
FuoHexHaxNAc
0
,
+
+
B
FucHexHaxNAc
HexNAc
\
/ +
I
+
NeuGc
HaxHexNAc
\ 374 4
I
100 953
+
NeuGcHsxHexNAc
+
d
C
T+Ac Y O C 4
lu
+
NeuAcHexHexNAc
x
Figure 15. A-fragment ion region of the spectra giving the molecular ion data shown in Figure 14: (A) brain, (B) liver, (C) lung.
942
-
32 Structural Analysis of Oligosaccharides
surface glycollpidd llpooligosaccharides
uoutermembrane"
- mycollc aclds
nner plasma membrane Figure 16. A chemical model of the mycobacterial cell wall. The two major components are the mycolyl arabinogalactan-peptidoglycan complex and the lipoarabinomannan/lipomannan. The amount and exact chemical nature of the surface glycolipids are species- and/or strain-specific.
quantitative as well as qualitative differences can be readily explored in experiments on knockout mice.
32.10.6 Case Study 6-MS
Analysis of Mycobacterial Glycoconjugates
The carbohydrate-rich bacterial cell wall presents a structural challenge of a very different nature to the case studies above. Although the available sample amount for analysis is less of a problem in comparison to the situation with bioactive mammalian or helminth glycoproteins, the extremely diverse range of glycoconjugates, as well as novel saccharide residues and non-saccharide substituents that may be present, exerts a high premium on detailed structural characterization. Studies of mycobacterial cell wall components (Figure 16) exemplify recent trends in mass spectrometric analysis in this area of biopolymer research. Sequencing of the species- and strains-specific surface glycolipids, including the glycopeptidolipids and the acyltrehalose-containing lipooligosaccharides, requires the kind of sequence-informative fragment ions afforded by FAB-MS analysis of permethyl and/or peracetyl derivatives, in addition to precise molecular weight determination [13, 141. On the other hand, structural elucidation of the two highly heterogeneous homo-polymeric components, namely the mycolyl arabinogalactan (AG) and the lipoarabinomannan (LAM), requires different strategic approaches. In the case of AG, initial efforts were directed towards GC-MS and FAB-MS analysis of the small fragments generated by partial hydrolysis. The deduced structural motifs were then pieced together to give the model shown in Figure 17 based
I
t
A&
I
Rha
Q
GICNAC
I
-1
Peptldoglycan
ddacidhyddysis
I Arabinogalactan I
I
~n
r%F!
Phosphatldyllnositol mannosldes
I
Mannan Core
I
1
I
Figure 17. Structural model of LAM and AG. A G is drawn without the mycolic acids attached to the terminal Ara residues. The molecular weight of LAM has been determined by MALDI-TOF on both native and permethylated samples. The arabinan circle in both LAM and AG represents structural details not yet defined. Detailed characterizations of the various arabinan motifs were based on MS/chemical analysis of fragments obtained through acetolysis, mild acid hydrolysis and endoarabinase digestion as indicated. The branched Aras motif is common to both AG and LAM whereas the linear Arao terminal motif is found only on LAM. An Arazzmotif as drawn is deduced to be present on A G [ 151.
Ads
;-
Galactan
I
Arabinan
Lipoarabinomannan
J
Arablnan
I
acetolysis I mild acid hydrolysis -I
Cap
944
32 Structural Analysis of Oligosaccharides
on FAB-MS analysis of larger pieces generated by very mild hydrolysis of permethylated samples [ 151. Importantly, any branching position in addition to the reducing end linkage is “visualised” by the mass difference between a methyl group and exposed hydroxyl group (which can be further tagged by deuteromethyl or deuteroethyl) as a consequence of hydrolyzing off the branch(es). Thus, as illustrated, arabinan oligomers with no additional branched point were detected as Ara 5, 6, 7, 8 and then Ara 17 since Ara 9-16 cannot be generated by partial hydrolysis with only one clip at the reducing end. The success of this approach relies on the ability of MS in detecting small mass difference at high mass range, i.e. high resolution. This important information cannot at present be obtained via direct MS analysis of non-derivatized samples. Similarly, although the approximate molecular weight distribution of intact LAM can be determined by MALDI-TOF and has been used effectively to confirm the truncation in size of LAM due to ethambutol drug inhibition [16], detailed structures of LAM can only be determined through analysis of fragments derived through both chemical and enzymatic degradation ~71. The capability of MS for examining crude mixtures (see for example Case Study 6 ) , affording precise mass values for each component present, enables rapid screening of novel modifications otherwise cryptic to conventional analysis such as the inositol cap on some LAMS [17]. Likewise, whilst the introduction of ES and MALDI has made the determination of the intact molecular weights of lipopolysaccharides (LPS), lipooligosaccharides, and other polysaccharides feasible, these techniques merely complement more detailed studies of fragments generated via partial degradation which are usually most effectively examined by FAB-MS. Thus, complete structural characterisation of LPS still involves painstaking and dedicated MS analysis at the level of the repeating units (0-antigen), core units, and lipid A with and without deacylation, just as is the case with the various structural motifs of LAM and AG (Figure 17). Finally, it should be mentioned that the LC-ES-MS methodology now widely used for detecting eukaryotic glycopeptides in proteolytic digests (see Case Study 2) has proven successful in analyzing mycobacterial glycoproteins [ 181. In this study, which provides the first firm chemical evidence for the presence of protein glycosylation in mycobacteria, the neutral loss and daughter ion scanning in ES-MS provided a very sensitive means of detection of minor glycopeptides in proteolytic digests.
32.11 Concluding Remarks As exemplified by the case studies described above, FAB-, MALDI- and ES-MS are powerful techniques for glycopolymer structure analysis. Each has its own strengths. For example, MALDI-MS .would be the method of choice for molecular weight profiling of polysaccharide mixtures (Case Study l), FAB-MS is ideally suited to screening for non-reducing epitopes in biological samples (Case Study 5),
References
945
whilst nano-ES-MS-MS on the Q-TOF provides the most sensitive means of sequencing peptides and glycopeptides (Case Study 4). However it is important to bear in mind that, with an appropriate choice of experimental strategy, many structural problems can be successfully addressed with any one of the three ionization methods. Thus, for many researchers, access to instrumentation and expertise might be more important than factors such as relative sensitivities or whether collisional activation is required to generate fragment ions. Advances in biopolymer mass spectrometry in the past 20 years have been truly breathtaking. We hope that the information provided in this Chapter will encourage readers to delve more deeply into the vast recent literature of mass spectrometric glycopolymer analysis and to use their new-found knowledge to tackle the next generation of structural problems. References 1. M. Barber, R.S. Bordoli, R.D. Sedgwick and A.N. Tyler, J. Chew. Soc. Commun., (1981) 325327. 2. A. Dell, H.R. Morris, H. Egge, G. Strecker and H.V. Nicolai, Curbohydr. Rex, 115 (1983) 4152. 3. A. Dell Adv. Curbohvdr. Chem. Biochem., 45 (1987) 19-72. 4. J.B. Fenn, M. Mann, C.K. Meng, S.F. Wong, d.M. Whitehouse, Mass Spectrom. Rev., 9 (1990) 37. 5. M. Karas, A. Ingendoh, U. Bahr. and F. Hillenkamp Biomed. Environ. Muss Spectrom., 18 (1989) 841. 6. H.R. Morris, T. Paxton, A. Dell, J. Langhorne, M. Berg, R.S. Bordoli, J. Hoyes, R.H. Bateman. Rupid Commun. Mass Spectrom., 10 (1996), 889-896. 7. H.R. Morris, T. Paxton, M. Panico, M. McDowell, A. Dell. Muss Spectrometry ojBioloqica1 Materials Vol. 2 (eds B. Larsen & C. McEwan) Marcel Dekker, New York, (1998), pp 53-80. 8. A.O. Chizhov, A. Dell, H.R. Morris, A.J. Reason, S.M. Haslam, R.A. McDowell, 0,s.Chizhov. and A.I. Usov, Carbohydr. Rex, 310 (1998), 203-210. 9. A. Dell, H.R. Morris, R.L. Easton, M. Panico, M. Patankar, S. Oehninger, R. Koistinen, H. Koistinen, M. Seppala, M. and G.F. Clark J. Bid. Chem., 270 (1995) 24116-241266. 10. S.M. Haslam, G.C. Coles, E.A. Munn, T.S. Smith, H.F. Smith, H.R. Morris, H.R. and A. Dell J. Biol. Chem., 271 (1996) 30561-30572 11. P. Teng-umnuay, H.R. Morris, A. Dell, M. Panico, T. Paxton, and C.M. West, J. Biol. Chem., 273 (1998) 18242-18249. 12. S.M. Haslam, G.C. Coles, A.J. Reason, H.R. Morris and A. Dell Mol. Biochem. Parasitol., 93 (1998) 143-147. 13. K.-H. Khoo, R. Suzuki, H.R. Morris, A. Dell, P.J. Brennan, and G.S. Besra, Carbohydr. Res., 276 (1995) 449-455. 14. K.-H. Khoo, D. Chatterjee, A. Dell, H.R. Morris, P.J. Brennan, P.J. and P. Draper, J. Bid. Chem., 271 (1996) 12333-12342. 15. G.S. Besra, M. McNeil, M., K-H. Khoo, A. Dell, H.R. Morris and P.J. Brennan, Biochemistry 34 (1995) 4257-4266. 16. K.-H. Khoo, E. Douglas, P. Azadi, J.M. Inamine, G. Besra, P.J. Brennan and D. Chatterjee, J. Bid. Chem., 271 (1996) 28682-28690. 17. K.-H. Khoo, A. Dell, H.R. Morris, P.J. Brennan, D. Chatterjee, J. Biol. Chem., 270 (1995) 12380- 12389. 18. K.M. Dobos, K.-H. Khoo, K. Swiderek, P.J. Brennan, P.J. and J.T. Belislie, J. Bucteriol., 178 (1996) 2498-2506. 19. B. Domon and C.E. Costello Glycoconj. J . , 5 (1998) 397-409.
Carbohydrates in Chemistry and Biology Editor Beat Ernst, Gerald W. Hart, Pierre Sinay copyright OWILEY-VCH Verlag GmbH, 2000
33 Conformational Analysis in Solution by NMR S. W. Homans
33.1 Introduction Of the functions that have been ascribed to oligosaccharides [ 11, their role in molecular recognition appears to be a repetitive theme in many biological processes. Recent work on the molecular genetics of glycosylation has indicated that oligosaccharides exhibit a particularly prominent role in the development of multicellular organisms [2-91. Morever, it has been known for many years that oligosaccharides are important for the invasion, infectivity and survival of parasites [ 101 and pathogens [ 111 in host cells. There is therefore compelling evidence to suggest that a role in cell-cell communication might be the primary function of oligosaccharides. This might explain the apparent lack of function of these moieties when examined at the level of a protein or indeed an individual cell. In order to understand the molecular basis of oligosaccharide-mediated recognition phenomena, we need to have knowledge of the structure and dynamics of the free carbohydrate ligand in addition to the ligand-receptor complex. In this Chapter, I shall review current knowledge on the former. A discussion of ligand-receptor complexes can be found elsewhere in this volume.
33.2 Solution Conformations of Oligosaccharides 33.2.1 The NMR Technique
Since oligosaccharides in general fail to crystallize, the only technique that is able reliably to offer information on the structure of oligosaccharides in solution at atomic resolution is nuclear magnetic resonance (NMR) spectroscopy [ 12-1 51. There are three NMR parameters that are relevant to the structural analysis of oli-
948
33 Conformational Analysis in Solution by N M R
gosaccharides. The chemical shift refers essentially to the frequency at which each discrete resonance line in the spectrum can be found. This frequency is dependent upon the precise chemical environment (hence the term chemical shift) of the nucleus that gives rise to the resonance, since this environment has the capacity to shield or deshield the nucleus from the full effects of the applied magnetic field. The chemical shift contains a wealth of information on molecular structure, but in view of the complexity of the factors that contribute to it, it is only possible to make practical use of this information in a few simple systems. Nevertheless, chemical shifts can offer useful information on carbohydrate structure when interpreted in a semi-quantitative sense [16-281. In addition to the chemical shift, each NMR resonance line typically exhibits a multiplet structure or ‘splitting’. This arises from the sensitivity of a given nuclear environment to the spin-state of nearby covalently bonded nuclei and is transmitted via the bonding electrons, becoming essentially unmeasurable over more than three bonds, at least in saturated systems. The threebetween nuclei is of considerable practical imporbond coupling (denoted 35nn’) tance, since the dihedral angle formed by the outermost pair of bonds influences the magnitude of the splitting. It was shown by Karplus [29] many years ago that an = A cos2 8 + Bcos 8 C (where A , B and C empirical relationship of the form 35nn’ are constants) can be used to derive angular information from three-bond couplings. A third parameter is known as the nuclear Overhauser effect (NOE). This parameter is not manifest in the NMR spectrum directly, but can be observed as a change in the intensity of the resonance corresponding to one nucleus when the spin-state populations of another resonance are perturbed by selective application of radio frequency energy [30]. This change in intensity is related, inter alia, to the inverse sixth power of the distance between the nuclei. Under the appropriate circumstances the NOE can therefore be used to measure internuclear distances up to -0.5 nm.
+
33.2.2 Conformational Parameters in Oligosaccharides Oligosaccharides are comprised of linear or branched monosaccharide units that are invariably linked from the C-1 position of one monosaccharide unit (the ‘glycon’) to one of several positions on the neighboring monosaccharide unit (the ‘aglycon’). With few exceptions, the chemical nature of the coupling between the monosaccharide units is an ether linkage (C-0-C), which is termed the glycosidic linkage. Therefore two rotatable bonds exist about the glycosidic linkage (Figure 1). In pyranoses (the types of monosaccharide that will almost exclusively be discussed here) the geometry of each monosaccharide ring can essentially be thought of as being fixed on the timescale of the NMR experiment, usually in the ‘chair’ (4C1) configuration. Therefore the primary sites of conformational variation are the two glycosidic torsion angles defined by the atoms H-1-C-1-0-1-C-X and C-1-0-1C-X-H-X, where C-X and H-X represent aglyconic atoms. These torsion angles are denoted +H and $H in IUPAC convention. In addition there is a third dihedral angle about the rotatable C-5-C-6 bond in each monosaccharide residue defined by H-5-C-5-C-6-0-6. This dihedral angle is of particular importance for conforma-
33.2 Solution Conformations of Oligosaccharides
949
Figure 1. Conformational torsion angles in oligosaccharides. For NMR studies, these angles are usually defined as Cp = H1-C1-01-CX, I) = Cl-01-CX-HX and o = H5-C5-C6-06, where CX and HX are aglyconic atoms.
tional studies when substitution occurs at the C-6 position and is given the symbol OH. Henceforth, the subscripts on +H, QH and OH will be dropped for convenience. 33.2.3 Conformational Restraints Since the internal geometry of each monosaccharide ring is essentially fixed, the solution conformation of an oligosaccharide can be defined by the torsion angles 4, and O. These angles can in principle be determined experimentally by measurement of conformational restraints from one monosaccharide across the glycosidic linkage to its neighbour. Thus, one or more NOEs can usually be measured from H1 of the glycon to aglyconic protons proximal to the glycosidic linkage, and two 13C-' H three-bond coupling constants can be measured across the glycosidic linkage. Unfortunately, even when three such NOEs can be measured (usually the maximum number that can be observed) together with two coupling constants, the number of restraints is such that it can be impossible to distinguish between models involving a single conformation about the glycosidic linkage rather than a model involving substantial conformational flexibility [3 11. Additional conformational restraints are required to distinguish between these two models, and much effort has been expended in recent years to achieve this goal.
+
33.2.4 I3C Isotopic Enrichment
In order to obtain additional conformational restraints in oligosaccharides, it is useful or even essential to work with the glycan in 13C-enriched form. This is by no means a trivial task. Although stable isotopic enrichment of proteins is now commonplace, this is often relatively straightforward by use of the relevant organism that over-expresses the protein in question together with isotopically enriched media. While it is possible to grow mammalian cells using such media and thus to
950
33 Conformational Analysis in Solution by N M R
prepare isotopically enriched glycoproteins [32], the cleavage of the glycans from the latter is not an efficient way to generate isotopically enriched material. It is usually more efficient to use a synthetic or chemoenzymatic strategy [33-361. The latter is particularly attractive in view of its simplicity on the milligram scale which is ideal for NMR studies. In particular, once the relevant isotopically enriched building blocks have been obtained, it is straightforward to prepare oligosaccharides with a variety of different labelling regimes to suit a particular NMR technique. In general, the protocols for the chemoenzymatic synthesis of 13C-enriched oligosaccharides can mirror those used for the synthesis of their natural-abundance 13C counterparts. However, it is of course necessary not only to have available the relevant I3C-enriched monosaccharide precursors, but also the nucleosidediphosphate derivatives that are the correct donor substrates of the relevant glycosyltransferase. Since the only monosaccharide that is available in fully 13C-enriched form at reasonable cost is glucose, a good deal of synthetic work might be necessary in order to obtain the relevant monosaccharides and their derivatives for the chemoenzymatic synthesis. As an example Figure 2 shows the protocol used in our own laboratory for the chemoenzymatic synthesis of sialyl-Lewisx,NeuSAca2-3Galpl4(Fucal-3)GlcNAc, which required 38 steps [34].
33.2.5 Additional Conformational Restraints Exchangeable Protons Conventional H NMR experiments on oligosaccharides are typically recorded in D20 solvent, in order to avoid swamping proton resonances from the oligosaccharide with the much higher concentration of protons (-1 10 M) in pure water. Under these circumstances, the hydroxyl and amide protons exchange rapidly with the deuterated solvent and become invisible in proton NMR spectra. While highly efficient solvent suppression pulse sequences have been developed recently which enable oligosaccharide proton NMR spectra to be recorded in H20 without substantial interference from the solvent resonance [37], it is still impossible to observe the hydroxyl protons at ambient temperature since they exchange too rapidly with the solvent. Pioneering work by Poppe and van Halbeek [38-401 showed that the hydroxyl proton exchange rate could be slowed sufficiently to enable the observation of discrete resonances in aqueous solution at temperatures lower than - 15 "C. This important development has enabled the measurement of NOEs to and from exchangeable protons, thus increasing dramatically the number of available conformational restraints in certain instances. One of the earliest applications of such NOEs concerns the study of the solution conformation of Neu5Aca2-6Gal~l-4Glc [41]. These authors also demonstrated that the observation of hydroxyl proton resonances can be very useful in demonstrating the existence of long-lived hydrogen bonds in solution. Such hydrogen bond restraints can be very powerful in the conformational analysis of oligosaccharides, since unlike the NOE, the presence of a hydrogen bond restrains the relevant atoms to a narrow range in the region of 0.25
-
33.2 Solution Conjiwmutions of Oligosuccharides OH OH
951
OH
O-"DP
OH
OH OH
OH
OH
I
O
NHAc H
W
OHE & O *
Galactosyltransferase
NHAc
=
O
OH
/
*
OH
OH
06
Milk Fucosyltransferase
I
OH
NHAc
OH
NHAc
OH
C H ; F - G D P OH
b *
0 -
H O
NHAc
NHAc
on
OH
C H ~ @ ~ ~ OH
Figure 2. Scheme for the chemoenzymatic synthesis of SLe" in 13C-enrichedform. The donor substrates UDP-Gal, PNP-NeuSAc and GDP-Fuc are typically prepared synthetically from uniformly "C-enriched glucose, giving rise to full 13C-enrichment in the glycan moiety of the donor. This scheme works efficiently on the milligram scale providing adequate material for NMR studies.
nm. Hydrogen-bonded hydroxyl protons present significantly smaller exchange rates in comparison with those that are not hydrogen bonded, and also possess significantly smaller temperature coefficients (i.e. the change in chemical shift with temperature). In addition, hydrogen bonded hydroxyl protons often exhibit scalar coupling constants to the adjacent non-exchangeable proton that differ substantially from the -5.5 Hz coupling that is observed when the hydroxyl group is free to rotate. In the case of Neu5Aca2-6Gal~1-4Glc,Poppe et al. [41] observed anomalous values for these parameters for NeuSAc OH8, NeuSAc OH7 and Glc OH3, confirming earlier reports of a hydrogen bond between Neu5Ac OH8 and the ring oxygen or carboxyl group of the Neu5Ac residue [38], and suggesting the presence of a hydrogen bond between Glc OH3 and the ring oxygen of the Gal residue. One
952
33 Conformational Analysis in Solution by N M R
further approach for defining hydrogen bond connectivities is to make use of the deuterium isotope effect on I3C chemical shifts. At low temperatures (248-268 K) and neutral pH values, the exchange of hydroxyl protons in water is slow enough to observe these short-range effects, which arise from the influence of the O-D group on the chemical shift of the parent carbon resonance. This effect is most pronounced for hydroxyl groups involved in long-lived hydrogen bonds, and can readily be detected as a 'splitting' of the carbon resonance into two signals in 50%H20/50%D20 solution [42-441. Isotopic enrichment is clearly not a prerequisite for the observation of hydroxyl protons in oligosaccharides. However, use of enriched material does simplify the attenuation of the strong H20 resonance and moreover permits spectral editing in a third I3C dimension that can be very useful in overcoming resonance overlap that is typically observed in the hydroxyl region of the 'H spectrum [45]. Heteronuclear Overhauser Effects It has long been recognized that useful structural information can be obtained from heteronuclear 13CC1H}Overhauser effects [46-48, 30, 49, 501, and it follows that such measurements might be of value in the conformational analysis of oligosaccharides. A principal difficulty with this approach is that NOES from H to 13C are of significant intensity only for quaternary carbons [41]-protonated carbons do not exhibit a substantial NOE by virtue of the efficient dipolar relaxation by the attached proton and consequent leakage of the NOE. A further complication is that an indirect NOE often exists from the source proton through the attached proton to the carbon, and since this NOE is opposite in sign to the direct effect and often larger, I3C{'H} NOEs in carbohydrates are usually negative [51]. This effect can readily be demonstrated from full-relaxation matrix simulations on a simple disaccharide (Figure 3 ) . A suitable means by which the l3CC1H}NOE intensity can be increased to more useful levels is to perdeuterate the aglycon. The indirect effect via protons is thus abolished, and permits the direct trans-glycosidic 13C{'H} NOE to be observed without interference. Given the substantial potential values of 13C{'H} NOEs for the conformational analysis of oligosaccharides, it is worthwhile considering the appropriate experimental regime for their measurement. Na'ively, one would choose steady-state NOE experiments where the source proton is saturated by an irradiating field, since theoretically it can be shown that these NOEs reach a value more than ten times greater than the transient NOE method. However, steady-state methods involve detection of 13C, whereas by comparison H detection offers approximately eightfold enhancement of signal-to-noise [50]. Given the difficulties with selective saturation of protons in very crowded regions of carbohydrate spectra, the transient method, which is at the heart of two-dimensional methods [46,47], is the method of choice. The applications of 13CC1H}NOEs for the conformational analysis of oligosaccharides have thus far been quite limited. However, they have been measured at natural abundance in Neu5Aca2-6Galp1-4Glc [41] and in I3C, 2H enriched Galpl-4Glc [51].
'
33.2 Solution Conformations of Oligosaccharides
-0.5
953
9 0
1
2
3
4
5
6
Mixing time (s)
0.6 h . c1I
B
c1
0.4
E
. I
0.2
0.0
Mixing time (s) Figure 3. Theoretical full-relaxation matrix simulations of H{ I3C} NOES across the glycosidic linkage in GalP1-4[U-'3C]Glc (top panel) and GalP1-4[U-13C,2H]Glc(bottom panel). Traces show Glc C-4, (0) Glc the NOE intensities to Gal H-1 vs mixing time in a HOESY experiment for (0) C-5 and (0)Glc C-6.
1 3 ~ - ' 3 Coupling-Constants ~
In recent years the value of long-range 13C-13C coupling constants for the conformational analysis of oligosaccharides has been realized [ 52-55, 56-58]. A principal difficulty with the measurement of these parameters is the requirement for 13C-enriched material-unlike heteronuclear 13C-' H measurements where the -1% natural abundance of I3C is not a substantial sensitivity barrier, 0.01% of
954
33 Conformational Analysis in Solution by N M R
molecules will contain two 13Cnuclei. For this reason most applications of 13C-13C coupling constant measurements have concerned bacterial polysaccharides or small oligosaccharides where isotopic enrichment can be achieved either biosynthetically or by chemoenzymatic synthesis. The most convenient approach for the measurement of these couplings appears to be the 'LRCC' method developed by Bax and co-workers for application to proteins [ 591, and several Karplus parametrizations have been reported with which to convert the relevant coupling constant into angular information for conformational analysis [56, 57, 601. Dipolar Couplings Recently, work on the inherent magnetic alignment of proteins in very high magnetic fields (>14 T) has permitted the measurement of residual dipolar couplings between NMR active nuclei [61-631. These couplings, which average to zero for an isotropically tumbling macromolecule, present a small but finite value due to the small net alignment in the magnetic field. Importantly, residual dipolar couplings provide long-range structural information since their magnitude depends, inter aka, on the inverse cube of the distance between the two nuclei and the function (3 cos20 - l), where 0 is the angle between the relevent bond vector and the principal axis of the alignment tensor [61]. The long-range structural information derives from the fact that 0 for each bond vector is referenced to the same axis system, and hence the angles in disparate regions of the molecule are directly related. Since the original observation of magnetic alignment in proteins, more recent work has demonstrated that it is possible to obtain alignment two orders of magnitude greater by use of dilute liquid-crystalline solvents [ 641. These solvents comprise dihexanoylphosphatidylcholine (DHPC) and dimyristoylphosphatidylcholine (DMPC) in aqueous solution, which form disc-like micelles ('bicelles') above the transition temperature (35 "C) that align in a magnetic field [65]. Macromolecules that are not spherically symmetric, when dissolved in these solutions, become partially aligned by virtue of their hydrodynamic properties (Figure 4). Below the transition temperature (27 "C) the solution becomes isotropic, and the dipolar coupling is no longer observed. Thus it is straightforward to measure dipolar couplings for each bond vector by comparison of the splittings at 35 "C, which will be the sum of the scalar and dipolar couplings, with those at 27 "C, which will exhibit only the scalar couplings. An example of the measurement of 13C-'H dipolar couplings from heteronuclear single quantum correlation spectra is shown in Figure 5. The dipolar couplings can be measured to reasonable accuracy in this manner, but for higher accuracy J-modulated or IPAP methods can be used [62, 661. The measured dipolar couplings are related to 0 by the following expression:
where S is the generalized order parameter for internal motion of the bond vector PQ, po is the magnetic permeability of vacuum, yp and yQ are the magnetogyric ratios of P and Q, h is Planck's constant, Y ~ Qis the distance between P and Q, A , and A , are the axial and rhombic components of the alignment tensor A, and 0 and
33.3 Experimental Restraints in Conformational Analysis
955
C
I
Bo
Figure 4. Diagrammatic illustration of the partial alignment of an anisotropic macromolecule in a liquid crystalline solution of phospholipid bicelles. The bicelles align spontaneously with respect to the applied magnetic field (Bo),and the macromolecule gains a small net degree of alignment by virtue of its physicochemical properties in the spaces between the bicelles.
4 are cylindrical coordinates describing the orientation of the vector P-Q in the principal axis system of A [64]. It is straightforward to extract the relevant angular information from this dependence, which gives rise to knowledge of the orientations of the relevant bond vectors with respect to a single fixed axis, thus providing longrange structural information that hitherto has been lacking in NMR studies.
33.3 Experimental Restraints in Conformational Analysis 33.3.1 Restraining Protocol
The principal purpose of the collection of the parameters described above is as restraints in conformational analysis. These restraints are incorporated in molecular mechanical simulations as pseudo-energy functions which apply an energy penalty if the theoretical parameter deviates from the experimental value according to some prescribed formula. However, care must be exercized in applying experimental restraints in conformational analysis. This is because in general it is impossible to exclude motional averaging about the glycosidic linkages (see Section 33.4). Under these circumstances, the relevant NMR parameter represents an average over a number of possible different configurations of the molecule, and if the restraints are applied without consideration of this possibility (i.e. assuming the molecule is ‘rigid’), there is every possibility that the resulting structure will be a ‘virtual conformation’ that bears little resemblance to reality [67, 681. Fortunately this problem has been recognised very early in structural studies of macromolecules, and protocols have been devised to deal with it. Biharmonic Restraints
In the case of distance restraints derived from NOE measurements or from hydrogen-bond connectivities, it is usual to apply a restraining function in the form of a
956
33 Conformational Analysis in Solution by N M R 654
66
a
/i
73
77
78 79 80 q,
, . , I , , . , I , , . . I , , , , I , , . . , . , . . I , . , , I , , , , I , , , , I , . , , I , , , . I . , ,
4.5
4.4
4.3
4.2
4.1 4.0
3.9
3.8
3.7
3.6
3.5
F2 (PPm)
0
4.5
4.4
4.3
4.a
4.1
4.0
3.9
0
3,s
3;7
3,6
3i5
F2 ( m m )
Figure 5. Proton coupled ‘H-I3C HSQC spectra of uniformly I3C-enrichedGalal-4Gal~1-4Glcin a 7.5% solution of DHPC:DMPC (1 :2.9) at (a) 27 “C (isotropic phase) and (b) 35 “C (liquid crystalline phase). The difference in ‘Jc,H for Gala H-5 is shown in each case.
biharmonic potential [69]. That is, there is no energy penalty if the theoretical distance lies within prescribed distance bounds, whereas an increasing penalty function is applied if the distance is outwith these bounds. Typically, the NOE restraints are semi-quantitatively categorized into ‘weak’, ‘medium’ and ‘strong’, with corre-
33.3 Experimental Restraints in Conformational Analysis
957
sponding distance bounds of 1.SA < r < 2.7A, 1.8A < r < 3.3A and 1.SA < r < 5 A respectively. In this manner the molecule is free to adopt any conformation without additional energy penalty provided all theoretical distances are within the relevent bounds. The relatively loose bounds are supposed to take account of the fact that the measured NOE may correspond to a motionally averaged distance. Incorporation of angular restraints is more difficult. Unlike the NOE, where the measurement of this parameter demonstrates at least that the relevant atoms must be close in space for some of the time, a given spin-coupling constant may correspond with a single, fixed angle in one extreme, or may represent an average over 360" rotation at the other extreme. Although methods have been proposed to overcome this limitation, in general it is undesirable to apply angular terms as primary restraints in conformational analysis. Time-Dependent Restraints
A principal limitation of conventional biharmonic restraints is that their presence can mask transitions between conformational states that are widely separated on the potential surface. Thus, if during the course of a transition from one low-energy state to another, distance restraint is violated (the distance between two restrained atoms exceeds 5 A, for example), then a substantial energy barrier to this transition will be created. In order to overcome this problem, restraints can be imposed in a so-called 'time-dependent manner' [70]. The basic concept is that the theoretical average distance over a defined part of the simulation, rather than the theoretical instantaneous distance, is required to satisfy certain bounds during the simulation. By use of this protocol, violations of conventional distance bounds are permitted, as long as the running average satisfies these bounds. Thus transitions between different low-energy minima are more likely to occur in simulations using the time-averaging protocol. Remarkably, to our knowledge very few studies on glycoconjugates have utilized time-averaged restraints [71, 721.
33.3.2 Dynamical Simulated Annealing
In principle, conformational restraints can be incorporated into conventional energy minimization algorithms in order to obtain a single, fixed, 'minimum energy' conformation that is consistent with available experimental data. However, this approach is not very efficient, and moreover by virtue of the local minimum problem, it is very likely that the resulting structure will not be the global minimum energy configuration. A much more efficient approach is termed dynamical simulated annealing [69]. This can be thought of as a hybrid between a molecular dynamics simulation and energy minimization. The idea is to simulate the dynamics of the molecule at a relatively high temperature (typically 750 K) over a period of time in order efficiently to sample a substantial region of conformational space. The simulation is continued while the system is then slowly cooled to a very low temperature. Unlike conventional minimization, the system has sufficient thermal energy so there is a tendency for the molecule to escape local minima (provided the cooling is slow
958
33 Conformational Analysis in Solution by N M R
enough) and to ‘fall’ to a lower energy minimum. Once the system has cooled to around 5 K, the simulation is terminated with a conventional energy minimization step. Throughout the simulation the experimental restraints are applied, such that the molecule is constantly driven to a minimum energy conformation that is hopefully consistent with all experimental data. In order that the restraints do not limit the accessible regions of conformational space in the early stages of the simulation at high temperature, their strength is typically scaled for a period of time until the temperature falls to around 300 K. Moreover, a series of simulations is usually performed with a randomized starting structure as input, in order further to explore conformational space and to ensure that the final structure is not dependent on the starting geometry. This typically results in one or more families of structures that are essentially consistent with the applied distance restraints.
33.4 Analysis of Oligosaccharide Dynamics Oligosaccharides do not exist as ‘rigid’ entities in solution. While each monosaccharide residue can be thought as being fixed in a certain conformation (at least for pyranoses) on the NMR timescale, there may be substantial torsional oscillations about the glycosidic linkages. In extended structures, these oscillations will be additive, and the manifold of conformations is solution can span considerable regions of conformational space. Characterization of the solution dynamics is very important from the point of view of binding affinity to receptors. To appreciate why this is the case it is convenient to consider a simple ligand-protein association involving a flexible ligand (A) in comparison with a rigid ligand (B). The configurational entropy (i.e. number of available degrees of freedom) of the former is greater than the latter, and hence the Gibbs free energy of the former will be lower by virtue of the relation G = H - TS (where H is the enthalpy and T is the absolute temperature). Thus, the combined free energy of the uncomplexed components will be lower in the case of the flexible ligand and, since the free energy of binding A& is the difference between the free energy of the complexed (which will be the same irrespective of the dynamics of the ligand, assuming it is rigidly held in the binding site) and the uncomplexed components, the free energy of binding of the flexible ligand will be lower than that of the rigid ligand. This in turn will give rise to a lower affinity by virtue of the relation AGO = -RTlnK,, where AGO is the standard free energy of binding, R is the gas constant and K, is the association constant. Thus in terms of inhibitor design, it is important to consider the dynamics of the ligand, i.e. a rigid ligand that adopts the bound-state conformation in solution is likely to have optimal affinity. The characterization of oligosaccharide dynamics by NMR is not trivial. All of the conformation-sensitive NMR parameters are averaged by the motion, and it is not possible directly to deconvolute the nature or number of conformational states that contribute to this average. NMR relaxation parameters are directly sensitive to motion if on the appropriate timescale [73, 741, but again cannot be interpreted in
33.5 A Case Study on NeuSAcd-3Gal~I-4Glc
959
the absence of a model for the motion [75]. Thus we are forced to rely on methods that simulate the regions conformational space that an oligosaccharide can access in solution, and two methods are in general use. 33.4.1 Monte-Carlo Simulations In this method [76-781 the starting point is an arbitrary conformation of the system which is perturbed slightly by, for example, a torsional rotation. The internal energy of the system is calculated and compared with the energy before the perturbation. If the energy has decreased then the new conformation is accepted. If the energy has increased, the increase is compared with the thermal energy k T is order to decide whether the new conformation is accepted. The value of exp(-AU/kT) is compared with a random number between 0 and 1, where AU is the increase in energy. If exp(-AU/kT) is larger than the random number, the new conformation is accepted. Otherwise a new conformation is generated, and the process is repeated. In this manner the system under investigation explores conformational space. Since perturbations that raise the energy are sometimes allowed, the procedure permits the escape of the molecule from local minima. 33.4.2 Molecular Dynamics Simulations The molecular dynamics method directly simulates the motions of all the atoms in the system, by using Newton’s laws of motion [79]. Since the intenal energy of a given conformation of the system can be derived from a conventional molecular mechanics approach, the force on each atom can be determined from the derivative of the energy along different directions of space. The motion of the atom can in turn be determined from the acceleration which is a known quantity given the force on each atom and its mass. The system is usually started in an arbitrary conformation near 0 K, and the velocities of the atoms are then increased so that the average kinetic energy corresponds with a desired temperature. One of the advantages of the molecular dynamics method is that the temperature can be raised or lowered at will by coupling to a temperature bath, which thus permits energy minimization by simulated annealing (Section 33.3.2). Moreover, since the time-scale of the internal motions is known from the simulation, it is possible to back-calculate time-averaged theoretical NMR parameters for comparison with experiment (Section 33.5.4).
33.5 A Case Study on NeuSAca2-3Galp1-4Glc As an example of the use of some of the techniques described above in the conformational analysis of oligosaccharides, a case study of the solution properties of the trisaccharide NeuSAca2-3GalS 1-4Glc will be described.
960
33 Conformational Analysis in Solution by N M R
33.5.1 Resonance Assignments in NeuSAca2-3Galj31-4Glc The first step in the conformational analysis of the oligosaccharide is the determination of complete proton and carbon resonance assignments. These have been reported previously for this particular trisaccharide [ 801. In systems with unknown assignments, the presence of uniform 13C-enrichment permits the application of conventional HCCH-COSY and HCCH-TOCSY experiments [81, 821, which invariably give complete resonance assignments in an efficient manner.
33.5.2 ROE Connectivities Although NeuSAca2-3Galfil-4Glc contains only 29 non-exchangeable protons, the proton NMR spectrum is remarkably complex, with most resonances concentrated within 0.4 ppm. Despite the fact that complete 'H and 13C resonance assignments are available, this overlap renders impossible analysis of the conventional H-' H NOESY spectrum. Isotopic 13C-enrichment permits the acquisition of threedimensional I3C-edited spectra at high sensitivity. Since the rotational tumbling time of the trisaccharide is close to the point where the homonuclear 'H-'H NOE is zero, it is necessary to acquire three-dimensional 13C edited ROESY-HSQC spectra. It is convenient to examine such spectra as a series of two-dimensional planes. In NMR of proteins, it is usual to examine the Fl/F3 ('H/'H) plane, but due to severe overlap of proton resonances, it is more useful to examine F2/F3 (I3C/'H) planes in oligosaccharide spectra. A typical F2/F3 plane from the ROESY-HSQC spectrum of NeuSAca2-3Galp1-4Glc is shown in Figure 6 [56]. This illustrates all the ROE connectivities derived from Gal H-1. Many of these correspond with intraresidue ROEs that do not contain conformational information across the glycosidic linkages. The remainder (crosspeaks plotted in boldface) represent ROE connectivities across the GalPl-4Glc glycosidic linkage. The ROE connectivities to Glc H-4 and Glc H-6 are anticipated on the basis of the known conformational preferences about the glycosidic linkage. However, substantial ROEs are also observed to Glc H-3 and H-5. These ROEs are extremely difficult to observe in conventional homonuclear H NMR spectra due to extensive overlap, and given their substantial intensity, they cannot arise from a single conformation about the Galb1-4Glc linkage that simultaneously gives rise to ROEs to Glc H-4 and H-6. Thus these measurements alone indicate that a degree of flexibility exists about the Galpl-4Glc glycosidic linkage. In particular, the ROEs to Glc H-3 and H-5 demonstrate the existence of the 'anti' conformation about the glycosidic linkage, i.e. where the glycosidic torsion angle $ adopts a value of -180" [83-861. By examination of other F2/F3 planes in the ROESY-HSQC spectrum, a total of seven transglycosidic ROE connectivities is observed for this glycan. These can be used as conformational restraints in the determination of the 'global minimum' energy configuration of the glycan .
'
'
33.5 A Cuse Study on Neu5Acc12-3Gul/lI-4Glc
961
F2
(PP4 62 64 66 68
3c
70 72 74
,
76
78
Gal H3
80 wpGlc H4
82 I""I""I""I""I""I""I""I""I~~"I""I""~
4.3
4.1
3.9
3.7
3.5
3.3
l H F3 (PPm) Figure 6. Two-dimensional F2/F3 ( I3C/'H) plane derived from three-dimensional ROESY-HSQC spectrum of NeuSAca2-3Gal~1-4Glc.This plane shows all intra- (normal face) and inter-residue (boldface) ROE connectivities to Gal H-1 .
33.5.3 'Global Minimum' Conformation of NeuSAcaZ3Gal~l-4Glc The 'global minimum' energy conformation of the glycan can be determined by dynamical simulated annealing calculations with the ROE connectivities described above as time-averaged conformational restraints. In general it is desirable to compute a series of such calculations with different (pseudo-random) conformations of the glycan as input. In principle, it is possible to begin with random coordinates, but this introduces a substantial complication since it is necessary to ensure that the correct chirality is preserved, and moreover the correct ring geometry must be defined. In practice, the chirality and ring geometry is known, and hence it is more convenient to generate a series of random structures with defined chirality and ring geometry but with pseudo-randomized torsion angles about each glycosidic linkage [87]. A set of such pseudo-random structures can be generated by use of a dynamical quenching procedure.
962
33 Conformational Analysis in Solution by N M R
NeuNA
Figure 7. Stereo view of the family of structures derived from a dynamical simulated annealing calculation on NeuSAca2-3Galfi1-4Glc. The three families are labelled ‘A’, ‘B’ and ‘C’ respectively.
The result of the dynamical simulated annealing calculations is a set of structures most of which satisfy the applied conformational restraints. Those that do not are discarded, and the remaining structures typically form ‘families’ with similar conformations (Figure 7). These data therefore indicate that different conformations satisfy the experimental ROE restraints simultaneously, and thus provide a crude picture of motional dynamics. A more detailed analysis of dynamics can be obtained from molecular dynamics simulations.
33.5.4 Conformational Dynamics of NeuSAca2-3Galp1-4Glc In order to probe the conformational dynamics of the glycan, one of the low-energy structures derived from the simulated annealing procedure is chosen as input to a restrained molecular dynamics simulation. The conformer with the lowest overall energy can arbitrarily be chosen for this purpose. In principle, it would be desirable to perform free dynamics simulations with explicit inclusion of solvent water in order to obtain an accurate picture of the solution dynamics of the glycan. However, the accuracy of current forcefield parametrizations for oligosaccharides is insufficient over the complete potential surface to generate an accurate model of solution dynamics. Moreover, the inclusion of solvent water molecules severely restricts the length of time over which the molecular dynamics simulation can simulated due to the very large number of computations that are required per step. Thus it is practical only to simulate the effects of solvent water in part by use of an appropriate dielectric constant. In adopting this approach, it must be remembered that a restrained molecular dynamics simulation will be performed which will include data from solution NMR studies, including hydrogen-bonding restraints where appropriate. It is therefore not necessary for the simulation conditions to reproduce accurately these solution properties. It can be argued that the application
33.5 A Case Study on Neu5Aco12-3Gu1~1-4Glc
2 O
O 2
1
‘-180
180
I
Phi (deg)
963
i
-180
180
Phi (deg)
+
Figure 8. Instantaneous values of vs $ for (a) NeuSAca2-3Gal and (b) GalP1-4Glc glycosidic linkages derived from a 5 ns in vacuo MD simulation of Neu5Aca2-3Gal~l-4Glc.
of experimental restraints will of course modify the available conformational space explored by the glycan during the simulation. However, in the absence of wellparametrized forcefields for oligosaccharides there is little option but to modify the behavior of the forcefield in this manner, and the use of time-dependent restraints minimizes the influence of such restraints in e.g. preventing excursions to other local minima via a substantial energy barrier. The result of a 5 ns restrained MD simulation for Neu5Aca2-3GalP1-4Glc is shown in Figure 8. It can be seen that each glycosidic linkage exhibits considerable conformational freedom, and in particular the GalPl-4Glc glycosidic linkage is seen to adopt the ‘anti’ conformation for a considerable period of time. The validity of this simulation can be further assessed by back-calculating the theoretical ROES from the MD simulation for comparison with experimental values, using the appropriate formalism for the computation of the time-averaged ROE. The backcalculation of theoretical parameters that are used as restraints in the simulation is not a circular argument, since such restraints are typically applied as a biharmonic function with lower and upper bounds that differ substantially, such that the theoretical ROE can in principle vary over a very wide range. Moreover, other conformation-sensitive parameters described above such as long range I3C- ‘H and I3C13Ccoupling constants can be back-calculated from the simulation for comparion with experimental values that are not used as restraints in the simulation. As seen in Table 1, the theoretical results for Neu5Acu2-3Gal~l-4Glcagree well with experimental values, suggesting that the dynamical properties defined by the simulations are a good approximation to the true solution dynamical behavior. 33.5.5 Short-range vs Long-range Restraints
Despite the good agreement between theoretical and experimental parameters in Table 1, a valid criticism of these studies (or indeed all NMR studies on macromolecular conformation until recently) is that all of the conformational parameters are short-range in nature. Thus by virtue of the inverse sixth power distance dependence of the NOE(ROE), distance restraints are typically limited to 0.5 nm or
964
33 Conformational Analysis in Solution by N M R
Table 1. Experimental ROE intensities and long-range coupling constants for I3C Neu5Aca23Galpl-4Glc vs theoretical values computed from a 5 ns MD simulation. ROE Connectivity
ROE Neu5Ac H-3ax-Gal H-3 Neu5Ac H-8-Gal H-3 Gal H-1-Glc H-4 Gal H-1-Glc H-6 Gal H-1-Glc H-6’ Gal H-1-Glc H-3 Gal H-1-Glc H-5
Jcc Gal C-3-NeuSAc C-3 Gal C-2-NeuSAc C-2 Gal C-4-Neu5Ac C-2 Gal C-l-GlcP C-5 Gal C-2-GlcaIP C-4 Gal C-l-Glca/P C-3
ROE Intensity
(%)l
Expt.
Theor.’
1 .o 0.4 4.2 0.4 0.5 0.4 0.5
1.5 0.6 3.9 0.6 0.1 0.4 0.6
Coupling (Hz) Expt.3
1.9 1.8
Theor.
1.9 2.6 <1
1.6 1.8 1.1 1.6 2.6 1.1
3.5 5.1 4.7
3.1 4.9 4.5
JHC
Gal H- 1-Glc C-4 Gal C- 1-Glc H-4 Neu5Ac C-2-Gal H-3
Experimental and theoretical intensities shown are for the Glcp anomer only, after correction for the mole fraction of this anomer. Calculated with a rotational correlation time of 0.13 ns. 3Error in these measurements estimated as k0.5 Hz.
less, and the long-range coupling constants provide conformational information on two bond vectors separated by a single covalent bond. It could be argued that such restraints satisfy the local order but do not define long-range order in the molecule. Thus, for extended structures of which polysaccharides are a prime example, the overall geometry of the molecule could be in error. This difficulty has recently been addressed by measurement of residual dipolar couplings as described above (Section 33.2.5). Typical such couplings, measured for NeuSAca2-3Gal~1-4Glcusing the J-modulated HSQC method [88], are given in table 2. These couplings can be applied as restraints in dynamical simulated annealing calculations, in order to obtain a family or families of structures which are consistent with the long-range order imposed by these restraints. The result for Neu5Aca2-3Gal~1-4Glcis two families of structures that differ in the conformation about the GalPl-4Glc linkage (Figure 9). Importantly, both of these structures have glycosidic torsion angles that map into low-energy regions of conformational space predicted from MD simulations involving ‘local’ restraints, therefore further suggesting that the latter provide an
33.5 A Cuse Study on N e u S A c d - 3 G u l ~ l - 4 G k
965
Table 2. Residual 'H-I3C Dipolar couplings for Neu5Aca2-3Galp1-4Glc in a 7.5% (w/v) solution of DHPC:DMPC (1 :2.9 w/w)in D20, pD 7.2, containing 100 mM KC1. Bond Vector
Residual dipolar coupling (Hz)'
GlcP H-1-C-1 Glcp H-2-C-2 Glcp H-3-C-3 GlcP H-4-C-4 GalP H-1-C-1 GalP H-2-C-2 GalP H-3-C-3 GalP H-4-C-4 GalP H-5-C-5 Neu5Acu H-4-C-4 Neu5Acu H-5-C-5 Neu5Acu H-7LC-7 Neu5Aca H-8-C-8
$7.4 +9.5 +9.6 +7.7 +11.5 $11.9 $9.2 -1.4 +9.5 f3.2 +4.8 -13.4 -11.1
' Values obtained by non-linear least-squares fitting of experimental intensities from J-modulated HSQC experiments. Estimated average error in the measurements is k 0 . 5 Hz.
NeuNAc
Gal
8 r
L
'-180
J 180
Phi (deg)
O ' L 2
k
-
'-180
l 180
Phi (deg)
Figure 9. (top) Family of structures derived from dynamical simulated annealing calculation on Neu5Aca2-3Ga1fi1-4Glc with residual dipolar coupling restraints given in Table 2. (bottom) Resulting values of and $ (open circles) for the NeuSAca2-3Gal and Galpl-4Glc glycosidic linkages indicated on the plots of Figure 8.
966
33 Conformational Analysis in Solution by N M R
adequate representation of the dynamic behavior of the glycan in solution. Clearly, further work is required to calculate time-averaged residual dipoar couplings from MD simulations for comparison with experimental values.
33.6 Conclusions Since early studies on oligosaccharides which treated these moieties as essentially ‘rigid’ bodies [89-931, a wealth of more recent data has indicated that oligosaccharides enjoy considerable motional freedom about the glycosidic linkages in solution. These conclusions are derived from improved computational procedures and better molecular mechanical forcefields, but the availability of additional experimental conformational restraints has also had a particularly significant impact. In particular the possibilities for the synthesis of oligosaccharides in 13C-enriched form using chemoenzymatic methods simplifies considerably not only the task of defining the solution behavior of the free glycan, but also the conformation while bound to a protein receptor. A description of techniques for the study of the latter can be found elsewhere in this volume.
References A. Varki, Glycobiology, 1993, 3, 97-130. E. Ioffe, P. Stanley, Proc. Natl Acad. Sci. U.S.A., 1994, 91, 728-732. P. Stanley, E. Ioffe, Faseb Journal, 1995, 9, 1436-1444. E. Ioffe, Y. Liu, P. Stanley, Proc. Nut1 Acad. Sci. U.S.A., 1996, 93, 11041-11046. P. Stanley, T. S. Raju, M. Bhaumik, Glycobiology, 1996, 6, 695-699. D. Chui, M. OhEda, Y. F. Liao, K. Panneerselvam, A. Lal, K. W. Marek, H. H. Freeze, K. W. Moremen, M. N. Fukuda, J.D. Marth, Cell, 1997, 90, 157-167. 7. J. J. Priatel, M. Sarkar, H. Schachter, J. D. Marth, Glycobiology, 1997, 7, 45-56. 8. M. Bhaumik, T. Harris, S. Sundaram, L. Johnson, J. Guttenplan, C. Rogler, P. Stanley, Cancer Rex, 1998, 58, 2881-2887. 9. T. S. Raju, P. Stanley, J. Bid. Chem., 1998, 273, 14090-14098. 10. M. A. J. Ferguson, Phil. Trans. R. Soc. Lond. Ser. B, 1997, 352, 1295-1302. 11. K. Aktories, Curr. Topics Microbiol. Immunol., 1992, 175, 1-143. 12. J. F. G. Vliegenthart, L. Dorland, H. van Halbeek, Adv. Carbohydr. Chem. Biochem., 1983, 41, 209-374. 13. S. W. Homans, Progr. N M R Spectr., 1990,22, 55-81. 14. H. van Halbeek, Meths. Enzymol., 1994,230, 132-168. 15. H. van Halbeek. Curr. Ouinion Str. Biol.. 1994. 4. 697-709. 16. J. F. G. Vliegenthart, L. Dorland, H. van Halbeek, Adv. Carbohydr. Cliem. Biochem., 1983, 41, 209-374. 17. P. E. Jansson, L. Kenne, G. Widmalm, Carbohydr. Res., 1987, 168, 67-77. 18. G. M. Lipkind, A. S. Shashkov, Y. A. Knirel, E. V. Vinogradov, N . K. Kochetkov, Carbohydr. Res., 1988, 175, 59-75. 19. A. S. Shashkov’, G. M. Lipkind, Y. A. Knirel, N. K. Kochetkov, Mag. Res. Chem., 1988, 26, 735-147. 20. P. E. Jansson, L. Kenne, G. Widmalm, Pure Appl. Chem., 1989, 61, 1181-1192. 1. 2. 3. 4. 5. 6.
References
967
P. E. Jansson, L. Kenne, G. Widmalm, Curhohydr. Rex, 1989, 193, 322-325. P. E. Jansson, L. Kenne, G. Widmalm, CLzrhohydr. Res., 1989, 188, 169-191. G. M. Lipkind, A. S. Shashkov, N. K. Kochetkov, Curhohydr. Res., 1990, 198, 399-402. H. Baumann, P. E. Jansson, L. Kenne, G. Widmalm, Carhohydr. Res., 1991, 211, 183-190. P. E. Jansson. L. Kenne, G. Widmalm, A n d . Bichenz., 1991, 199, 1 1 17. P. E. Jansson, L. Kenne, G. Widmalm, Actu Chem. Scandin., 1991, 45, 517-522. N. K. Kochetkov, E. V. Vinogradov, Y. A. Knirel, A. S. Shashkov, G. M. Lipkind, Bioorg. Khim., 1992,18, 116-125. 28. N. E. Nifantev, A. S. Shashkov, G. M. Lipkind, N. K . Kochetkov, B. Jann, K. Jann, Glycoconj. J.; 1993, 10, 335-335. 29. M. Karplus, J. Am. Chem. Soc., 1963, 85, 2870-2871. 30. D. Neuhaus, M. P. Williamson, The Nwlear Overliuuser EfSect in Structurul und Conformationul Analysis, Verlag Chemie, New York 1989. 31. T. J. Rutherford, J. Partridge, C. T. Weller. S. W. Homans, Biochemistry, 1993, 32, 1271512724. 32. C. T. Weller, J. Lustbader, K. Seshadri. J. M. Brown, C. A. Chadwick, C. E. Kolthoff, S. Ramnarain, S. Pollak, R. Canfield, S. W. Homans, Biochemistry, 1996, 35, 8815-8823. 33. Y. Ichikawa, Y.-C. Lin, D. P. Dumas, G.-J. Shen, E. Garcia-Junceda, M. A. Williams, R. Bayer, C. Ketcham, L. E. Walker, J. C. Paulson, C.-H. Wong, J. Am. Chem. Soc., 1992, 114, 9283-9298. 34. M. A. Probert, M. J. Milton, R. Harris, S. Schenkman, J. M. Brown, S. W. Homans, R. A . Field, Tetruhed. Lett., 1997, 38, 5861-5864. 35. A. K. Misra, J. M. Brown, S. W. Homans, R. A. Field, Curhohydr. Lett., 1998, 3, 217-222. 36. H. Shimizu, J. M. Brown, S. W. Homans, R. A. Field, Tetrahedron, 1998, 54, 9489-9506. 37. T. L. Hwang, A. J. Shaka, J. Magn. Reson., 1995, A 112, 275-279. 38. L. Poppe, H. vanHalbeek, J. Am. Chem. Soc., 1991, 113, 363-365. 39. L. Poppe, H. van Halbeek, J. Mugn. Reson., 1992, 96, 185-190. 40. L. Poppe, H. van Halbeek, Nuture Struct. B i d , 1994, 1, 215-216. 41. L. Poppe, R . Stuikeprill, B. Meyer, H. van Halbeek, J. Biomol. N M R , 1992,2, 109-136. 42. J. Reuben, J. Am. C’hem. Soc., 1984, 106, 6180-6186. 43. J. C. Christofides, D. B. Davies, J. A. Martin, E. B. Rathbone, J. Am. Chem. Soc., 1986, 108, 5738-5743. 44. E. Alvarado, T. Nukuda, T. Ogawa, C. E. Ballou, Biochemistry, 1991, 30, 881--886. 45. R. Harris, T. J. Rutherford, M. J. Milton, S. W. Homans, J. Biomol. N M R , 1997, 9, 47-54. 46. P. L. Rinaldi, J. Am. Clzem. Suc., 1983, 105, 5167-5168. 47. C. Yu, G. C. Levy, J. Am. Chem. SOC.,1984,106, 6533-6537. 48. K. E. Kover, G. Batta, J. M a p . Reson., 1988, 79, 206-210. 49. D. Neuhaus, C. P. M. Vanmierlo, J. Mugn. Reson., 1992, 100, 221-228. 50. K. Stott, J. Keeler, Mug. Res. Chem., 1996. 34, 554-558. 51. G. R. Kiddle, R. Harris, S. W. Homans, J. Biomol. N M R , 1998, 11, 289-294. 52. J. M. Duker, A. S. Serianni, Carbohydr. Rex, 1993, 24Y, 281-303. 53. T. Church, I. Carmichael, A. S. Serianni, Curbohydr. Res., 1996, 280, 177-186. 54. A. S. Serianni, Abstr. Am. Chem. Soc., 1996, 211, 10-CARB. 55. T. J. Church, I. Carmichael, A. S. Serianni, J. Am. Chem. Soc., 1997, 119, 8946-8964. 56. M. J. Milton, R. Harris, M. Probert, R. A. Field, S. W. Homans, Glycuhiology, 1998, 8, 147153. 57. Q. W. Xu, C. A. Bush, Carhohydr. R e x , 1998, 306, 335-339. 5 8 . S. K. Zhao, G. Bondo, J. Zajicek, A. S. Serianni, Curhohydr. Res., 1998, 309, 145-152. 59. A. Bax, D. Max, D. Zax, J. Am. Chen?. Soc., 1992, 114, 6923-6925. 60. B. Bose, S. Zhao, R. Sterutz, F. Clovar, P. €3. Bordo, G. Bordo, B. Hertz, I. Carmichael, A. S. Seriarri J. Am. Chem. Soc., 1998, 120, 1 1 158-1 1173. 61. J. R. Tolman, J. M. Flanagan, M. A. Kennedy, J. H. Prestegard, Proc. Natl. Acud. Sci., 1995, 92, 9279-9283. 62. N. Tjandra, S. Grzesiek, A. Bax, J. Am. Chern. Soc., 1996, 118, 6264-6272. 63. N. Tjandra, A. Bax, J. M a p . Reson., 1997, 124, 512-515. 64. N. Tjandra, A. Bax, Science, 1997,278. 1 1 11-1 114.
21. 22. 23. 24. 25. 26. 27.
-
968 65. 66. 67. 68. 69. 70. 71.
33 Conformational Analysis in Solution by N M R
C. R. Sanders 11, J. P. Schwonek, Biochemistry, 1992, 31, 8898-8905. M. Ottiger, F. Delaglio, A. Bax, J. Mugn. Reson., 1998, 131, 373-378. 0. Jardetzky, Bioch. Biophys. Actu, 1980, 621, 227-232. D. A. Cumming, J. P. Carver, Biochemistry, 1987, 26, 6664-6676. M. Nilges, A. M. Gronenborn, A. Briinger, G. M. Clore, Protein Eng, 1988, 2, 27-38. A. E. Torda, R. M. Scheek, W. F. van Gunsteren, J. Mol. Biol., 1990,214, 223-235. D. G. Low, M. A. Probert, G. Embleton, K. Seshadri, R. A. Field, S. W. Homans, J. Windust, P. J. Davis, Glycobiology, 1997, 7, 373-381. 72. R. Harris, G. R. Kiddle, R. A. Field, B. Ernst, S. W. Homans, J. Am. Chem. SOC.,1999, 121, 2546-2551. 73. M. Vignon, F. Michon, J. P. Joseleau, K. Bock, Macromolecules, 1983, 16, 835-838. 74. Q. W. Xu, C. A. Bush, Biochemistry, 1996,35, 14512-14520. 75. G. Lipari, A. Szabo, J. Am. Chem. SOC.,1982, 104,4546-4559. 76. N. Metropolis, A. W. Rosenbluth, M. N. Rosenbluth, A. H. Teller, E. Teller, J. Chem. Phys., 1953,21, 1087-1092. 77. R. Stuike-Prill, B. Meyer, Eur. J. Biochem., 1990, 194, 903-919. 78. T. Peters, T. Weimar, J. Biomol. NMR, 1994, 4, 97-116. 79. H. J. C. Berendsen, J. P. M. Postma, N. F. van Gunsteren, A. DiNola, J. R. Haak, J. Chem. Phys., 1984,81, 3684-3690. 80. L. Lerner, A. Bax, Curbohydv. Res., 1987, 166, 35-46. 81. A. Bax, G. M. Clore, P. C. Driscoll, A. M. Gronenborn, M. Ikura, L. E. Kay, J. Muyn. Reson., 1990, 87, 620-627. 82. L. Yu, R. Goldman, P. Sullivan, G. F. Walker, S. W. Fesik, J. Biomol. N M R , 1993, 3, 429441. 83. G. M. Lipkind, A. S. Shashkov, N. K. Kochetkov, Curbohydr. Res., 1985, 141, 191-197. 84. L. Poppe, C.-W. von der Lieth, J. Dabrowski, J. Am. Chem. SOC.,1990, 112, 7762-7771. 85. K. Bock, J. 0. Duus, S. Refn, Curbohydr. Res., 1994, 253, 51-67. 86. J. Dabrowski, T. Kozar, H. Grosskurth, N. E. Nifantev, J. Am. Chem. SOC.,1995, 117, 55345539. 87. S. W. Homans, M. Forster, Glycobiology, 1992,2, 143-151. 88. G. R. Kiddle, S. W. Homans, FEBS Lett., 1998, 436, 128-130. 89. K. Bock, J. Arnarp, J. Lonngren, Eur. J. Biochem., 1982, 129, 171-178. 90. S. W. Homans, R. A. Dwek, D. L. Fernandes, T. W. Rademacher, FEBS Lett., 1982, 150, 503-506. 91. J . R. Brisson, J. P. Carver, Biochemistry, 1983, 22, 3680-3686. 92. J. R . Brisson, J. P. Carver, Biochemistry, 1983, 22, 3671-3680. 93. S. W. Homans, R. A. Dwek, D. L. Fernandes, T. W. Rademacher, FEBS Lett., 1983, 164, 231-235.
Carbohydrates in Chemistry and Biology Editor Beat Ernst, Gerald W. Hart, Pierre Sinay copyright OWILEY-VCH Verlag GmbH, 2000
34 Oligosaccharide Conformations by Diffraction Methods Serge Pkrez, Cutherine Gautier, and Anne Imherty
34.1 Introduction
Diffraction by single crystals is by far the most powerful experimental method for the characterization of the atomic arrangements in molecules. X-ray and neutron, are used since their wavelengths are of the same order of magnitude as the interatomic distances, typically around an Angstrom unit. The electron and nuclei are the scatterers of the X ray and neutron incident radiation, respectively. The single crystals are usually grown by slow evaporation of saturated solution under controlled environments. Ideally their dimensions should be in the order of 0.2 to 0.5 mm and over 1.0 mm, in the respective case of studies from X-ray and neutron diffraction. Irradiation of the crystal by a monochromatic beam leads to constructive interferences, known as diffraction, of the scattered waves in specific directions. The diffraction patterns, that are recorded on electronic devices, consist of a series of Bragg reflections. Their positions and intensities, respectively, contain the details on the unit cell dimensions, space group symmetry, and atomic positions. Unit cells are the building block of the crystals. In order to determine the crystal structure, both the amplitude and phase of the diffracted wave in every Bragg reflection are required. While the measured intensities are proportional to the square of the amplitude, the phase of the reflection relative to that of the incident beam is unknown as it cannot be recorded experimentally. There are several ways to calculate the phases of some important reflections, among them direct methods which are now used routinely to solve crystal structures. Using Fourier transform, the amplitude and phase information is used to synthesized electron density maps that reveal the atomic content of the unit cell. Least square refinement methods are then used to minimize the discrepancy between the measured and calculated structure amplitudes. From a knowledge of the final atomic coordinates, the molecular shape is revealed and such structural features as bond lengths, bond angles, torsion angles, hydrogen bonds, intermolecular distances are directly computed.
970
34 Oligosaccharide Conformations by Diffruction Methods
Carbohydrates were among the first organic compounds to be investigated by X-ray crystal structure analysis: pentaerythritol [ 11 and a-D-glucosamine hydrochloride and hydrobromide [2]. The work developed slowly as only eight crystal structures were described over the next fifteen years; among them to elucidation of sucrose.NaBr.2H20, by Beevers and Cochran in 1947 [3]. The number of X-ray diffraction studies of carbohydrate increased slowly thereafter and the crystal structures of sucrose and a-D-glucose were analyzed by neutron diffraction in the sixties by Brown and Levy [4, 51. Very recently, the crystal structure elucidation of the cellodextrins [6-8] were the first example of the use of synchrotron radiation, and exemplifies how single crystals having minute dimensions can be successfully investigated.
34.2 General Analysis The Cambridge Structural Data Base (CSDB) contains over 160,000 entries in the form of structural data related to geometry, configuration, conformation, and packing of molecular crystals for organic and metal-organic compounds [9]. As a consequence of the automation of experimental measurements along with the development of crystal structure analysis for non-centrosymmetric space groups, there has been a significant increase in the number of reported crystal structures of carbohydrates between the years 1980-1 995. The number of entries dealing with carbohydrate crystal structures in the CSDB reaches almost 4000. About 1000 among these entries do not list any atomic coordinates and cannot be considered as structurally informative, in terms of conformations and configurations. Despite the fact that it is possible to determine the absolute configuration of an optically active molecule from the effects of anomalous scattering, such a level of structural characterization is not performed routinely. Determination of absolute configuration generally involves scattering from relatively heavy atoms. It can nevertheless be determined from molecules containing a significant fraction of oxygen atoms providing that efforts are given to obtain accurate diffracted intensities. Among the 4000 structural investigations of carbohydrates, only 150 dealt with the determination of the absolute configuration. Caution should be taken to the few crystal structures that have been published with the wrong enantiomer. Most of the recently reported crystal structures use the a priori knowledge of the configuration of the constituting monosaccharide units. Crystalline data can be readily found for many members within the classification of the carbohydrates that encompasses (i) the aldose and kestoses which are the monomers, (ii) the alditol, or sugar alcohol which differ from the aldoses in having members of the series being in meso-configurations, (iii) the cyclitols which make up a family of nine compounds, the configurations of which differ by the axial or equatorial disposition of the hydroxyl groups around the chair-shaped cyclohexane ring, (iv) the anhydro sugars which are formed by the elimination of water between hydroxyls of the pyranoses and furanoses thereby forming fused bicylcic or tricyclic
34.2 Generul Analysis
971
ring structures, (v) the carbohydrate acids which are related to the alditols and aldoses, (vi) the oligosaccharides. The disaccharides result from the condensation of a reducing group [C-1 OH in aldose, C-2 OH in ketose] with another hydroxyl. The family of cyclodextrins is characterized by excellent crystals and therefore many structures analyses have been reported. Typical examples of these compounds are cyclic 1-4 linked a-D-glucopyranosides with 6 , 7 and 8 monosaccharides residues. Being shaped like truncated cones, with the inside voids occupied by solvent or guest molecules, these cyclic oligosaccharides are amenable to crystallization, and exhibit only three types of packing modes. A very large number of crystals structures of the cyclodextrins and their complexes have been reported, including some neutron diffraction analysis. Larger cyclodextrins, namely with 10 glucose and 14 glucoses in the ring, have also been crystallized and display distorted ring shape. Very recently, crystal structure of large cycloamylose compound, consisting of 26 glucose residues, have been solved [lo]. The large ring is folded into a structure similar to the one of V-amylose. This cyclodextrin field has been extensively reviewed several times [ 111 and it will now be covered in the present article. There are 55 crystal structures of unsubsituted disaccharides and a dozen of crystal structures of the fully (or almost fully) acetylated disaccharides. When looking at larger compounds, there are 17 trisaccharide crystals structures and only 4 tetrasaccharides (Table 1). One linear oligomaltose has been crystallized and analyzed as a poly-iodide complex, that of p-nitrophenyl-a-D-maltohexopyranoside [ 121. The malto-hexaose molecules form a double helical anti-parallel stranded structure which enclosed the poly-iodide chain in away similar to that found in cyclodextrin inclusion compounds. Both the conformation and the relative orientation of the single stranded chains are completely different from those found in the crystalline arrangement of a small fragment of amylose, the structure of which has been determined from the combined used of fiber X-ray diffraction and electron single crystal diffraction [13]. Crystal structure have been solved for most of the pentoses and hexoses, in one or more of the isomeric forms or as a 1-0-methyl derivatives. A significant amount of crystal structures of carbohydrates have been determined from neutron diffraction experiments. From these highly accurate structural determination, standard molecular dimensions of the constituting units have been established (Table 2). These data are an update of the ones published previously by Jeffrey & Taylor [ 141 and, in a similar way, they can serve as a basis for parameterizations of molecular mechanics force fields. There is an obvious reluctance of carbohydrates to crystallize in a form suitable for X-ray or neutron diffraction studies. This is particularly true for aldose and ketose containing carbohydrates as a configurational mixture of four isomers, aand P-pyranoses and a- and p-furanose, is likely to occur. Configurational heterogeneity in solution tends to inhibit crystallization. For this reason 1-0-methyl derivatives, which cannot epimerize, are likely to crystallize more readily. In some instances, the a and epimers can co-crystallize, and more than 10 examples of such co-crystallization can be found. The a and P ratio may be dependent on the temperature and solvent of crystallization, and may not be reproducible between independent investigators. One extreme example is provide by the crystal struc-
912
34 Oligosuccharide Conformations by Diffruction Methods
Table 1. Crystal structures of trisaccharides and tetrasaccharides. Trisaccharide Name
Common name
Code
aFuc( I-2)PGal( I-3)GlcNAc aGlcA-4-O-Me( 1-2)PXyl( 1-4)PXyl PGal( 1-4)[ aFuc( 1-3)] PGlcN Ac- 0-Me Manu(l-3)ManP( 1-4)GlcNAc uGlc(l-4)aGlc(l-2)PFru
Blood group B Aldotriuronic acid Lewix X Fragment N-glycan Erlose (mono et trihydrate)
aGlc(1-4)aGlc( 1-4)OGlc-0-Me aGlc(1-6)aGlc( 1-4)a/fiGlc aGal( 1-6)aGlc( l-2)PFmf fiMan(l-4)PMan(l-4)aMan PGlc(1-4)PGlc( I-4)PGlc-0-Me aGlc( 1-2)PFruf( 1-2)PFruf aGlc( 1-2)PFruf( I-6)PFruf aGlc( 1-3)PFruf(2-l)aGlc
Ma1totrioside Panose Raffinose Mannotnose Cellotrioside I-Kestose 6-Kestose Melezitose (2 forms) Planteose Cellotriose
nd GURXPX 10 nd MPYAGL HAHXUJ HEGXOG DUDXOP KOYZAZ RAFINOOI COFMEPIO TAQYAL KESTOS CELGIJ MELEZTO1 MELEZT02 PLANTEIO ACCELLlO
Common name
Code
Ref
STACHYOI PEKHESOI WIMNOV
(481 [49]
ZILTUJ
PI
_ _ _ _ _ _ _ ~ ~ ~
Ref.
~
aGal( 1-6)PFruf(2- 1)aGlc PGIc( 1-4)PGlc( 1-4)PGlc peracetylated Tetrasaccharides Name aGal( 1-6)aGal( I-6)ctGlc( I-2)PFruf aGlc(1-2)fiFruf( l-2)PFruf(l-2)PFruf
Stachyose Nystose Schyzophyllan fragment PGIC(~-~)[PG~C(~-~)]PGIC(~-~)PG~C peracetylated PGlc(I-4)PGlc( 1-4)PGlc( 1-4)PGlc Cellotetraose
[5OJ
171
ture of the disaccharide lactulose, as the crystals contains an mixture of isomers in which the fructose moiety is p-D-fructofuranose, a-D-fructofuranose and p-Dfructopyranose in the ratio 74.5/10/15.5 [ 151. The reluctance of carbohydrate to crystallize in a form suitable for X-ray diffraction studies, is more pronounced for compounds having molecular weights ranging from 1000 to 5000. This is true but for the exception of cyclic compounds such as cyclodextrins and cycloamyloses. One of the reasons may be the lack of sufficient amount of material available for crystal growth. The other reason is that the techniques of growing organic crystals of medium size bio-molecules has not parallel the revolution of growing protein and viruses crystals. Since the beginning of the 90s an increasing number of crystal structures have been reported for glycoproteins and protein-carbohydrate complexes. The resolu-
34.3 Crystalline Conjormations of Disaccharide Moieties
973
Table 2. Standard molecular dimensions for pyranosides. Distances are given in A and angles in '.
'
U-D-4C1
p-D-4c~
u-L- c4
p-L- I cq
379 1.52(2) 1.520(17) 1.520(18) 1.526(17) 1.412(17) 1.413(17) 1.440(16) 113.9(14) 11 l.3(11) 116(3) (3x4) 108 (7) 185 fvug 72( 10) 194 fvug
184 1.5 17(19) 1.522( 15) 1.521(16) I.S26(16) l.394( 17) 1.422( IS) 1.433(14) 111.9(13) 107.4(12) I I5(2) 177(3) -80(11)
35 1.519(16) 1.5 16( 17) 1.514(19) 1.523(16) 1.408(15) 1.413( 13) 1.440(13) 113.8(16) 112.0(13) 115(3) -62(4) - 104(8) 6 fvug -73(12) 2 9 f r ~ g
12 1 .5 1S( 12) 1 .5 1 8(9) 1.529(9) 1.521( 14) l.389( 11) 1.428(9) 1.437(7) 11 1.2(11) 107.7(6) 115(2) 177(2) 79P)
tion of the first reported structures was rarely sufficient to provide reliable conformational information. Significant and rapid progresses arising from the use of synchrotron radiation are providing, now, access to highly resolved structures.
34.3 Crystalline Conformations of Disaccharide Moieties 34.3.1 The Disaccharides
Important in their own right, disaccharides take great importance as the shortest components of the family of oligo, polysaccharides and complex carbohydrates. As such a particular attention is given to their conformational properties because their molecular shapes are considered to be important determinants of their properties and those of their larger parents. The disaccharides result from the condensation of a reducing group [C-1 OH in aldose, C-2 OH in ketose] with another hydroxyl. For hexose in the pyranose shape, this linkage may be 1+n where n is 1 to 6, except 5. If two reducing groups are involved, the disaccharide is non-reducing as in sucroses and trehaloses. For disaccharide moieties, the main conformational determinants are: the ring shapes, the orientations of the hydroxyl groups, and the relative orientations of the monosaccharide units at the glycosidic linkage. Ring shapes can be defined in terms of reference conformations (chair, C; twist T; boat, B; envelope, E; skew, S) or by the so-called puckering parameters [ 161. The exocyclic primary alcohol groups can adopt a number of low-energy conformations. They are usually in staggered ar-
974
34 Oligosaccharide Conformations by Difraction Methods
axial-axial 1-1
1-2
1-1
+
1-2
axial-equatorial 1-3
1-4
1-6
1-4
=4
=4
h
0
axial-hanose
equatorial-equatorial 1-2
1-1
qoq0+ 1-3
w 0 J a o
1-2
1-6
equatorial-furanose
hanose-furanose
1-4
2-1 and 2-6
Figure 1. Schematic representation of the different type of linkages observed in crystal structures of disaccharides and oligosaccharides. Sugars with 'Cd ring shape such as L-fucose have not been represented.
34.3 Crystalline Confbrmations of Disaccharide Moieties
975
rangements that correspond to local minima. In the case of pyranoses, primary hydroxyl groups most frequently occupy two positions, avoiding interactions between 0 - 4 and 0-6. However, each of the secondary hydroxyl groups can rotate almost freely. The relative orientation of two consecutive monosaccharide units in a disaccharide is customarily described by the torsion angles CD and Y around the glycosidic bonds. @ represents the torsion angle about the C(anomeric)-0 bond, whereas Y represents the torsion angle about the 0-Cx bond. The sign of the torsion angles is given in accordance with the IUPAC-IUB Joint Commission of Biochemical Nomenclature [ 171. The consideration of the axial/equatorial nature at the glycosidic linkage provides a useful framework for a classification of the disaccharide moieties, independently of the remaining and of the surrounding of the oligosaccharidic molecule. Using such a classification, all the unsubstituted components of the linear oligosaccharide structures have been reported in Table 3 . For each class, the nature of the glycosidic linkage: l+n is indicated, along with the REFCODE of the corresponding oligosaccharidic structure, its trivial/usual name, description of the disaccharide, the magnitude of the angles [(o)), Y ,z] at the glycosidic linkage, and the occurrence (if any) of inter-residual hydrogen bonds. As for six membered ring containing disaccharides, axial-axial, axial-equatorial and equatorial-equatorial are found. The families involving axial-furanose, and equatorial-furanose linkage to a hexopyranose are also indicated, along with the furanose-furanose cases. Proper references to the original (or most recent) crystal structure work is also provided. In order to have a comprehensive vision of the spatial occurrence of all these conformations as a function of their belonging to a given class, a schematic representation of their crystallographic conformations at the glycosidic linkage, has been set with a superimposition onto the low energy contours that have been computed for a prototypical motif using molecular mechanics calculations (Figure 2). Those contours correspond to conformation of the prototypical disaccharide having energy values of 2 kcal/mol and 6 kcal/mol with respect to the lowest energy minimum. As clearly indicated in Fig. 2a, b, and c, the chosen classification and representations appear to be quite relevant to describe the ensemble of solid state conformations found in crystalline oligosaccharides and their analogs. It is worth noticing that there is no representative of the equatorial-axial class, even though such type of glycosidic linkage can be found in several carbohydrate containing molecules and macromolecules. Whereas all the crystalline conformations lie within the 5 kcal/mol energy contour, it is noteworthy to observe a limited dispersion about the glycosidic 0 angles compared to that observed about Y , as an expression of the influence of the exo-anomeric effect on the establishment of preferred conformation in crystalline oligosaccharides. The exo-anomeric effect arises due to the particular bonding sequence C-5-0-5C- 1-0-g-C-x' in glycopyranosides and disaccharides and influences the conformation about the glycosidic torsion angle @ [ 181. In the case of axial type of linkage (typically as in 4 C D-a ~ configuration) only one staggered conformation is preferred with @ being in the vicinity of 60". As for the equatorial type of linkage (typically as in 4C1 D-p configuration); two staggered conformations are preferred (CD = 60" and -60") of which that corresponding to @ = -60" is favored due further stabilization occurring from non-bonded interactions. The values of @ generally varies between
a,a-Allo-trehalose, CaC12, 5 H 2 0 3,3’ deoxy-Arabino-trehalose. H 2 0 a,a-Trehalose, 2 H 2 0 a,a-Trehalose a,a-Trehalose, 1/2(CaBr2) H 2 0 a,a-Galacto-trehalose a,a-tetrachloro-Galacto-trehalose
[52] (531 [54] [55] [56] [ 571
ALTRCA LETTEJ TREHALlO DEKYEX TRECAB YOXFUM YODSOZ
[58]
a,a-Allo-trehalose, 6H20
[51]
YOXFOG
Molecule
1-1
Ref
Code
=4
1-4
1-2
Atom
1-1
axial-axial
a/Axial-axial linkage between two pyranose rings
aAllo(1- 1)aAllo aAra(I-1)aAra aGlc( 1- 1)aGlc aGlc( 1-1)aGlc aGlc( 1-1)aGlc aGal( 1-1)aGal aGal(1-1)aGal
aAllo(1-1)aAllo
Disaccharide
110.4 110.2 115.3 114.4 115.7 113.3 113.2 116.2 114.8
z
74.7 73.1 45.1 64.5 75.1 60.8 77.0 66.3 77.3
(D
Table 3. Details of the conformations at the glycosidic linkages for disaccharides and oligosaccharides in crystalline state.
75.5 74.8 57.1 75.6 61.6 60.1 77.0 62.4 77.3
Y
H-bond
%
0
$ s
6’ s
2
2
b
4-
m
-4
W
LEUCROOI
CITSTHlO
1-4
[61]
[60]
[59]
Galabiose
P-Leucrose, 2H20
Man, a (1-2) Mana-0-Me
a,P-Trehalose, H 2 0
Blood group B trisacch. H type 1, 1/2 H 2 0
[25]
[33] [62]
[63] Mana (1-2) PGlc [34] Aldotriuronic acid trisacch., 3H20
TIQDUS
nd TIYYOP
RESMOR GURXPXlO
1-1
1-2
Molecule
1-6
Ref
1-3
axial-equatorial
Code
1-4
1-2
Atom
1-1
blAxia1-equatorial linkage between two pyranose rings
FABYOW10
1-2
1- 5
aFuc(1-2)PGal aFuc( 1-2)SGal aFuc(1-2)PGal aMan( 1-2)PGlc aGlcA-4-O-Me( 1- 2)PXyl
aGlc(l-1)PGlc
Disaccharide
115.8 116.7 116.6 118.0 116.0
113.7
z
117.5
aGal(1-4)aGal
114.0 115.4
aMan(1 -2)aMan aGlc(1-5)PFrup
-66.0 -92.7 -92.6 59.0 78.8
68.8
-91.3 -174.8 -174.8 -148.6 -81.9
-93.4
Y
157.7
98.1
0
136.1 -94.0
64.2 68.8
0 - 6 . . .O-6
H-bond
HO-3.. .O-5’
0-2g. . .HO-2f
n
1-6
1-4
1-3
aGlc( 1-4)Glc aGal(lL6)Glc aGlc( 1-6)aGlc
alP-Panose
a/P-Melibiose, H 2 0 a/P-Panose
KOYZAZ
76.4 70.7
aGlc( 1-4)aGlc aClc(I-4)PGlc aGlc( 1 -4)uGlc
6‘-iodo phenyl a-Maltose Methyl 0-Maltoside Methyl P-Maltrotrioside. 3 H 2 0
IPMALT MMALTS DUDXOP
111.7 112.0
aGlc( 1-4)aGlc aGlc( 1-4) PGlc aGlc( 1-4)aGlc
a-Ma1tose P-Maltose, 1 H 2 0 Phenyl a-Maltoside
MALTOT MALTOSll PHMALT
,,
-63.4 74.7
aGlc( 1-4)aGlc
p-nitrophenyl-Maltohexaoside, BaI2 27H20
FOXSUG20-b
MELIBMlO KOYZAZ
aGlc( 1-4)aGlc aGlc( 1-4)aGlc
Erlose, H 2 0 Erlose 3 H 2 0
HAHXUJ HEGXOG
0
107.2 68.7 51.1 112.2 95.7 93.2 118.2 103.1 45.4 94.0 104.7 79.9 106.5 116.1 121.7 108.5 110.0 72.5 109.9 82.3 82.7 96.8
117.0 115.0 116.7 113.3 119.2 119.6 114.1 113.3 119.2 125.5 112.1 112.3 115.7 120.1 117.9 116.9 116.5 112.7 117.6 114.7 115.5 113.9
aMan( 1-3)PMan wGlc(1-3)aGlc aGlc( I-3)bFrup
Mana( 1-3)ManP( 1-4)GlcNAc Methyl a-Nigeroside P-Turanose
MPYAGL MOGLPR TURANS
,
56.2 -72.5 -76.7 60.5 99.9 98.3
116.4 115.1 115.1 114.1 116.1 116.3
aGal(1-3)PGal uFuc( 1-3)GlcNAc
Blood group B trisacch. Lewis X trisacch., 9 H 2 0
nd nd
Table 3 (continued
167.3
- 174.2
-163.5 -120.8 -117.3 -126.1 -142.6 -128.4 -164.8 -129.5 -133.7 -151.8 - 129.6 -118.0 -107.7 -139.4 -138.9 -155.0 -109.1 -148.9 -151.8 -134.8
0 - 3 . . .HO-2’
0 - 3 . . .O-2’ 0 - 3 . . .O-2’ 0 - 3 . . .O-2’ 0 - 3 . . .O-2’ 0 - 3 . . .O-2’ 0 - 3 . . .O-2’ 0 - 3 . . .O-2’ 0 - 3 . . .O-2’ 0-3.. .O-2’ 0 - 3 . . .O-2’ HO-3.. .O-2’ HO-3.. .O-2’ 0 - 3 . . .HO-2’ 0 - 3 . . .HO-2’
HO-3.. .O-2’
- 129.9 - 150.0
HO-4.. .O-2’ HO-4.. .O-2’
60.0 139.2 139.0 97.0 104.2 111.6
YEPNUC
1711
[40] [48]
KDO disacchac., 2Na+ H 2 0
Raffinose, 5 H 2 0 Stachyose, 4 H 2 0
WACHOX
SOPROS
LAMB10 WAGBOV VIZFUF CHONDM
1-2
1-3
Code
[74] [75] [76] [77]
[73]
[72]
Ref
equatorial-equatorial
1-1
1-2
1-1
P-Laminarabiose, H 2 0 Methyl P-Laminarabioside, H 2 0 Neocarrabiose beta monohydrate Chondrosine monohydrate
a-Sophorose, H 2 0
P,P-Trehalose, 2 H 2 0
Molecule
c/Equatorial-equatorial linkage between two pyranose rings
*Two other torsion angles at the linkage: 56.3 63.3
2-8
RAFINOOI STACHYOI
-93.6 -85.7 94.5 -87.8
118.2 117.5 116.5 116.5 PGlc( I-3)PGlc PGlc( 1-3)PGlc a-L-3,6AnGal( 1-3)PGlc PGlcA( 1-3)aGalNAc
-78.9
113.7
PGlc( I -2)aGlc
-75.6
CD
71.8 85.2 64.9 58.1
115.2
z
112.0 113.1 111.1 115.50
PGlc(l-l)PGlc
Disaccharide
aGal(1-6)ctGlc -63.1 aGal( 1 -6)aGal 87.1 nGal( 1-6)aGlc -62.1 aKD0(2-8)aKDO*
77.7 76.0 141.9 55.4
-139.8
-75.5
Y
-170.8 -172.4 -175.1 131.9
HO-4.. .05’ HO-4.. . 0 5 ’ HO-2. . .O-5‘ HO-4.. .05’
H-bond
HO-7.. .OIA’
\D
\D 4
2
s:
2. R
1-4
PGlc(l-4)PGlc
P-Cellobiose P-Cellobiosyl-nitromethane Methyl P-Cellobioside, methanol p-Cellotetraose, 1/2 H20
Methyl P-Cellotrioside, EtOH,H20
a-Chitobiose P-Chitobiose, 3H20
CELLOB02 WEHTEI MCELOB ZILTUJ
TAQYAL
ACHITM10 BCHITTIO
[87] [88]
PGal(1-4)aGlcNAc PGa1(1-4)aGlc PGal(1-4)aGlc pGal( 1-4)PGlc PGaI(1-4) PGlc pMan( I-4)aMan PMan(1-4)PMan PMan(1-4)aMan PGlc(1-4)PGlc PGk( 1-4)PGlcPGlc(1-4) PGlc-0-Me PGlc(I-4)PGlc
a-N-acetyllactosamine, H 2 0 a-Lactose, CaC12 7H20 a-Lactose, 1H20 P-Lactose P-Lactosylurea, 2H20 a-Mannobiose a-Mannotriose, 3H20
ACLACT LACCCB LACTOS03 BLACTO REMVUA DIHTUJ COFMEPlO
PGlcNAc(1-4)aGlcNAc PGlcNAc(1-4)PGlcNAc
,f
PXYV 1-4)PXYI PMan(1-4)aGlcNAc pGal( 1-4)GlcNAc
Aldotriuronic acid trisacch., 3H20 Mana(l-3)ManP(l-4)GlcNAc Lewis X trisac., 9H20
GURXPXIO MAYAGL
Table 3 (continued)
-
113.8 114.6 117.0 117.7 116.3 115.9 117.2 116.5 117.2 115.0 117.4 115.6 116.1 116.5 115.8 116.7 115.9 116.2 116.3 117.5 116.3 117.3 117.2 116.6 116.8 117.6 117.3 117.2 117.6 116.3 117.1
-145.5 -75.7 -80.0 -70.5 -88.1 -76.9 -93.5 -70.9 -88.1 -96.0 -71.9 -93.9 -76.3 -93.0 -91.1 -95.3 -94.8 -93.2 -98.3 -93.2 -91.9 -93.7 -96.7 -96.8 -93.4 -96.3 -91.5 -91.7 -95.5 -76.9 -90.3 -
-79.6 -130.7 -104.6 -107.7 -139.5 -136.7 -143.5 -131.5 - 159.4 -148.1 -131.6 - 150.4 - 132.3 -139.8 -160.7 -143.9 -142.9 -141.6 150.8 - 149.0 - 152.4 -141.5 -141.2 -141.9 -141.5 - 150.6 -151.8 - 152.0 - 150.6 -106.9 -162.3
HO-3.. .O-5’
HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’ HO-3.. .O-5’
HO-3.. .05’
GENTBS
[90]
[89]
P-Gentiobiose
Gal P(1-4) Man, 2H20
axial-furanose
1-2
1-6
1-3
KESTOS CELGIJ MELEZTOl MELEZTO2 PEKHESO 1 PLANTEI 0 HAHXUJ HEGXOG
Code
Ref
1-Kestose 6-Kestose, H20 Melezitose, H 2 0 form I Melezitose, H20 form I1 Nystose, 4H20 Planteose, 2H20 Erlose, H20 Erlose, 3H20
Molecule
q0e
1-2
dllinkage between a pyranose ring axial and a furanose ring
1-6
DICMEH
aGlc( 1-2)PFru aGlc( 1-2)PFru aGlc( 1-2)PFru aGlc(1-2)PFru aGlc( 1-2)PFru aGlc( l-2)PFru aGlc( 1-2)PFru aGlc( 1-2)PFru
Disaccharide
PGlc(lL6)PGlc
PGa1(1-4)aMan 60.7
0
0
84.6 89.6 99.8 109.6 102.3 108.2 104.5 98.0 109.9
119.4 121.3 119.4 115.3 118.3 118.8 116.5 117.7 116.2
58.6
-83.2
7
113.6
117.1
H-bond 0-2g.. .O-lg 05-g.. .HO-6f
Y
-65.9 -168.0 -30.6 -43.4 -18.6 -26.2 -32.6 -55.7 -39.8
155.1
-109.9
00
2
53.
e&
S-
ai;
E;.
a k
k
IMATUL PLANTE10
1-6
[97] [46]
[44] 1451
1-4
1-4
BOBKUYlO PAJNUJ
Code
equatorial-h a n o s e
[15] [98]
Ref
P-Lactulose 0-Lactulose, 3 H 2 0
Molecule
PGal( 1-4)PFru PGa1(1-4)PFru
Disaccharide
aGlc(1 -6)PFru aGal(1-6)PFru
aGlc( I-3)PFru aGlc( 1-3)PFru
Melezitose monohydrate form I Melezitose monohydrate form I1
P-Isomaltulose, H 2 0 Planteose, 2H20
aGlc( 1-2)PFru aXyl( l-2)PFru
-64.6 63.5
0
113.1 116.1
z
115.5 111.2
115.9 116.9
118.6 116.6 118.1
120.9 117.1 4-CI-Gal(l-2)1,6di-Cl-Fru 119.2 aGlc( 1-2) PFru 114.3 aGlc( 1-2)PFru 116.7 aGlc( I-2)PFru
aGlc(1-2)PFru aGlc( 1 -2)PFru
Sucrose, sarcosine Sucroxyl, H 2 0
Raffinose, 5H20 Stachyose, 4 H 2 0 Tri-chloro-galactosucrose Sucrose Sucrose, NaBr2 2 H 0 Sucrose, i(2 NaI 3H20)
e/linkage between a pyranose ring equatorial and a furanose ring
MELEZTOI MELEZT02
[95] [96]
NEHCAE HAHYUK
1-3
[40] [48] 1911 [92] [93] [94]
RAFINOOl STACHYOl KANJOY SUCROS04 DINYOOlO n.d
Table 3 (continued)
~
-82.1 -79.9
Q
76.9 58.5
78.4 91.6
82.1 109.3 91.4 107.8 99.8 79.9 79.6 84.0 108.2 96.9
-164.4 -170.3
Y
143.6 172.5
143.1 153.9
12.0 -47.3 -162.2 -44.8 -46.1 -66.6 -61.6 -20.6 -46.9 -37.2
0 - 5 g . . .HO-3f
H-bond
0-2x.. .HO-lf 0 - 2 ~, ..HO-lf
HO-2g.. .0-3f
0-2g.. .HO-lf
3
8g.
5
b
z
2
F'
5
3
5
9
8-
2L
'.
8
0, 2
G.
Q
h
h,
00
Q
[42] [49]
[43]
KESTOS PEKHESO1
CELGIJ
2-6
Ref
1-2
Code
2-1 and 2-6
fhnose-furanose
6-Kestose, H 2 0
1-Kestose Nystose, 3H20
Molecule
f/Linkage between two furanose rings
w
179.2 -63.3 -175.9 67.9
Disaccharide
PFm(1-2)PFru PFru( 1-2)PFru PFru(l-2)PFru PFru(2-6) PFru 116.4 117.1 115.3 118.0
t
-41.1 55.9 -47.1 -55.8
a,
-169.6 -133.3 -165.5 145.7
Y
H-bond
n
%
s
2E
0
i,
984
34 Oligosaccharide Conformations by Diflraction Methods 360 300
240
o ! -120 -60
0
60
120
180
240
1
I
-120 -60
Y
,
0
I
I
I
60
,
,
,
,
120
180
24(
6o Y I2O
I8O
240~
Y
360
120
300
60
0
240
CD
0
0
180
-60
120
-120
60
-1 80 -240
0
-240 -180 -120
I
-60
Y
0
60
120
-60
Figure 2. Iso-energy maps for the linkage between two pyranose linked in an axial-axial configuration (a), in a axial-equatorial configuration (b), in a equatorial-equatorial configuration (c) or linkage between a furanose and a pyranose with an axial glycosidic oxygen. The iso-contours represent approximately 2 and 6 kcal/mol as calculated using the MM3 software for aMan(1-2)Man (a), aMan(1-3)Man (b), cellobiose in (c) and sucrose (d). Conformations observed in the crystal structures have been superimposed on the maps using the following code: circle (I-l), square (l-2), triangle (1-3), diamond (1-4) and cross (1-5). The black symbols represent the conformation of an analog with an hetero atom at the position of the glycosidic oxygen. Appropriate transformation has been carried on the value of the @ and Y angle for the aim of comparison: the inverse of the value was taken for L-sugar, for 1-3 linkages, +120" or -120" was added to the value.
34.4 Hydrogen Bonding in Crystalline Oligosaccharides
985
40" and 120" for 1-x axial linkages, and between -100" and -65" for l - + x equatorial linkages. Obviously, the magnitude of the exo-anomeric effect is not strong enough to drive the CD angle to one single conformation. It can be counterbalanced by the occurrence of inter-residue hydrogen bond or other favorable interatomic interactions. Not unexpectedly, the dispersion of the observed conformations about the torsion angle Y is much wider. A somehow similar set of observations can be made in the case of disaccharide segments belonging to the axial-furanose family (Fig. 3d), which encompasses most of the sucrosyl containing molecules. In such cases, a double exo-anomeric effect, occurring from the C-S-O-5-C- 1-O-g-C-x'-O-5' sequence could be even more influential in the establishment of glycosidic conformations. Whereas the dispersion in CD angles goes from 60" to loo", the Y torsion angles span more than 200". Most of these observations have been rationalized throughout molecular modeling investigations. 34.3.2 The Analogs (S, C, N , . . ..) The replacement of the interglycosidic atom in a disaccharide by S, C, NH, . . . generates a class of interesting analogues, namely thio-disaccharides, C-disaccharides, diglycosylamines, which all constitute potential non-hydrolyzable epitopes and substrates analogues. Similarly, sulfur-analog can be obtained by substitution of the ring oxygen. The crystal structures of those analogs that have been reported, are listed in Table 4. The X-ray structure of methyl a-thiomaltoside showed that the effect of replacing the glycosidic oxygen atom by sulfur on the geometry and conformation of the pyranose rings is minimal [ 191. However, the magnitude of glycosidic valence angle 7 (100.3") is larger than the value usually found for C-S-C fragments; as such it follows the general trend observed in the corresponding O-glycosides. The same value has been observed when both the glycosidic oxygen and the ring oxygen of the non-reducing unit of maltosc are substituted by sulfur [20]. The glycosidic C-1-S and S-C-4' bonds are 1.83 A length and this results in a larger separation of the two pyranose rings. The torsional angles at the thio-glycosidic linkage are 0-C-lS-C-4' = 89" and C-l-S-C4'-CS' = - 1 16.8", they correspond to a low energy conformation within a potential energy surface that is similar to that computed for maltose. In C-glycosides, a methylene group (C-g) replaces the glycosidic oxygen atom and the bonding sequence C-5-0-5-C- 1-C-g-Cx' replaces the original sequence present in the parent O-glycosidic molecules. It is therefore expected that in the crystalline conformation, the anomeric and exo-anomeric effects will no longer be driving forces in these molecules. The conformation of C-gentiobioside [21] in the solid state is characterized by torsion angles CD = 5S.9", Y = 175.1' and o = 63.9". There is indeed a striking difference between C-gentiobioside and its O-counterpart since there is a 120" difference between the magnitude of the CD angle in both molecule. The in uacuo potential energy calculations for C-gentiobioside indicates that the lack of influence of the exo-anomeric effect is not responsible for the occurrence
N-analog
RIWZIG S-analog CIBYAN
C-analog EABRAA
Code
a,@-N-trehalose octaacetate aGlc( 1-1)PGlc
aGlc( 1-4)aGlc aGlc( 1-4)aGlc PGlc(1-2)aGlc
Methyl a-thiomaltoside di-thio-maltose, CH30H 5 S-kijobiose
[19] PO01 [201
~ 3 1
aGlc(1-6)aGlc
C-isomaltose heptaacetate
PI
aGlc(l-2)PFru fiGlc(l-6)PGlc
Disaccharide
C-sucrose octaacetate Methyl-C-gentiobioside
Molecule
[22] PI1
Ref
61.6
-63.9
w
57.6 59.8
89.0 80.7 79.3
100.3 100.4 114.4 119.9 118.9
49.2
68.1 55.9
4
114.6
111.3 113.6
T
Table 4. References and selected structural information for the crystal structures of disaccharides analogs.
-78.8 -85.9
-116.8 -125.7 -140.8
64.4
-77.2 175.1
cp
H06.. .06’
H-bond
34.5 Packing Features
981
of such crystalline conformation; it may simply result from steric interaction of the substituents at the inter-residue linkage. Similarly, the crystalline conformation of C-sucrose, as dictated by torsion angles @ = -54" and Y = 44.1" is significantly different from that of its 0 analog [22]. Aminosugars are potent reversible inhibitors of glycosidases and can be used for many therapeutic agents providing that they exhibit sufficient stability. In diglycosylamines, where a N H group replaces the glycosidic oxygen atom, the basicity of the such glycosylamine nitrogen is expected to be decreased by the combined electron withdrawing effect of the two neighboring oxygen atoms. The crystalline and molecular features of the peracetylated a , 0-glucopyranosylamine, the N-analog of per-acetylated a, p trehalose, has been solved [23]. The hydrogen atom covalently linked to the nitrogen at the glycosidic linkage is well located, yielding to a R configuration for the nitrogen. The substitution of 0 by NH at the glycosidic linkage induces a significant perturbation. The glycosidic C-N-C bond angles in 6" larger than that of a, p trehalose, and a lengthening of the 0-5-C-1 bond is also observed. This feature was predicted to occur from calculations and is attributed to a stabilizing delocalization of the nitrogen lone pair into an antibonding C-0 orbital ( n N + ( ~ c - 0 '[24]. ) The torsional angles at the glycosidic linkages (57.6", -78.8") and (59.8", -85.9") are slightly different from the conformation found in a, p trehalose monohydrate [25] even though the potential energy surface computed for the N-analogue exhibits the same aspects than the one calculated for a, p trehalose, except that the accessible area is larger.
34.4 Hydrogen Bonding in Crystalline Oligosaccharides The carbohydrates offer rich examples to analyze the hydrogen bonding in crystals, from which rules can be extracted to be further used in molecular modeling situations. Most of the basic rules have been established throughout the analysis of high accuracy X-ray analysis and most evidently from those crystalline structures that have been derived from neutron diffraction investigations. Neutron diffraction determines nuclear coordinates whereas X-ray diffraction refers to maxima of the electron density distribution. A systematic investigation of the differences in bond lengths from X-ray and neutron diffraction analyses of the same crystal structure gave significant differencFs for d,-dN for C-H and 0 - H bonds, respectively -0.096(7) and -0.155(10) A [26]. This is the reason why the X-ray X-H bond lengths have to be normalized to standard, neutron diffraction, values for the interpretation of hydrogen bonding in molecular crystals. Essentially, the rules governing the establishment of hydrogen bonds obey to two basic concepts [27]: i) maximize the hydrogen-bond interactions throughout the participation of all hydroxyl groups and as many ring and, to a lesser extend, glycosidic oxygen atoms as possible. These features implies both two and three-center bonds.
988
34 Oligosaccharide Conformations by Diffraction Methods
ii) maximize cooperativity by forming as many finite and infinite chains of hydrogen bonds as possible. Several patterns have been identified by Jeffrey [28] and the crystal structure can be roughly divided equally between them. Class A: encompasses those structures where the cooperative effect is maximized by the formation of infinite chains or spirals of hydrogen-bonded hydroxyls. In class B, a more limited cooperative effect is obtained throughout the occurrence of finite chains with the acetal oxygen atoms acting as chain terminators. Class C, in which the maximum cooperative effect is achieved with all the hydroxyl groups, except one, which forms a separate two- or three-centered hydrogen bond to a ring or glycosidic oxygen. In class D, the infinite chains are retained and the ring and glycosidic oxygen atoms are included in the hydrogen bonding scheme throughout the occurrence of three-center bonds. In almost all cases, hydroxyl groups act as both hydrogen bond donors and acceptors. Compared with the other hydroxyl groups, the anomeric hydroxyls tend to be stronger donor and weaker acceptors. As for ring oxygen and glycosidic oxygen, stereochemical and packing reasons may prevent these atoms to be acceptors. Because there are more acceptors than donors in carbohydrates, it is not surprising to find a significant occurrence of three-centered hydrogen bonds. Hydration is a fairly common features of crystalline oligosaccharides, which may result from their molecular shapes that do not provide efficient packing. Therefore, voids from the water molecules are left in the crystalline structures. It should be clearly stated that the analysis of crystalline oligosaccharides cannot provide a limited insight into the preferred hydration effect. Whereas water molecules may influence the occurrence of a given conformation, these water molecules may be expelled upon formation of the crystalline arrangement. In the hydrate structures, the water molecules always donate two hydrogen bonds; they may accept one or two.
34.5 Packing Features As for their crystalline structure, more than 95% of the carbohydrates crystallize in non centro-symmetric structures. The rare examples of centro-symmetric space groups deal with some linear or cyclic polyalcohol along with some pyranoses and furanoses. In order to adopt such features, these molecules either have an internal symmetry or crystallize as racemic. Figure 3 displays the histogram of the occurrence of space groups in carbohydrate crystal structures, from which it is obvious that only four space groups, i.e. P212121, P21, PI and Ct account for more than 80% of the observed space groups. Despite the wealth of potential structural information, there is a lack of comprehensive investigation of the molecular principles underlying the formation of the oligosaccharide crystals. Only few reports have tried to analyze the anisotropy of intermolecular interactions in these crystals. A typical way of exploring the packing
34.5 Packing Features
989
space groups Figure 3. Histogram of the distribution of space groups among the crystal structures of oligosaccharides.
arrangement is to evaluate the intermolecular energy between one molecule, i.e. the reference molecule, and all its neighbors. This energy is evaluated by taking into account the intermolecular hydrogen bonds as well as the non-bonded interactions. The number of 'close' contacts corresponding to interactions distances less than 1.5 times the sum of the van der Waals interacting atoms may be evaluated. The estimation of electrostatic component is more straightforward as it may depend upon the way the atomic charges are evaluated along with the cut-off distance used in the calculations. Since most of the oligosaccharide structures solved up to now concern neutral carbohydrates, such a point has not be given much attention. The packing arrangements found in crystal structures of oligosaccharides are consistent with a high packing density, as each molecule is usually surrounded by 12 neighbors. Among these neighbors, which occur in pair, a certain anisotropy exist, which in many cases can be correlated to the dimensions of the unit cell, and/or the morphology of the crystals. From the differences in the magnitude of the intermolecular energies can be identity supramolecular elements such as molecular chains and/or molecular layers, that constitute the basis of the molecular layers. From the limited number of packing of oligosaccharide structures which have been investigated so far, the low energy molecular layers correspond to the few which underline the formation of the space groups cited above.
990
34 Oligosaccharide Conformations by Diflruction Methods
Figure 4. The molecular and crystal structure of Lewis X [29]. Graphical representation of the two trisaccharides constituting the asymmetric unit along with the water molecules hydrating them. Nine of the water molecules are part of the asymmetric unit content and the other ones are related by symmetry element.
34.6 Selected Examples Lewisx. Despite their well recognized biological role, the first crystal structure of a histo-blood group carbohydrate-dependent antigen, i.e. Lewis' [ PGall4[aFucl31PGlcNac was reported in 1996 [29] (Figure 4). Starting from chemically synthesized LeX trisaccharide methyl glycoside, single crystal could be grown from a slow evaporating solution of Lex, water/ethanol mixture. Over a period of 2 years, more than 20 crystals having dimensions suitable for X-ray investigations were obtained and investigated. One single crystal having dimensions of 0.5 x 0.25 x 0.05 mm was of a sufficient quality to diffract and yielded enough reflections to solve and refine the structure to a reliability index of 0.05. Le' crystallizes in the monoclinic space group P21, with unit cell parameters u = 12.147(6), b = 27.552(9), c = 8.8662(6) A and P = 91.71"(5). In such a unit cell, the asymmetric unit contains two independent LeXmolecules, and an unusually high number of water molecules. All hydrogen atoms of the 0-H groups and the water molecules could be located in this crystal structure, allowing a straightforward assignment of hydrogen bonding scheme.
34.7 Crystalline Conjovmations o j Oligosuccharides Complexed with Lectins
99 1
The two crystallographically independent LeX molecules differ in their overall conformations, and these differences are essentially located at the glycosidic torsion angles at the PGall4GlcNAc linkage for which angle (D differs by 10". Neither of the two trisaccharides exhibits any intramolecular hydrogen bonds. A strong interaction exists between the fucose and galactose residue, but only non polar van der Waals contacts are involved, each ring presenting its most hydrophobic face to the other one. Conformational studies using NMR and/or molecular modeling generally agree on a single conformation for LeXin solution, corresponding closely to the one found in the crystal structure. The nine water molecules present in the asymmetric unit are arranged in a cluster-like fashion. They establish hydrogen bonds to other water molecules within the cluster and to the surrounding carbohydrate molecule. They are arranged to fill up an empty space in the crystal packing. Six and seven water molecules, respectively, are involved in the hydration of the two trisaccharides. The crystal structure displays an extremely dense network of hydrogen bonds. Thirty six hydrogen bonds are observed in the asymmetric unit, 30 of them implying water molecules. Each trisaccharide is involved in 21 hydrogen bonds, the ration per glycosidic residue varying from 5 to 8. Such a high number of hydrogen bonds can be correlated to (i) the high hydration level and (ii) the peculiar folding of the oligosaccharides, burying the hydrophobic faces of the residue and presenting the hydrophilic faces to the external part. The analysis of the packing indicates a well defined hierarchy of intermolecular contacts, some of them suggesting how LeX-LeXinteractions may occur in biological conditions providing the molecular basis for cell-cell recognition event. Cellodextrins. The quest for the structural elucidation of the crystalline allomorphs of cellulose has stimulated interest in the resolution of the crystal structures of the low molecular weight oligomers. In the case of cellulose 11, it has been established that the cellodextrins having a degree of polymerization of four and higher, give X-ray powder diffractograms that are similar to those of the polysaccharides. Furthermore, powders of methyl P-cellotrioside, and higher members, give diffraction patterns having the cellulose I1 features. The crystal structure of methyl P-cellotrioside was solved as a monohydrate 0.25 ethanolate, using synchrotron data collected on crystals of dimensions 0.43 x 0.33 x 0.04 mm [6]. B-D-cellotetraose crystallizes in the triclinic space group PI with two independent molecules and one water molecule in the unit cell having dimensions a = 8.045( 12), b = 9.003(9), c : 22.51(2) A, a = 89.66(7), P = 94.83(13), y = 115.80(4)' [7]. Because of the very small dimensions of the crystals (0.40 x 0.15 x 0.015 mm) the X-ray diffraction data were collected at room temperature, using beam line at the European Synchrotron Radiation Facility in Grenoble. A monochromatic radiation with a wavelength of 0.925 A was used, and the data collection was achieved on image plate detectors. At about the same time were reported the results of an independent investigation that was performed using a combination of conventional Xray and synchrotron sources on crystals having dimensions 0.4 x 0. x 0.1 mm, [8]. In that case, the reported unit cell dimensions were a = 8.023(1), b = 8.951(2), c = 22.445(2) a = 89.26(2), P = 85.07(l ) , y = 63.93( l)', this corresponds to a different setting of the crystallographic axis.
A,
992
34 Oligosuccharide Conformations by Diflruction Methods
The least-squares superposition of the C and 0-atoms ofthese two structural investigations, has a root mean square deviation of 0.05 A for the non hydrogen atoms. Some several discrepancies are noticed in the positions of 0-H hydrogen atoms and consequently in hydrogen bonding scheme. The way H atom positions were located in the two investigations may explained such discrepancies as in one case they were located in ‘theoretical’ positions whereas in the other one, they were located from differences Fourier analyses. Among the many structural and conformational insights that such investigations are providing, one would like to emphasize the striking finding that the two crystallographically independent cellotetraose molecules are significantly different. These structural differences are not only observed with the ring puckering parameters, but also with the glycosidic @ and Y torsion angles. Whereas one molecule is in a ‘standard’ conformation; the other one displays a significant conformational strain. The influence of mode of packing of these two molecules in the crystal has been invoked to explain such a difference, even though no significant molecular mechanic calculations have yet been performed to elucidate the reasons underlying such significant deviations from normality (Figure 5). When a subcell is constructed of the two central D-glucopyranoses in the two independent molecule, the obtained cell dimensions are identical to those of the cellulose I1 allomorph. Based on this subcell, a new model for cellulose I1 has been proposed [81.
34.7 Crystalline Conformations of Oligosaccharides Complexed with Lectins Since the beginning of the 90’s an increasing number of crystal structures have been reported for glycoproteins and protein-carbohydrate complexes. The resolution of the first reported structures was rarely sufficient to provide reliable conformational information. Significant and rapid progresses arising from the use of synchrotron radiation are providing access to highly resolved structures. For example, the protein-carbohydrate literature provides structural data for 15 protein/cellobiose, 10 protein/lactose, 35 protein/maltose 4 protein/sucrose and 1 proteinla-a trehalose complexes crystal structures. Forces in protein-carbohydrate complexes should be more varied than in the fairly homogenous small molecule oligosaccharides crystals. The protein geometry reduces the influence of preferred packing modes, this is particularly true for hydrolytic enzymes that induces significant distortions both of the ring geometry and the conformations at the glycosidic linkages. Other bias may occur, resulting from the low-resolution of the structure, or from the fact that the crystallographic refinements are often guided with force fields. For these reasons, we are restricted the presentation to the class of lectin-oligosaccharide crystal structures, where there is no distortion occurring from hydrolysis and for which the crystallographic resolution is generally very high. Among proteins that interact non covalently with carbohydrates, lectins bind mono- and oligosaccharides reversibly
34.7 Crystalline Conformations of Oligosaccharides Complexed with Lectins
993
j I
Figure 5. The molecular structure of p-D-cellotetraose [S]. Two orthogonal views of the unit cell.
and specifically, while displaying no catalytic or immunological activity. More than 200 of crystal structures of lectins have been solved, most of them as complexes with carbohydrate ligands. From a data base of three-dimensional structures of lectins (http://www.cermav.cnrs/databank/lectins)Table 5 has been set, to illustrate the extraordinary wealth of information which is available about oligosaccharides. One can found information about simple disaccharides (maltose, sucrose,. . . .) and investigate how their ‘bio-active’ conformation may be similar/different from the one display in molecular crystals. Presumably more informative are those complexes involving biologically important oligosaccharides for which no structural information was available. For example these are: (i) the sialic acid containing oligosaccharides (sialyllactose, sialoglycopeptide, . . .), important antigens (T antigen, histo blood group antigen such as Lewis antigens and their sialylated or sulfated
Sialyllactose Sialogycopeptide Sialyllactose Galpl,3GalNAc Lactose Gal+ 1,3-GalNAc-a-O-benzyl Lactose T-antigen C-lactose N-acetyllactosamine aMan(lL3)uMan aMan( lL6)aMan
Agglutinin I (WGA)
Agglutinin II(WGA) Agglutinin Ricin (RCA) Agglutinin (ACA) Lectin (PNA)
Concanavalin A (ConA)
Lectin Lectin (DBL) Lectin (EcorL)
Soybean agglutinin (SBA)
Isolectin IV (GS4)
Isolectin I (LOL-I)
Isolectin I1 (LOL-11)
Lectine (LcL) Agglutinin (GNA)
Wheat germ
Wheat germ Maclura pomfera Ricinus communis Amaranthus caudatus Arachis hypogaea
Canavalia ensformis
Dioclea grandiflora Dolichos bijorus Erythrina corallodendron
Glycine max
Griffonia simplicfolia
Lathyrus ochrus
Lathyrus ochrus
Lens culinuris Galanthus niualis
Lewis B Lewis Y aMan(1-3)PMan( 1-4)GlcNAc Biantennary octasaccharide glycopeptide N2 fragment of lactotransferine Sucrose aMan(IL3)aMan Mannopentaose
N-ace tyllactosamine N-acetyllactosamine
Pentasaccharide from N-glycan aMan(lL6)[aMan(l-3)]aMan Forssman disaccharide Lactose
uMan(lL6)[aMan(l-3)]aMan
Glycan
Protein
Origin
1WGC [ 1011 2CWG [ 1021 2WGC [ 1011 lJOT [lo31 2AAI [ 1041 1JLX [ 1051 2PEL [ 1061 2TEP [ 1071 lBZW [I081 lCIW [lo91 IQDO [I101 lQDC /110] ICVN [ I l l ] IONA [ 1121 ITEI [I131 l D G L [114] l L U l [115] lLTE [116] IAXl [117] 1AX2 [117] 2SBA [ 11S] ISBD, ISBE, lSBF [I191 1LED [ 1201 lGSL [120] lLOG [121] 1LOF [ 301 lLGC [31] lLGB [31] lLES [ 1221 INIV [123] IJPC [I241
PDB code and ref
Table 5. References and selected structural information for the crystal structures of lectin-oligosaccharide crystal structures.
Galectin-1
Galectin-2 Galectin-7 Galectin-7 Congerin-I Sialoadhesin Pertussis toxin Cholera toxin
Heat-labile Enterotoxin Verotoxin- 1 Enterotoxin B
Coat protein
Hemagglutinin Tailspike protein
Bufo arenarum
Homo sapiens
Staphylococcus aureus
Murine Polyomavirus
Influenzae virus Phage P22
Escherichia coli
3’ Sialyllactose Salmonella octasaccharide Salmonella nonasaccharide
Lactose Receptor GB3 Lactose GM3 trisaccharide 3’ Sialyllactose Disialylated oligosaccharide
Mannose 6-P receptor Galectin-1
Bos taurus Bos taurus
Conger myriaster Mus Musculus Bordetella pertussis Vibrio cholerae
aMan(I -3)aMan Biantennary glycopeptide 3’sialyl Lewis X 3’sulfo Lewis X 4’sulfo Lewis X Mannopentaose N-acetyllactosamine Biantennary oligosaccharide N-acetyllactosamine Thio-digalactose Lactose Lactose N-acetyllactosamine Lactose 3’ Sialyllactose 3’ Sialylgalactose G M 1 pentasaccharide
Daffodil lectin Mannose binding protein A MBP-A mutant CL-K3
Narcissus pseudonarcissus Rattus rattus Rattus norvegicus
Q
5 lTYW [142]
i! !E
x
5
9 5 f?-:
00
.a
c*i
lNPL [ 1251 2MSB [ 1261 2KMB [ 1271 3KMB [I271 4KMB [127] 1C39 [ 1281 lSLT [ 1271 ISLA, ISLB, ISLC [I291 1CAN 1A78 lHLC [130] 4GAL [131] SGAL [131] l C l L [132] lQFO [I331 lPTO [134] lCHB [I351 2CHB, lCTl [32] 1LTT [ 1361 IBOS [137] 1SE4 [I381 1SE3 [138] lSID [139] lSIE [139] lVPS [ 1401 l H G G [141] lTYU, lTYX
996
34 Oligosaccharide Conformations by Difraction Methods
forms.), (iii) moieties of glycolipids such as G M l and GM3, (iv) oligosaccharides belonging to N-linked glycans, biantennary glycopetide, a N2 fragment of lactotransferine, (v) fragments of polysaccharides from the cell wall of pathogenic bacteria (Salmonella octa-and nona-saccharide). . . . Whereas the study of the conformations about the glycosidic torsion angles indicated a somehow limited flexibility in molecular crystal (vide infra) a different pictures emerges for the flexibility of oligosaccharides interacting with lectins. Figure 6 depicts the distributions of glycosidic torsion angles within three disaccharide segments: aMan(1-3)Man, PGlcNAc(1-2) Man and aNeuAc(2-3)Gal. In the case of the aMan(1-3)Man segment, the observed conformations are essentially located around a @ value of 80", with an excursion in the vicinity of 140". May be more interesting is the observation that a remote low energy area (located at @ = 90" and 3 ' ' = 310") can be occupied, as observed in the crystalline complex between Latyrus ochrus and a biantennary glycan [30]. The study of the dispersion of conformations observed for the disaccharide segment PGlcNAc( 1-2) Man, provides another illustration of the occurrence of conformations in remote energy well of the potential energy surfaces. The location of this well is 120" away from what would correspond to the stable conformation driven by the exo-anomeric effect in the case of an equatorial type of linkage. Such examples are observed in the crystalline complexes involving the isolectin I1 of Latyrus ochrus, complexed with high molecular oligosaccharides such as a biantennary octasaccharide [30], a glycopeptide or a N2 fragment of lactotransferin [31]. The aNeuAc(2-3)Gal offers an extreme case of conformational flexibility as it can be ten fold more flexible than the other disaccharides. Here again, the conformation corresponding to the establishment of the exo-anomeric effect CD = 60" is adopted in several case. Such a stabilizing influence can be easily overridden as exemplified by the occurrence of several low energy conformations having @ in the vicinity of -60"; this is observed for the GMl pentasaccharide interacting in the combining site a cholera toxin [32].
34.8 Concluding Remarks With this article, we have set out to provide enough information to initiate the general reader into the world of oligosaccharide structures as derived from diffraction methods. Oligosaccharide structures, numeric data and detailed reference lists have been presented, which may be useful to those directly involved in structural glycoscience. Some keys conformational features of oligosaccharides, have been dissected using the disaccharide moiety as a basic reference entity. This allowed us to provide a useful classification, both of the disaccharides and their analogs. Some of the general rules which dictate the formation of hydrogen bonding and packing patterns in crystalline oligosaccharides have been presented. They provide an efficient template to decipher those very complex structures. It must certainly be emphasized that despite the wealth of potential structural information available, there is a lack of comprehensive investigation of the molecular principles underlying the formation of oligosaccharide crystals.
References 9 N 0
03
0
N
::e 0
P 0
2
I
L
991
998
34 Oligosucchuride Conformutions by Dcjruction Methods
There has been a significant stagnation of the number of oligosaccharide structure analysis reported over the last decade. Usually, the minute amount of available pure oligosaccharides along with their natural reluctance to crystallize in a form suitable for X-ray diffraction studies may explain such a stagnation. It is nevertheless expected that progress in the synthesis or biosynthesis of carbohydrates as well as development in X-ray diffraction sources will be sufficient to overcome the paucity of structural and crystalline data on oligosaccharides. Interest is focusing on the more complex carbohydrate molecules, as diffraction methods extend to the field of protein-carbohydrate complex. Such a field benefits from the developments of methods for crystallizing these protein-based complexes as well as from the rapid progresses arising from the use of synchrotron radiation which is providing access to highly resolved structures. The conjunction of these new developments is likely to provide exciting new results and a greater insight into the biological mechanisms of this fascinating class of biomolecules.
References 1. F.J. Llewellyn, E.G. Cox, T.H. Goodwin, J. Chem. Soc. 1937, 883-894. 2. E.G. Cox, G.A. Jeffrey, Nature 1939, 143, 894-895. 3. C.A. Beevers, W. Cochran, Proc. R. Soc. London Ser. A. 1947, 190, 276-285. 4. G.M. Brown, H.A. Levy, Science 1963,141,921-924. 5. G.M. Brown, H.A. Levy, Science 1965, 147, 1038--1041. 6. S. Raymond, B. Hennissdt, D.T. Qui, A. Kvick, H. Chanzy, Carbohydr. Rex 19y5,277, 209229. 7. S. Raymond, A. Heyraud, D. Tran Qui, A. Kvick, H. Chanzy, Macromolecules 1995, 28, 2096--2100. 8. K. Gessler, N. Krauss, T. Steiner, C. Betzel, A. Sarko, W. Saenger, J. Am. Chem. Soc. 1995, 117, 11397-11406. 9. F.H. Allen, 0. Kennard, Chemical Design Automation News 1993, 8, 31-37. 10. K. Gessler, I. Uson, T. Takaha: N. Krauss, S.M. SMith, S. Okada, G.M. Sheldrick, W. Saenger, Proc. Nut. Acad. Sci. USA 1999, 96,4246-425 1. 11. W. Saenger, J. Jacob, K. Gessler, T. Steiner, D. Hoffman, H. Sanbe, K. Koizumi, S.M. Smith, T. Takaha, Chem. Rev. 1998, 98, 1787-1802. 12. W. Hinrichs, W. Saenger, J. Am. Chem. Soc. 1990. 112, 2789-2796. 13. A. Imberty, H. Chanzy, S. Perez, A. Buleon, V. Tran, J. Mol. Biol. 1988,201, 365-378. 14. G.A. Jeffrey, R. Taylor, J. Comput. Chem. 1980, 1, 99-109. 15. G.A. Jeffrey, R.A. Wood, P.E. Pfeffer, K.B. Hicks, J. Am. Chem. Soc. 1983,105, 2128-2123. 16. D. Cremer, J.A. Pople, J. Am. Chem. Soc. 1975,97, 1354-1358. 17. IUPAC-IUB, Arch. Biochem. Biophys. 1971, 145, 405-621. 18. R.U. Lemieux, S. Koto, D. Voisin In: Anomeric effect: origin and consequence, ACS symposium Series No. 87. Szarek, W. A. and Horton, D., Ed.; Amer. Chem. Soc.: Washington DC, 1979, pp 17-29. 19. S. Perez, C. Vergelati, Acta Cryst. 1984, B40, 294--299. 20. T. Weimar, U.C. Keis, J.S. Andrews, B.M. Pinto, Carbohydr. Res 1999, 315, 222-233. 21. A. Neuman, F. Longchambon, 0. Abbes, H. Gillier-Pandraud, S. Perez, D. Rouzaud, P. Sinay, Carbohydr. Rex 1990, 195, 187-197. 22. D.J. O’Leary, Y. Kishi, J. Org. Chem. 1993,58, 304-306. 23. A. Imberty, J. Gruza, N. Mouhous-Riou, B. Bachet, S. Perez, Carbohydr. Res. 1998, 311, 135-146. 24. I. Tvaroska, J.P. Carver, J. Chem. Phys. 1996, 100, 11305-11313. 25. T. Taga, Y . Miwa, Z. Min, Acta Crystallogr 1997, C53, 234-256.
References
999
26. F.H. Allen, Actu Cryst. 1986, B42, 515-522. 27. G.A. Jeffrey, Actu Cryst. 1990, B46, 89-103. 28. G.A. Jeffrey, W. Saenger Hydrogen bonding in biological structures; Springer-Verlag: Berlin, 1991. 29. S. Perez, N. Mouhous-Riou, N.E. Nifant'ev, Y.E. Tsvetkov, B. Bachet, A. Imberty, Clycobiology 1996, 6, 537-542. 30. Y. Bourne, P. Rouge, C. Cambillau, J. Biol. C h m . 1992, 267, 197-203. 31. Y. Bourne, J. Mazurier, D. Legrand, P. Rouge, J. Montreuil, G. Spik, C. Cambillau, Structure 1994, 2, 209-219. 32. E.A. Merritt, S. Sarfaty, M.G. Jobling, T. Chang, R.K. Holmes, T.R. Hirst, W.G. Hol, Protein Sci. 1997, 6, 1516-1528. 33. A. Otter, R.U. Lemieux, R.G. Ball, A.P. Venot, 0. Hindsgaul, D.R. Bundle, Eur. J. Biochem. 1999,259, 295-303. 34. R.A. Moran, C.F. Richards, Actu Cryst. 1973, B29, 2770-2783. 35. V. Warin, F. Baert, R. Fouret, C. Strecker, C. Spik, B. Fournet, J. Montreuil, Curbohydr. Res. 1979: 76, 11-22. 36. T. Taga, E. Inagaki, Y. Fujimori, S. Nakamura, Curbohydr. Res. 1993, 240, 39-45. 37. T. Taga, E. Inagaki, Y. Fujimori, S. Nakamura, Curbohydr. Res. 1994, 251, 203-212. 38. W. Pangborn, D. Langs, S. Perez, Int. J. Bid. Mucromol. 1985, 7, 363-369. 39. G.A. Jeffrey, D.-B. Huang, Carbohydr. Rrs. 1991,222, 47-55. 40. G.A. Jcffrey, D.-B. Huang, Carbohydr. Res. 1990, 206, 173-182. 41. W. Mackie, B. Sheldrick, D. Akrigg, S. Perez, Znt. J. Bid. Mucromol. 1986, 8, 43-51. 42. G.A. Jeffrey, Y.J. Park, Actu Cryst. 1972, B28, 257-267. 43. V. Ferretti, V. Bertolasi, G. Gilli, C.A. Accorsi, Acta Cryst. 1984, 40, 531-535. 44. D. Avenel, A. Neuman; H. Gillier-Pandraud, Acta Cryst. 1976, B32, 2598-2605. 45. J. Becquart, A. Newman, H. Gillier-Pandrdud, Curbohydr. Res 1982, 111, 9-21. 46. D.C. Rohrer, Actu Cryst. 1972, B28, 425-433. 47. S. Perez, F. Brisse, Actu Cryst. 1977, B33, 2578-2584. 48. G.A. Jeffrey, D.-B. Huang, Curbohydr. Res. 1991,210, 89-104. 49. G.A. Jeffrey, D.B. Huang, Curbohydr. Res. 1993,247, 37-50. 50. K. Noguchi, E. Kobayashi, K. Okuyama, S. Kitamurd, K. Takeo, S. Ohno, Curbohydr. Res. 1994,258, 35-47. 51. A. Linden, C.K. Lee, Acta Cryst. 1995, C51, 1007-1012. 52. J. Ollis, V.J. James, S.J. Angyal, P.M. Pojer. Curbohydr. Res. 1978, 60, 219-228. 53. C.-K. Lee, L.L. Koh, Curbohydr. Res. 1994, 254, 281-287. 54. G.M. Brown, D.C. Rohrer, B. Berking, C.A. Beevers, R.O. Gould, R. Simpson, Actu Cryst 1972, B28, 3145-3158. 55. G.A. Jeffrey, R. Nanni, Curbohydr. Res. 1985, 137, 21-30. 56. W.J. Cook, C.E. Bugg, Curbohydr. Res. 1973, 31, 265 ~275. 57. A. Linden, C.K. Lee, Actu Cryst. 1995, C51, 1012-1016. 58. C.-K. Lee, A. Linden, Curbohydr. Rex 1994, 264, 319-325. 59. T. Srikrishnan, M.S. Chowdhary, K.L. Matra, Curhohydr. Res. 1989, 186, 167-175. 60. J. Thiem, M. Kleeberg, K.-H. Klaska, Curbohydr. Res. 1989, 189, 65-77. 61. G . Svensson, J. Albertsson, C. Svensson, C. Magnusson, J. Dahmen, Curbohydr. Res. 1986, 146, 29-38. 62. D.K. Watt, D.J. Brasch, D.S. Larsen, L.D. Melton, J. Simpson, Carbohydr. Ras. 1996, 285, 1-15. 63. L. Eriksson, R. Stenutz, G. Widmalm, Actu Cryst. 1997, C53, 1105-1107. 64. A. Neuman, D. Avenel, F. Arene, H. Gillier-Fandraud, J.-R. Pougny, P. Sinay, Carbohydr. Res. 1980, 80, 15-24. 65. A. Neuman, D. Avenel, H. Gillier-Pandraud, Acta Cryst. 1978, B34, 242-248. 66. F. Takusagawa, R.A. Jacobson, Actu Cryst 1978, B34, 213-218. 67. M.E. Gress, G.A. Jeffrey, Actu Cryst. 1977, B33, 2490-2495. 68. I. Tanaka, N. Tanaka, T. Ashida, M. Kakudo, Actu Cryst. 1976, B32, 155-160. 69. S.S.C. Chu, G.A. Jeffrey, Actu Cryst. 1967, 23, 1038-1049. 70. M.E. Gress, G.A. Jeffrey, D.C. Rohrer, Actu Crystullogr. 1978, B34, 508-512.
1000
34 Oligosaccharide Conformations by Diffruction Methods
V. Mikol, P. Kosma, H. Brade, Carbohydr. Res. 1994,263, 35-42. C.-K. Lee, L.L. Koh, Actu. Cryst. 1993, C49, 621-624. J. Ohanessian, F. Longchambon, F. Arene, Actu Cryst. 1978, BM, 3666-3671. H. Takeda, N. Yasuoka, N. Kasai, Curbohydr. Res. 1977,53, 137-152. K. Noguchi, K. Okuyama, S. Kitamura, K. Takeo, Carbohydr. Res. 1992,237, 33-43. D. Lamba, A.L. Segre, S. Clover, W. Mackie, B. Sheldrick, S. Perez, Curbohydr. Res. 1990, 208,215-230. 77. M. Senma, T. Taga, K. Osaka, Chem. Lett. 1974, 1415-1418. 78. F. Longchambon, J. Ohanessian, H. Gillier-Pandraud, D. Duchet, J.-C. Jacquinet, P. Sinay, Actu Cryst. 1981, B37, 601-607. 79. C.E. Bugg, J. Am. Chern. SOC.1973, 95, 908-913. 80. J.H. Noordik, P.T. Beurskens, P. Bennema, R.A. Visser, R.O. Could, Z. Kristallogr. 1984, 168, 59-65. 81. K. Hirotsu, A. Shimada, Bull. Chem. SOC.Jpn. 1974, 47, 1872-1879. 82. M.M. Olmstead, M. Hu, M.J. Kurth, J.M. Krochta, Y.L. Hsieh, Acta Cryst. 1997, C53, 915916. 83. B. Sheldrick, W. Mackie, A. D., Curbohydr. Res. 1984, 132, 1-6. 84. S.S.C. Chu, G.A. Jeffrey, Actu Cryst., 1968, B24, 830-838. 85. P. Koll, M. Petrusova, L. Petrus, B. Zimmer, M. Morf, J. Kopf, Curbohydr. Res. 1993, 248, 37-43. 86. J.T. Ham, D.G. Williams, Acta Cryst. 1970, B26, 1373-1383. 87. F. Mo, L.H. Jensen, Acta Cryst. 1978, B34, 1562-1569. 88. F. Mo, Actu Ckem. Scand. 1979, A33, 207-218. 89. C. Burden, W. Mackie, B. Sheldrick, Acta Cryst. 1986, C42, 177-179. 90. F. Arene, A. Neuman, F. Longchambon, C. R. Acud. Sci. Paris 1979, (2288, 331-334. 91. J.A. Kanters, R.L. Scherrenberg, B.R. LeeAang, 3. Kroon, M. Mathlouthi, Cuvbohydr. Res. 1988,180, 175-182. 92. G.M. Brown, H.A. Levy, Acta Cryst. 1973, B29, 790-797. 93. C.A. Accorsi, F. Bellucci, V. Bertolasi, V. Ferretti, G. Gilli, Carbohydr. Rex, 1989, 191, 105116. 94. C.A. Accorsi, V. Bertolasi, V. Ferretti, G. Gilli, Curbohydr. Res. 1989, 191, 91-104. 95. R.V. Krishnakumar, S. Natarajan, Carbohydr. Res. 1996,287, 117-122. 96. T. Taga, E. Inagaki, Y. Fujimori, K. Fujita, K. Hara, Curbohydr. Res. 1992, 241, 63-69. 97. W. Dreissig, P. Luger, Actu Cryst. 1973, B29, 514-521. 98. G.A. Jeffrey, D.B. Huang, P.E. Pfeffer, R.L. Dudley, K.B. Hicks, E. Nitsch, Carbohydr. Res. 1992,226, 29-42. 99. T. Skrydstrup, D. MazCas, M. Elmouchir, G. Doisneau, C. Riche, A. Chiaroni, J.M. Beau, Chem. Eur. J. 1997,3, 1342-1356. 100. S. Mehta, K.L. Jordan, U.C. Kreis, T. Weimar, R.J. Batchelor, F.W.B. Einstein, B.M. Pinto, Tetrahedron Asym. 1994,5, 236772396, 101. C.S. Wright, J. Mol. Biol. 1990, 215, 635-651. 102. C.S. Wright, J. Jaeger, J. Mol. Biol. 1993,232, 620-638. 103. X. Lee, A. Thompson, Z. Zhang, H. Ton-that, J. Biesterfeldt, C. Ogata, Xu, L., R.A. Johnston, N.M. Young, J. Biol. Chem. 1998,273,6312-6318. 104. E. Rutenber, B.J. Katzin, S. Ernst, E.J. Collins, D. Mlsna, M.P. Ready, R. J.D., Proteins 1991,10,240-250. 105. T.R. Transue, A.K. Smith, H. Mo, I.J. Goldstein, M.A. Saper, Nut. Struct. Biol. 1997, 10, 779-783. 106. R. Banerjee, K. Das, R. Ravishankar, K. Suguna, A. Surolia, M. Vijayan, J. Mol. Biol. 1996, 259, 281-296. 107. R. Ravishankar, M. Ravindran, K. Suguna, A. Surolia, M. Vikayan, Cum. Sci. 1997, 72, 855861. 108. R. Ravishankar, A. Surolia, M.V.S. Lim, Y. Kishi, J. Amer. Chem. SOC.1998, 120, 1129711303. 109. R. Ravishankar, K. Suguna, A. Surolia, M. Vijayan, Acta. Crystullogr. 1999, D.55,13751382. 71. 72. 73. 74. 75. 76.
34.2 General Analysis
1001
110. J. Bouckaert, T. Hamelryck, L. Wyns, R. Loris, J. Bid. Chem. 1999,274, 29188-29195. 111. J.H. Naismith, R.A. Field, J. Biol. Chenz. 1996, 271, 972-976. 112. R. Loris, D. Maes, F. Poortmans, L. Wyns, J. Bouckaert, J. Biol. Chem. 1996, 271, 3061430618. 113. D.N. Moothoo, J.H. Naismith, Glycohiology 1998, 8, 173-181. 114. D.A. Rozwarski, B.M. Swami, C.F. Brewer, J.C. Sacchettini, J. Biol. Chem. 1998, 273, 328 18-32825. 115. T.W. Hamelryck, R. Loris, J. Bouckaert, M.-H. Dao-Thi, G. Strecker, A. Imberty, E. Fernandez, L. Wyns, M.E. Etzler, J. Mol. Bid. 1999,286, 1161-1177. 116. B. Shaanan, H. Lis, N. Sharon, Science 1991,254, 862-866. 117. S. Elgavish, B. Shaanan, J. Mol. Bid. 1998, 277, 917-932. 118. A. Dessen, D. Gupta, S. Sabesan, C.F. Brewer, J.C. Sacchetini, Biochemistry 1995, 34, 49334942. 119. L.R. Olsen, A. Dessen, D. Gupta, S. Sabesan, J.C. Sacchettini, C.F. Brewer, Biochemistry 1997, 36, 15073-1 5080. 120. L.T. Delbaere, M. Vandonselaar, L. Prasad, J.W. Quail, K.S. Wilson, Z. Dauter, J. Mol. Bid. 1993,230, 950-965. 121. Y. Bourne, P. Rouge, C. Cambillau, J. Bid. Chenz. 1990, 265, 18161-18165. 122. F. Casset, T. Hamelryck, R. Loris, J.R. Brisson, C. Tellier, M.H. Dao-Thi, L. Wyns, F. Poortmans, S. Perez, A. Imberty, J. Biol. Chem. 1995,270, 25619-25628. 123. G. Hester, C.S. Wright, J. Mol. Biol 1996, 262, 516-531. 124. C.S. Wright, G. Hester, Structure 1996, 4, 1339-1352. 125. M.K. Sauerborn, L.M. Wright, C.D. Reynolds, J.G. Grossmann, P.J. Rizkallah, J. Mol. Bid. 1999,290, 185-199. 126. W.I. Weis, K. Drickamer, W.A. Hendrickson, Nature 1992, 360, 127-134. 127. K.K. Ng, W.I. Weis, Biochemistry 1997, 36, 979-988. 128. L.J. Olson, J. Zhang, Y.C. Lee, N.M. Dahms, J.J.P. Kim, Journal of Biologica1 Chemistry 1999, 274, 29889-29896. 129. Y. Bourne, B. Bolgiano, D.I. Liao, G. Strecker, P. Cantau, 0. Herzberg, T. Feizi, C. Cambillau, Nature Struct. Biol. 1994, I , 863-870. 130. Y.D. Lobsanov, M.A. Gitt, H. Leffler, S.H. Barondes, J.M. Rini, J. Biol. Chem. 1993, 268, 27034-27038. 131. D.D. Leonidas, E.H. Vatzaki, H. Vorum, J.E. Celis, P. Madsen, K.R. Acharya, Biochemistry 1998, 37, 13930-13940. 132. T. Shirai, C. Mitsuyama, Y. Niwa, Y. Matsui, H. Hotta, T. Yamane, H. Kamiya, C. Ishii, T. Ogawa, K. Muramoto, Structure 1999, 7, 1223-1233. 133. A.P. May, R.C. Robinson, M. Vinson, P.R. Crocker, E.Y. Jones, Mol. Cell. 1998, I , 719-728. 134. P.E. Stein, A. Boodhoo, G.D. Armstrong, L. Heerze, S.A. Cockle, M.H. Klein, R.J. Read, Nut. Struct. Biol. 1994, 1, 591-596. 135. E.A. Merritt, S. Sarfaty, F. Van Den Akker, C. L’hoir, J.A. Martial, W.G.J. Hol, Protein Sci. 1994, 3, 166-175. 136. T.K. Sixma, S.E. Pronk, K.H. Kalk, B.A.M. Van Zanten, A.M. Berghuis, W.G.J. Hol, Nature 1992, 355, 561-564. 137. H. Ling, A. Boodhoo, B. Hazes, M.D. Cummings, G.D. Armstrong, J.L. Brunton, R.J. Read, Biochemistry 1998, 37, 1777-1788. 138. S. Swaminathan, W. Furey, J. Pletcher, M. Sax, Nature Struct. Bid. 1995, 2, 680-686. 139. T. Stehle, S.C. Harrison, Structure 1996, 4 , 183-194. 140. T. Stehle, S.C. Harrison, EMBO J. 1997, 16, 5139-5148. 141. N.K. Sauter, J.E. Hanson, G.D. Glick, J.H. Brown, R.L. Crowther, S.J. Park, J.J. Skehel, D.C. Wiley, Biochemistry 1992, 31, 9609-9621. 142. S. Steinbacher, U. Baxa, S. Miller, A. Weintraub, R. Seckler, R. Huber, Proc. Nut. Acad. Sci. USA 1996, 93, 10584-10588.
Carbohydrates in Chemistry and Biology Editor Beat Ernst, Gerald W. Hart, Pierre Sinay copyright OWILEY-VCH Verlag GmbH, 2000
35 Transfer NOE Experiments for the Study of Carbohydrate-Protein Interactions Thomas Peters
35.1 Introduction Many biological recognition events are based upon specific protein-carbohydrate interactions. For instance, self non-self discrimination in immune reactions, bacterial and viral adhesion to mammalian target cells as well as key steps in inflammatory processes involve protein-carbohydrate recognition as the heart of the matter at the molecular level [l-51. The broad interest in a detailed understanding of complexation reactions between carbohydrate ligands and corresponding receptor proteins certainly originates from considerations that go well beyond mere scientific questions. In most cases the biological recognition phenomenon is potentially coupled to pathophysiological conditions that could be cured by inhibition of the particular protein-carbohydrate interaction. Therefore, a strong driving force for research projects that address the molecular nature of protein-carbohydrate recognition reactions stems from potential pharmaceutical applications. A good example that has attracted much attention during recent years is the interaction between selectins and their carbohydrate ligands because these complexation reactions represent the first steps in the inflammatory cascade [6-lo]. In pathological situations such as myocardial infarction, transplantation, or rheumatoid arthritis, efficient suppression of the inflammatory cascade is very important. The design of potent inhibitors obviously requires precise knowledge about the underlying binding reaction, and more specifically about the bioactive conformation of the carbohydrate ligand [ 11-17]. In the case of the selectins, an X-ray structure of the carbohydraterecognition domain of E-selectin has been published [ 181. Attempts to co-crystallize E-selectin with its “natural” ligand, the sialyl Lewis’ tetrasaccharide (Figure 1), failed so far. Knowledge about the bioactive conformation of sialyl Lewis’ came from NMR spectroscopy utilizing experiments that are based upon the so called transfer NOE (trNOE) effect [ 19-23]. In general, the observation of transfer NOES allows an analysis of bound ligand conformations without requiring any knowledge about the complex protein NMR spectrum (for a comprehensive explanation of the trNOE effect see [24]).
1004
35 Transfer NOE Experiments
Neu5Ac
HO
Gal
~
GlcNAc
LOH
NHAc
OH
-OH HO
OH
Fuc
Figure 1. Chemical formula of sialyl Lewis' (sLeX),a-~-Neu5Ac-(2+3)-b-~-Gal-( 1 +4)-[a-~-Fuc(1+3)]-!3-D-GlcNAc.
35.2 The Transfer NOE Experiment Transferred NOEs were originally observed and their principles described more than twenty years ago [26, 271. The experiment is based on a chemical exchange between ligand molecules in solution and ligand molecules bound to large proteins. Various experimental implementations have been explored since then, ranging from one dimensional selective saturation transfer experiments [27, 281 to one and two dimensional transient NOE experiments [29, 301. The chemical exchange between free and bound ligand molecules leads to an averaging of physical properties characteristic for the free and for the bound state, respectively. One important physical parameter that distinguishes free and bound molecules is the so called motional correlation or tumbling time, zc. The motional correlation time zc is defined as the time that is required for a molecule to advance by one radian during translational and rotational diffusion. It is apparent that small molecules have short correlation times z, whereas large molecules such as high molecular weight proteins have long correlation times z,. As an approximation, Equation 1 relates the motional correlation time zc to the molecular weight of the molecule in question [24]:
with M , representing the molecular weight in kDa. This equation assumes isotropic overall molecular motion and neglects internal molecular motions but it is usually sufficient for a first estimate of z,. It is of fundamental importance to realize that the correlation time zc determines sign and size of the NOE [24]. Low or medium molecular weight molecules (MW < 1-2 kDa) have short tumbling times zc, and as a consequence such molecules exhibit positive NOEs, no NOEs, or very small negative NOEs depending on their molecular weight, shape and the field strength
35.2 The Transfer NOE Experiment
I
I
I
1005
I
small molecules
-0.5
-
-1.0
-
large molecules
0.1
1
10
100
Figure 2. Dependence of the NOE enhancement (q) on the motional correlation zc under steady state conditions [24]. The spectrometer frequency w was set at 500 MHz. The size of tetra- and pentasacchdrides usually leads to molecular tumbling times z, that cause the NOE to become zero or very close to zero.
applied. Large molecules such as proteins, polysaccharides or nucleic acids have long tumbling times T ~ ,and therefore exhibit strong negative NOEs reaching -100% at the extreme. The dependence of the NOE on the tumbling time and the field strength is depicted in Figure 2. It is obvious that the motion of a small molecule that binds to a large molecule is determined by the long correlation time of the large molecule during the life time of the complex. Upon dissociation of the complex the small ligand tumbles again with its own characteristic correlation time. During a normal NOESY experiment the large negative NOEs of the bound ligand will be transferred to the sharp signals of the free ligand. The NOEs that are observed under such circumstances are called transferred NOES (trNOEs). It is obvious that trNOEs contain information that is necessary to determine the conformation of the bound ligand, the so called bioactive conformation. One advantage of trNOE experiments is that they usually work at optimum with a ten to twenty fold excess of ligand, making it very easy to observe signals of free ligand molecules against the background of broad protein signals. The reason for the fact that the optimum is reached at excess ligand concentrations is inequality 2 [24]: Nb X CJb
>> Nf
X Of
(2)
and Nf are the number of ligand molecules in the bound (b) and the free (f) state, respectively. Cq, and q designate the cross relaxation rates of the ligand in the
Nb
1006
35 Transfer NOE Experiments
bound and free state. Cross relaxation rates are mainly a function of r p 6 , with r being the distance between two protons, and of the motional correlation time T~ (Equation 1). They determine sign and size of the NOE (cf. Figure 2). Since cross relaxation rates of large biomolecules such as proteins are much larger than cross relaxation rates of small molecules, inequality 2 is usually satisfied for a 10-20-fold excess of ligand. In fact, ofsometimes is so small that it does not play any significant role. This is the case for the turning point in Figure 2, where no NOEs can be observed. Usually, tetra- and pentasaccharides are of such a molecular weight (Equation 1) that NOEs are very close to zero. It is important to realize that the observation of trNOEs is only possible under certain conditions. In general, it can be said that trNOEs are only observable for to complexes with &-values approximately in the range between It follows that trNOE-experiments are ideally suited for the investigation of proteincarbohydrate complexes which are usually characterized by low binding affinities [31]. The reason for the limitations of the observation of trNOE lies in the nature of the NOE itself. Dipole-dipole relaxation is the source of NOEs, and therefore the observation of trNOEs depends critically on the kinetics of the associationdissociation process of a protein-carbohydrate complex [ 32-35]. In general, it is required that the dissociation of the complex is fast on the relaxation time scale. Whether the exchange process is fast or slow on the chemical-shift time scale plays no role for the observation of trNOEs, because it is always the signal of the free ligand that carries the information about the bound state. In other words, it is critical that a large amount of ligand molecules samples the binding pocket of the protein frequently enough during the mixing time of the NOESY experiment. This is visualized in Figure 3 . In practice, exchange frequencies of 50-100 Hz are sufficient to permit the observation of trNOEs.
35.3 Measurement of trNOEs The measurement of trNOEs is performed with exactly the same pulse sequences that are used for the measurement of NOEs. Therefore, the NOESY experiment is well suited to record trNOEs. As pointed out above, the right conditions for the measurement of trNOEs are found by varying the carbohydrate-protein ratio. This is usually done in a titration starting at ligand-protein ratios of approximately 4: 1 (for an example see [36])and than increasing the ratio until maximum trNOEs are observed. During titration it is usually sufficient to perform one-dimensional NOE experiments such as 1D-NOESY in order to save time. The mixing times used for the observation of trNOEs are much smaller than the mixing times required to obtain optimum NOEs for di-, tri-, or tetrasaccharides. This is illustrated in Figure 4 that displays typical NOE-curves in comparison to corresponding trNOE curves for the disaccharide a-~-Fuc-( 1-i6)-fL~-GlcNAc-OMefree and bound to Aleuria aurantia agglutinin (AAA) [37].
35.3 Measurement of trNOEs
1007
\
Figure 3. Schematic representation of the transfer NOESY (trNOESY) experiment. Ligands have to sample the binding site of the protein (shown in red) often enough during the mixing time in order to generate a transfer of NOES. It follows that dissociation of the protein-carbohydrate complex must be fast on the relaxation time scale. With mixing times ,z usually being 50-300 ms for trNOESY experiments, dissociation rates of ca. 50-100 Hz are sufficient.
Although trNOE-experiments are performed with a large excess of ligand the protein background obstructs the resulting NOESY spectra. Especially if a quantitative evaluation of trNOEs is desired, techniques should be applied to remove unwanted protein signals. The optimum solution is the use of a spin-lock filter that is applied after the first n/2 pulse in the NOESY sequence [38].During this time the protein signals undergo fast transverse relaxation whereas signals of the ligand survive because their transverse relaxation rates are negligible. If a quantitative analysis of trNOEs is planned trNOEs should be measured at different mixing times leading to trNOE curves (for an example see [39]). The trNOE step can also be combined with other types of NMR-experiments. For instance, the use of 3DTOCSY-trNOESY experiments and one-dimensional versions thereof has been demonstrated to relieve signal overlap [20]. Especially powerful is the combination of heteronuclear correlation experiments with trNOESY, such as the 3D-HSQCtrNOESY experiment [23]. Unfortunately, this usually requires the availability of 13C-labelledcarbohydrate ligands (see discussion below) that are time consuming and expensive to synthesize [23, 40-421. In general, any multi-dimensional NMRexperiment that contains a NOESY step can be used to detect trNOEs during this step. Once experimental trNOEs have been measured, a bioactive conformation may be derived.
1008
35 Transfer NOE Experiments
NOE in
t
n
eu
8Z
o -in
-20
-30
Figure 4. NOEs for free u-~-Fuc-( 1+6)-fi-~-GlcNAc-OMe (upper half of the graphics), and trNOEs for c~-L-Fuc-( 1+6)-p-~-GlcNAc-OMein the presence of Aleuriu uuruntiu agglutinin (lower half of the graphics), measured at 600 MHz as a function of the mixing time z,. Circles refer to proton pairs H6proRG'cNAc-H6pr~SGlcNAc and diamonds to H 1Fuc-H6pr~SG'cNAc , respectively. Precise experimental conditions have been described elsewhere [37]. Maximum NOEs for the free disaccharides are found around mixing times z, of 1000 ms, whereas maximum trNOEs are reached at mixing times lower than 300 ms.
35.4 Bioactive Conformations of Carbohydrate Ligands From trNOE Experiments As explained above, trNOEs carry the information about the geometry of the bound state of a ligand molecule binding to a receptor protein. Therefore, the bioactive conformation may be back-calculated from observed trNOEs. This analysis can be performed on different levels ranging from qualitative approaches to highly sophisticated protocols that take into consideration e.g. the binding kinetics and all protons of the receptor-protein binding site and the carbohydrate ligand (for recent reviews on these techniques see: [43, 441). The most simplistic approach would be to decide on the basis of the absence and/or presence of inter-glycosidic trNOEs which conformation is bound. From the examples presented in the following, it will become clear that this may easily lead to wrong conclusions mainly because of the probability of so called spin-diffusion processes where NOEs or trNOEs occur between protons that are not necessarily close in space. Only if spin-diffusion has been excluded by specific NMR experiments, this simple procedure is allowed. A semi-
35.5 Spin Difusion may Generate Misleading Distance Constraints
1009
quantitative method requires the acquisition of trNOE build-up curves that have been corrected for spin-diffusion. Since the initial slope of such buildup-curves is easily translated into inter-proton distances by the so called initial slope approximation (ISPA) [45] bound conformations can be deduced. Two circumstances limit the straightforward translation of initial slopes of trNOE build-up curves into threedimensional structures. First, inter-glycosidic trNOEs are sparse, and therefore several conformations may fulfill the observed trNOEs. Second, if more than one conformation is bound by the receptor protein “virtual” bioactive conformations may be obtained. In order to interpret data sets that are incomplete in terms of unambiguous structural information experimental restraints may be combined with MD or MMC simulations (see for instance [19, 22, 23, 461. Following such protocols a range of possible, or “allowed” conformations will be available. If a model for the carbohydrate-protein complex is accessible from X-ray data, or from e.g. homology modelling, extended protocols may be applied. One such approach is implemented in the program CORCEMA ([44] and references cited therein) that is based on full-relaxation matrix calculations taking into account the binding kinetics, and all protein protons that are located in the binding pocket of the protein. Unfortunately, often the structural information required for performing such complex calculations is not available.
35.5 Spin Diffusion may Generate Misleading Distance Constraints For large molecules such as proteins magnetization transfer in NOESY experiments not only takes place between protons that are close in space. A transfer of magnetization may also occur between protons that are separated by far larger distances than usually required for the observation of NOES. The reason is a phenomenon called spin diffusion which allows magnetization between two protons to be transferred via relay protons [24]. Spin diffusion becomes of increasing importance with increasing molecular weight and with increasing mixing times. Therefore, spin diffusion also plays an important role for the observation of trNOEs. It is clear that trNOEs that are due to spin diffusion cannot be used as distance constraints in the generation of bioactive conformations. To avoid spin diffusion short mixing times of less than 50 ms are required if no other precautions are taken. But usually at such low mixing times the intensity of trNOEs is so low that no meaningful analysis may be performed. An example will illustrate the misleading effects of spin diffusion artefacts in trNOESY spectra. The bioactive conformation of a (1 +6)-linked disaccharide, methyl 6-O-p-~galactopyranosyl-4-deoxy-2-deutero-4-fluoro-~-~-galactopyranoside bound to the Fab fragment of an anti-(1+6)-P-~-galactopyrananantibody had originally been analyzed on the basis of trNOEs that were not corrected for spin diffusion [47]. It was concluded that a significant conformational change about the (1+6)-glycosidic linkage occurs upon binding. The resulting bound conformation was not found in aqueous solution without the protein present. Later, the authors identified the key
1010
35 Transfer NOE Experiments
trNOEs that lead to the unusual bioactive conformation as spin diffusion artefacts [48]. They reached this conclusion by applying trROESY experiments and comparing the results to the trNOESY spectra. In trNOESY spectra spin diffusion leads to cross peaks that are of the same sign as cross peaks that are due to direct dipolar contacts, and therefore direct dipolar interactions cannot be distinguished from indirect (spin diffusion) interactions. In trROESY spectra cross peaks from indirect magnetization transfer are of opposite sign compared to direct effects, assuming that one “relay” proton mediates the indirect transport of magnetization. If more than one proton mediates spin-diffusion the sign of the corresponding cross peak changes in an alternating manner, but at the same time significantly losing intensity. Therefore, in such cases usually no cross peaks are observed. It follows that the detection of trROEs of opposite sign or of very weak intensity at positions where trNOEs were observed indicates spin diffusion. This is shown schematically in Figure 5a. In the case described above, trROE experiments showed that the critical cross peaks were due to spin diffusion, and therefore the model for the bound conformation had to be revised. Other groups found similar results [49, 501, and in general it can be stated that testing for spin-diffusion contributions is essential for the eluci-
ROESY
NOESY
-,*--A A
hA [
<3A
<3A
C
jc
AB
L
-v
,,_ _
AB
i C
-C
Figure 5. Schematic representation of three spins, A , B, and C interacting via direct dipolar contacts (black arrows) and/or spin diffusion (dashed arrows). a) Trace of a 2D NOESY spectrum (right) and a corresponding trace from a 2D ROESY spectrum (left). Spins A and B are close in space, and spin diffusion mediates magnetization transfer between protons A and C, that are not close in space. The 2D ROESY experiment allows unambiguous discrimination between direct and spin diffusion effects. b) Protons A and C are close in space, and in addition magnetization is transferred between the two protons via spin diffusion. The effect is a cancellation of the 2D ROESY signal. A cancellation can also occur if indirect magnetization transfer involves more than one relay proton (proton B in this case). Therefore, a discrimination between spin diffusion and direct dipolar interaction is not possible in this case.
35.6 The Conformation of Sialyl Lewis" Bound to E-selectin
1011
dation of bioactive conformations of carbohydrates (and other ligands). TrROESY experiments are a suitable tool to perform this test, but special constellations may exist that require additional experiments. In such cases direct and indirect (spin diffusion) dipolar interactions interfere and lead to a cancellation of trROEs (see Figure 5b) [39]. This observation would mimic the presence of spin diffusion and corresponding distance constraints would be removed, again leading to a false bioactive conformation. The problem here is to separate direct and indirect (spin diffusion) magnetization transfer. It has been shown, that so called QUIET-trNOESY experiments are well suitable for this task [51]. In these type of experiments spins of interest are inverted during the mixing time leaving only their mutual direct dipolar interaction and cancelling all other magnetization transfers [521. For small ligands such as carbohydrates binding to large receptor proteins such as antibodies the main source of spin diffusion are protons that are attached to the binding site of the protein [35, 39, 48, 51, 531. Therefore, band-selective inversion of the ring-proton region usually leads to spectra without spin-diffusion contributions from aliphatic or aromatic protein side chain protons [39]. Figure 5 displays a scheme that summarizes the occurrence of cross-peak patterns in the different type of experiments discussed above. Examples will illustrate the capabilities of the methodology described above. No attempt was made to review the field in total since several very good review articles have appeared during the recent years [54-56].
35.6 The Conformation of Sialyl LewisX Bound to E-selectin Several studies have been performed to elucidate the conformation of the sialyl Lewis' (sLe') epitope bound to E-selectin [ 19-23]. The selectins are membrane bound glycoproteins that are involved in the initial steps of the inflammatory cascade [6-lo]. It has been shown that these proteins mediate rolling of leukocytes by binding to the sLeXepitope on the surface of leukocytes. Clearly, this provides a key point of therapeutic interaction in the case of inflammatory diseases. In order to design potent mimics of sLeXthe bioactive conformation of the molecule must be known. In solution, the tetrasaccharide sLeXadopts several conformational states as this has been shown by many conformational analysis studies [ 19-23, 57-59]. Two main conformational families may be distinguished due to two different major orilinkage as this is depicted in Figure 6. entations of the a-~-NeuSAc-(2+3)-P-~-Gal The two conformational families are characterized by two inter-glycosidic NOES H3-Gal/H3ax-NeuSAc and H3-Gal/H8-NeuSAc, that are mutually exclusive. The precise distribution of the two orientations and the presence of other local minima in aqueous solution is still a matter of dispute. Upon binding to E-selectin only one of the two major conformational families generated by different orientations of the a-~-Neu5Ac-(2+3)-P-~.Gallinkage is recognized. The bound conformation is characterized by the observation of a trNOE between H3-Gal and HS-NeuSAc, whereas no interaction is observed between H3-Gal and H3ax-
1012
35 Transfer NOE Experiments
NeuSAc (Figure 6). This global result has been found by all conformational analyses of sLex bound to E-selectin performed so far [ 19-23]. Different protocols have been applied for the analysis of trNOEs, and therefore one would predict that different values are reported for the precise orientation of the a-~-NeuSAc-(2+3)-p-~Gal linkage in the bound state of sLe". Indeed, different values were found but nevertheless the gross orientation is similar in all cases. The latest study reported for the bioactive conformation of sLeX utilized fully I3C-labeled sLeX [23]. The 13C-enrichment made it possible to perform 3DNOESY-HSQC spectra. From these spectra additional inter-glycosidic trNOEs were extracted that cannot be obtained from homonuclear spectra because of severe spectral overlap. These additional trNOEs represent extra restraints for the con1-+4)-p-~-GlcNAc linkage and the a-~-Fuc-( 1+~)-P-Dformations of the p-~-Gal-( GlcNAc linkage but not for the a-~-Neu5Ac-(2-.3)-P-~-Gallinkage. Therefore, this study provides more integral data for the bioactive conformation of the Le" fragment than previous studies. The glycosidic torsion angles of the different bioactive conformations of sLeXbound to E-selectin reported in the studies mentioned above are compiled in Table 1. Stereo pictures of the different bioactive conformations of sLeXfrom Table I are shown in Figure 7. It has been shown that for biological activity the relative orientation of the fucose and the neuraminic acid residue are relevant [ 11, 121. Following this guide line, a potent mimic of sLeXhad been synthesized [ 11-13]. A conformational analysis of this sLeXmimic in the free and bound state [60] clearly showed that the bioactive conformation is very similar to the one derived originally for sLeX bound to E-selectin [19]. In none of the studies published so far the protein protons in the binding pocket were taken into account. For a more precise analysis this would be mandatory but it would also require knowledge about the orientation of the tetrasaccharide in the binding pocket. This information is currently not available, although several models for the binding of sLeXto E-selectin have been proposed. For a complete understanding of the carbohydrate recognition by E-selectin and consequently as a prerequisite for designing new drugs that block the sLeX/E-selectininteraction it would clearly be desirable to have a better knowledge about the orientation of sLeXin the binding pocket of E-selectin. It is interesting to compare the bioactive conformation of sLeXwhen binding to different receptor proteins. For instance, a different orientation of the a-~-NeuSAc(2+3)-p-~-Gallinkage was postulated for sLeXbound to L-selectin [22]. The biological implications of this finding remain to be elucidated, but certainly it would be interesting to collect more experimental data that demonstrate the presence of distinct bioactive conformations of the sLex ligand when binding to different receptor proteins. For instance, what will happen if totally different (from E-selectin) "carbohydrate recognition" proteins are employed? We therefore studied the binding of sLeXto the lectin Aleuria aurantia agglutinin (AAA) [61], a lectin from orange peel mushroom, that specifically recognizes fucose residues. Since this lectin is known to have binding specificity for fucose one could well assume that the Neu5Ac part of sLeXretains its flexibility even in the bound state, as this had been reported for other saccharide-protein complexes [50, 371. Surprisingly, this is not the case. Probably
I \
HO OH
1
OH
v,
H8-Neu5Ac
OH
NHAc
Figure 6. Two conformations of sLeXrepresenting the two major conformational families resulting from different orientations around the a-~-NeuSAc-(2+3)-P-~-Gal glycosidic linkage. Mutually exclusive inter glycosidic NOES occur.
~ 3 4 - 3 ~ 1 H3ax-Neu5Ac
/
HO
F
rl
3
3c
r:
$F'
Y
5
1014
35 Transfer NOE Experiments
Table 1. Torsion angles at the glycosidic linkages of bioactive conformations of sLeX bound to E-selectin 119, 22, 231 and to Aleuriu uuruntiu agglutinin (AAA) [61]. Reference
N(2+3)G
G( 1+4)GN
~~~
~ 9 1 [221 ~ 3 1 [611
-76"/+@ -58"-22" -43"/- 12" -61"/-4"
F(1+3)GN ~~
+39"/+12" +24"/+34" +45"/$19" +67"/+17"
+38"/+2@ +71"/+ 14" +29"/+4l" -25"/-28"
Figure 7. Different bioactive conformations of sLeX bound to E-selectin as reported in the literature [19, 22, 231. The bioactive conformation of sLeX bound to Aleuriu uuruntiu agglutinin (AAA) is also shown [61]. It is obvious that the latter conformation differs from the other conformations in the orientation of the fucose residue (compare Table 1).
because of unfavourable steric interactions with protein side chains, the Neu5Ac residue adopts a conformation that belongs to the same conformational family that also embraces the conformation of sLeXbound to E-selectin. As pointed out above, the inter-glycosidic trNOE H3-Gal/H8-NeuSAc is characteristic for this conformation [see Figure 61. Inspecting the data that are available for the bioactive conformation of sLeXbound to E-, L- or P-selectin [ 19-23] it is clear that in all of these conformations the galactose and the fucose pyranose rings have a stacked orientation giving rise to certain characteristic inter-glycosidic NOES H2-Gal/HS-Fuc, H2Gal/H6-Fuc, and H1-Fuc/NAc-GlcNAc. This orientation also corresponds to the
35.6 The Conformation of Sialyl Lewisx Bound to E-selectin
1.5
I
H3axN-
2.0 2.5
I
H2"
/
B
' f NHAc/
A
NHAc
1015
-' H3axN+ ,
B
3.0 3.5 4.0
ppm
5.0 4.5 4.0 3.5 3.0 2.5 2.0
1.5
5.0 4.5 4.0 3.5 3.0 2.5 2.0
1.5
Figure 8. 500 MHz NOESY spectrum of sLeX(left, 900 ms mixing time) compared to a 500 MHz trNOESY spectrum of sLe" bound to AAA (right, 150 ms mixing time). It is obvious that a number of inter glycosidic trNOEs are not observed (gray circles) where NOEs were observed (left). Important to notice is the absence of signal intensity for the contact between H2-Gal (H2G)and H5Fuc (HsF), a contact that would indicate stacking of fucose and galactose. An inter glycosidic trNOE is observed between protons HI-Fuc and H2-Gal. This trNOE is difficult to locate at the present expansion of the spectrum but the effect is clearly visible when inspecting the corresponding part of the trNOESY spectrum [61].
global minimum energy region of this glycosidic linkage, and is prevalent in aqueous solution. It follows that the hydrophobic side of the fucose residue is shielded from potential interactions with a protein, and it can be hypothesized that a fucosebinding lectin such as AAA will not recognize a fucose residue that is so well shielded. From trNOE experiments it is evident that AAA recognizes the fucose residue in sLex, but in a conformation that is considerably different from the global minimum and that does not obey the exo-anomeric effect. From the arguments given above this is understandable because recognition of the fucose would be hampered by the presence of the galactose-fucose stacking in the global energy minimum. The experimental evidence that led to this conclusion is clear cut. Upon binding to AAA the NOEs characteristic for the global minimum disappear. Instead, a trNOE is observed between H1-Fuc and H2-Gal (a NOESY and a trNOESY spectrum displaying the major features discussed are shown in Figure 8). This in1+3)-p-~-GlcNAc dicates that the above mentioned local minimum at the a-~-Fuc-( linkage is selected upon binding to AAA. The torsion angles at the (1+3)-glycosidic linkage are around $I = -25" and \1, = -28" (compare Table 1 and Figure 7). Since usually conformations that are recognized by proteins are "preformed" in aqueous solution we reinvestigated the solution conformation of sLeXand LeX to find out whether this "non-exo-anomeric" conformation is also present in aqueous solution. The presence of this minimum had been predicted recently on the basis of
1016
35 Transfer NOE Experiments
theoretical M D simulations and has also been discussed in a study that compared the conformational features of the disaccharide a-~-Fuc-( 1+3)-p-~-GlcNAc to its thio-analog [46]. But so far, no direct experimental evidence was available for the presence of this local minimum. The minimum is also predicted by MMC simulations that we performed for sLeXand Le". An accurate conformational analysis of LeXin aqueous solution reveals that the inter-glycosidic NOE between H1-Fuc and H2-Gal is indeed present although with a rather low intensity. For sLeXwe did not detect this NOE, probably because of the larger molecular weight and the charge of sLeXthat renders very weak negative NOES, as this was reported before [20, 57, 581. Nevertheless, our experiments allow the conclusion that a small portion of Le" and most likely of sLeXin aqueous solution is "preformed" in the local minimum conformation with dihedral angles close to = -25" and \Ir = -28". These findings clearly question the concept of rigidity for the LeXtrisaccharide core structure, and suggest that carbohydrate chains in general may carry different biological information that is encoded by different potential bioactive conformations. Depending on the cognate receptor protein different "conformational information" may be read out.
+
35.7 Interaction of Bacterial Lipopolysaccharide Fragments with Monoclonal Antibodies Much attention has been paid during the past ten years to the specific recognition of carbohydrate epitopes by monoclonal antibodies (mAbs) [62-661. Understanding these reactions at a molecular level will certainly help in generating new perspectives in diagnosis and therapy of related diseases. In our laboratory, we studied the interaction of synthetic lipopolysaccharide fragments with corresponding mAbs with NMR [39, 671. Several mAbs were generated against synthetic and isolated fragments of lipopolysaccharides (LPS) that are present on the surface of chlamydial bacteria [68].These parasites are responsible for a variety of diseases in humans and animals. During infection, antibodies are expressed against components in the outer membrane, with LPS as one of the major surface antigens. Here, we will focus on the disaccharide element a-Kdo-(2-+4)-a-Kdo that constitutes a common structural element of the core region of Gram-negative bacterial LPS in general [69].The conformational features of the binding of a-Kdo-(2+4)-a-Kdo-(2+O)-allyl (Figure 9) to two mAbs S25-2 and S23-24 will be described [39]. Compared to S25-2, S23-24 binds to disaccharide a-Kdo-(2+4)-a-Kdo-(2+O)-allyl with approximately 50-fold increased affinity, and the question arises if this also is reflected by different bioactive conformations that this disaccharide adopts binding to the two mAbs. It turned out that the acquisition of QUIET-trNOESY spectra was mandatory because interference of direct and spin-diffusion mediated magnetization transfer was observed. This is demonstrated in Figure 10 that depicts parts of trNOESY, trROESY and QUIET-trNOESY spectra. The critical cross peaks indicate short distances between protons attached to the carbon atom C8 of ring b and protons
35.7 Interaction of Bacterial Lipopolysaccharide Fragments
s/ 1
OH
HO
.
H
0 .
O
T
coo-
o
1017
H
coo-
..
1.
Figure 9. Chemical formula of the disaccharide a-Kdo-(2+4)-a-Kdo-(2+0)-allyl.
attached to C3 of ring c. The trROESY experiment suggests that these interglycosidic trNOEs are due to spin-diffusion because no intensitiy is observed at the critical positions (Figure lob). In contrast, QUIET-trNOESY experiments clearly show that only part of the interaction is due to spin diffusion (Figure 1Oc). The experiments show that direct dipolar interactions and spin diffusion occur at the same time, giving misleading results for the trROESY experiments. From a variety of NMR experiments it was concluded that protein protons are the major source of spin diffusion, and therefore doubly band selective experiments were performed where the regions of ring protons of 4.10-3.60 ppm and 2.17-1.67 ppm were inverted simultaneously during mixing. Buildup curves from such QUIETtrNOESY experiments were measured and translated into bioactive conformations using restrained MMC simulations. For a-Kdo-(2+4)-a-Kdo-(2+0)-allyl binding to S25-2 the analysis results in a rather limited part of conformational space that contains the bioactive conformation as this is shown in Figure 11. For the other antibody, mAb S23-24, it is impossible to deduce a single bound conformation. Obviously, this antibody recognizes two different bioactive conformations, with one being similar to the global minimum A (Figure l l ) of disaccharide a-Kdo-(2+4)-a-Kdo-(2+O)-allyl and the other being rather close to the conformation C (Figure 11) that is bound by the other antibody S25-2. The bioactive conformations found are in good agreement with the binding data. The antibody with higher affinity binds to a conformation that is highly populated in aqueous solution. This study underlines that one carbohydrate epitope may be recognized in different bioactive conformations by different receptor proteins (Figure 12). These results emphasize that the recognition of different bioactive conformations is an essential component of protein-carbohydrate recognition reactions. Indeed, other examples for this phenomenon have recently been published [66, 70, 711. As one example, the reader's attention should be drawn to studies that targeted the binding of C-glycosides to glycosidases. Using e.g. C-lactose as a non-hydrolizable substrate for Escherichia coli-p-galactosidase, it was shown that the enzyme binds to a high energy local minimum that represents the so called anti-conformation around the pseudo-glycosidic linkage [70]. From crystallographic studies it followed
1018
35 Transfer NOE Experiments
2.0
3axc 3eq"
c?
2.0
c?
C?
3
3ax" 3eq"
b I
8
2.0
Bax" t3eq"
c
0
4.2
4.0
3.8
3.6
Figure 10. Parts of 2D trNOESY (a), 2D trROESY (b), and 2D QUIET-trNOESY (c) of a-Kdo(2+4)-a-Kdo-(2+O)-allyl bound to mAb S25-2. The QUIET-trNOESY experiment was recorded with a 15 ms double-band selective 4 3 inversion pulse (inversion of regions 4.10-3.60 ppm and 2.17-1.67 ppm). Peaks within the inverted regions show an opposite sign (bold lines, c ) relative to the other cross peaks outside these regions. The mixing time was 250 ms for all experiments. A comparison of the spectra allows identification of spin-diffusion effects. Cross-peaks that are cancelled in the trROESY spectrum because spin diffusion and direct dipolar interactions take place at the same time (see discussion in the text) are marked with circles in the 2D trROESY spectrum (b). Reprinted with permission from Biochemistry [39].
that C-lactose binds to peanut lectin in a conformation that corresponds to the minimum energy conformation of 0-lactose [72]. Therefore, the selection of a high energy conformation of C-lactose by E. coltp-galactosidase may promote the cleavage of the glycosidic linkage by the enzyme [70]. At the present, there are too few examples to draw meaningful general conclusions about the biological significance of the recognition of different carbohydrate conformations by different receptor proteins. Certainly, in the future more such data will be collected for other biological relevant cases, and it will be a challenge to link the different bioactive conformations to different biological functions.
35.8 Conclusions and Future Directions -1 80 -
w
0
7
0
-180 180
180
-180
7
l
180
180
r ----I
I30-50% ---1 I50-70% 180
180 -180 180
0
1019
0
Figure 11. Contour plots showing the relative population of conformational space around the (2+4)-glycosidic linkage in a-Kdo-(2+4)-a-Kdo-(2iO)-allyl. The +/$-maps were divided into bins of lo" in and +-direction, and the number of conformations in each bin was counted. Then, contour levels were calculated relative to the highest populated bin (global minimum). The contour levels are color coded. Magenta encodes 1-10%, dark blue 10-30%, light blue 30-50%0, green 5070%, yellow 70-90%0, and red more than 90% of the number of conformations in the highest populated bin. (a) MMC simulation at 600 K. (b) MMC simulation at 2,000 K. (c) and (d) represent possible conformations of a-Kdo-(2+4)-u-Kdo-(2~O)-allyl bound to mAb S25-2. (c) all conformations from the MMC simulation at 2,000 K that satisfy only positive distance constraints (0.15% of the total number of conformations from the MMC simulation). (d) all conformations that satisfy positive and negative distance constraints (0.075% of the total number of conformations). Reprinted with permission from Biochemistry [39].
+-
35.8 Conclusions and Future Directions A major consequence from the studies reported above, and from other examples from the literature, it can be stated that the inherent flexibility of oligosaccharides as compared to e.g. globular proteins allows to encode different biological information for recognition reactions in a parallel manner. So far it appears that binding affinities for the recognition of a certain conformation parallel with the potential
1020
35 Transfer NOE Experiments
B
D
Figure 12. Stereo images of a-Kdo-(2+4)-a-Kdo-(2-+O)-allyl (relaxed view); (A) global minimum A; (B) local minimum B; (C) conformation C that is recognized by mAb S25-2; (D) local minimum D. Reprinted with permission from Biochemistry 1391.
energy of the conformation in aqueous solution, not bound to a protein. The way how nature takes advantage of this phenomenon is not known yet. In order to establish links between different bioactive conformations and biological functions, it will be important to study the thermodynamics and kinetics of the binding reactions in parallel. Microcalorimetry [31, 651 and surface plasmon resonance [e.g. 731 provide powerful tools to collect such data. For more detailed structural analyses it will be important to make use of new techniques that have emerged in protein NMR spectroscopy. The main breakthroughs are probably the introduction of TROSY spectroscopy [74], extending the size limit of biological macromolecules that can be subjected to NMR analyses, the creation of so called cross correlation experiments [75] that allow the direct measurement of dihedral angles, and the measurement of residual dipolar couplings [76] that complement data from NOESY spectroscopy. Unfortunately, all of these techniques require, or at least benefit from isotope enrichment which remains a difficult and expensive task for carbohydrates. Consequently, only few examples utilizing e.g. 13C-labelledcarbohydrates have been published so far [23, 40-421. It is
References
1021
obvious that the new tools that have been discovered for NMR spectroscopy will also greatly promote the analysis of protein-carbohydrate interactions. Interesting developments originate from experiments that use the trNOE effect to screen substance libraries for binding activity against receptor proteins. The first attempts to perform such bio-affinity protocols were rather successful [77, 781, and recently a new experimental strategy was introduced that allows a very robust and powerful screening of libraries, called STD-NMR [79, SO]. The new method is based on the principle of saturation transfer and can be combined with virtually any other NMR technique. Whereas trNOE experiments are well established tools to study bioactive conformations, especially of carbohydrate ligands, the use of bio-affinity NMR methods is still in its infants and promises more surprises in the near future.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 1I . 12. 13. 14. 15. 16. 17.
18. 19. 20. 21. 22. 23. 24. 25. 26.
T. A. Springer, Nature, 1990, 346, 425-434. T. Feizi, Curr. Opin. Struct. Biol., 1993, 3, 701-710. A. Varki, Glycobiology, 1993, 3, 97-130. R. A. Dwek, Chem. Rev., 1996, 96, 683-720. P. Sears, C.-H. Wong, Cell. Mol. Lifl. Sci. 1998, 54, 223-252. M. P. Bevilacqua, S. Stengelin, M. A. Gimbrone, Jr., B. Seed, Science, 1989, 243, 1160-1165. L. A. Lasky, Science, 1992: 258, 964-969. A. Levinovitz, J. Muhloff, S. Isenmann, D. Vestweber, J. Cell. Biol., 1993, 121, 449-459. M. Lenter, A. Levinovitz, A,, S. Isenmann, D. Vestweber, J. Cell. Biol., 1994, 125, 471-481. K. L. Moore, N. L. Stults, S. Diaz, D. F. Smith, R. D. Cummings, A. Varki, R. P. McEver; J. Cell. Biol., 1992, 118, 445-456. H. C. Kolb, B. Ernst, Pure Appl. Chem., 1997, 69, 1879-1884. H. C. Kolb, B. Ernst, Chem. Eur. J., 1997, 3, 1571-1578. K. E. Norman, G. P. Anderson, H. C. Kolb, K. Ley, B. Ernst, Blood, 1998, 91,475-483. T. F. Lampe, G. Weitz-Schmidt, C.-H. Wong, Angew. Chem., 1998,110, 1761-1764. T. Uchiyama, V. P. Vassilev, T. Kajimoto, W. Wong, H. Huang, C.-H. Wong, J. Am. Chem. Soc., 1995, 117, 5395-5396. E. E. Simanek, G. J. McGarvey, J. A. Jablonowski, C.-H. Wong, Chem. Rev., 1998, 98: 833862. P. Sears, C.-H. Wong, Angew. Chem. Int. Ed,, 1999,38, 2300-2324; Anyew. Chem., 1999, 111, 2446-247 1. B. J. Graves, R. L. Crowther, C. Chandran, J. M. Rumberger, S. Li, K.-S. Huang, D. H. Presky, P. C. Familletti, B. A. Wolitzky, D. K. Burns, Nature, 1994, 367, 532-538. K. Scheffler, B. Emst, A. Katopodis, J. L. Magnani, W. T. Wang, R. Weisemann, T. Peters, Angew. Chem., 1995, 107, 2034 -2037; Angew. Chem. Int. Ed. Engl., 1995, 34, 1841-1844. K. Scheffler, J.-R. Brisson, R. Weisemann, J. L. Magnani, W. T. Wang, B. Ernst, T. Peters, J. Biomol. N MR , 1997, 9, 423-436. R. M. Cooke, R. S. Hale, S. G. Lister, G. Shah, M. P. Weir, Biochemistry, 1994, 33, 1059110596. L. Poppe, G. S. Brown, J. S. Philo, P. V. Nikrad, B. H. Shah, J. Am. Chem. Soc., 1997; 119, 1727-1736. R. Harris, G. R. Kiddle, R. A. Field, M. J. Milton, B. Ernst, J. L. Magnani, S. W. Homans, J. Am. Chem. Soc., 1999,121, 2546-2551. D. Neuhaus, M. Williamson, The Nuclear Overhauser Efect in Strucutval and Conformational Analysis, VCH Wiley, New York, 1989. P. Balaram, A. A. Bothner-By, J. Dadok, J. Am. Chem. Soc., 1972, 94, 4015-4017. P. Balaram, A. A. Bothner-By, E. Breslow, J. Am. Chem. Soc., 1972, 94, 4017-4018.
1022 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.
35 Transfer NOE Experiments
G. M. Clore and A. M. Gronenborn, J. Mugn. Reson., 1982,48,402-417. G. M. Clore and A. M. Gronenborn, J. Mugn. Reson., 1983,53,423-442. N. H. Andersen, K. T. Nguyen, H. L. Eaton, J. Magn. Reson., 1985, 63, 365-375. N. H. Andersen, H. L. Eaton, K. T. Nguyen, Mugn. Reson. Chem., 1987,25, 1025-1034. E. Toone, Curr. Opin. Struct. Biol., 1994, 4 , 719-728. R. E. London, M. E. Perlman, D. G. Davis, J. Mugn. Reson., 1992, 97, 79-98. F. Ni, J. M a p . Reson., 1992, 96, 651-656. W. Lee, N. R. Krishna, J. Mugn. Reson., 1992, 98, 36-48. H. N. B. Moseley, E. V. Curto, N. R. Krishna, J. Mugn. Reson. Ser. B, 1995, 108, 243-261. F. Casset, A. Imberty, S. Perez, M. Etzler, H. Paulsen, T. Peters, Eur. J. Biochem., 1997, 244, 242-250. 37. T. Weimar, T. Peters, Angew. Chem. Znt. Ed. Engl., 1994, 33, 88-91; Angew. Chem., 1994, 106, 79-82. 38. T. Scherf, J. Anglister, Biophys. J., 1993, 64, 754-761. 39. T. Haselhorst, J.-F. Espinosa, J. Jimenez-Barbero, T. Sokolowski, P. Kosma, H. Brade, L. Brade, T. Peters, Biochemistry, 1999, 38, 6449-6459. 40. M. A. Probert, M. J. Milton, R. Harris, S. Schenkman, J. M. Brown, S. W. Homans, R. A. Field, Tetrahedron Lett., 1997, 38, 5861-5864. 41. D. G. Low, M. A. Probert, G. Embleton, K. Sheshadri, R. A. Field, S. W. Homans, J. Windust, P. J. Davis, Glycobiology, 1997, 7, 373-381. 42. H. Shimizu, J. M. Brown, S. W. Homans, R. A. Field, Tetrahedron, 1998, 54, 9489-9506. 43. F. Ni, Prog. N M R Spectr., 1994, 26, 517-606. 44. N. R. Krishna, H. N. B. Moseley, in Biological Magnetic Resonance, 17: Structure and Dynamics in Protein NMR, Eds. Krishna and Berliner. Kluwer Academic/Plenum Publishers, New York, 1999, 223-307. 45. A. M. Gronenborn, G. M. Clore, Progr. Nucl. Mugn. Reson. Spectrosc., 1985, 17, 1-32. 46. B. Aguilera, J. Jimenez-Barbero, A. Ferandez-Mayoralas, Carbohydr. Res., 1998, 308, 1927. 47. C. P. J. Glaudemans, L. Lerner, G. D. Daves Jr., P. Kovac, R. Venable, A. Bax, Biochemistry, 1990,29, 10906-10911. 48. S. R. Arepalli, C. P. J. Glaudemans, G. D. Daves Jr., P. Kovac, A. Bax, J. Mugn. Reson., 1995, 106, 195-198. 49. T. Weimar, S. L. Harris, J. B. Pitner, K. Bock, B. M. Pinto, Biochemistry, 1995, 34, 1367213680. 50. J. L. Asensio, F. J. Caiiada, J. Jimenez-Barbero, Eur. J. Biochem., 1995, 233, 618-630. 51. S. J. F. Vincent, C. Zwahlen, C. B. Post, J. W. Burgner, G . Bodenhausen Proc. Nut1 Acud. Sci. USA, 1997,94,4383-4388. 52. C. Zwahlen, S. J. F. Vincent, L. Di Bari, M. H. Levitt, G. Bodenhausen, J. Am. Chem. Soc., 1994,116,362-368. 53. F. Ni, Y. Zhu, J. Mugn. Reson. Ser. B, 1994, 102, 180-184. 54. T. Peters, B. M. Pinto, Curr. Opin. Struct. Biol., 1996, 6, 710-720. 55. A. Poveda, J. Jimenez-Barbero, Chem. SOC.Rev., 1998,27, 133-143. 56. J. Jimenez-Barbero, J. L. Asensio, F. J. Caiiada, A. Poveda, Curr. Opin. Struct. BioL, 1999, 9, 549-555. 57. J. Breg, L. M. J. Kroon-Batenburg, G. Strecker, J. Montreuil, J. F. G. Vliegenthart, Eur. J. Biochem. 1989, 178, 727-739. 58. Y. Ichikawa, Y.-C. Lin, D. P. Dumas, G-J. Shen, E. Garcia-Junceda, M. A. Williams, R. Bayer, C. Ketcham, L. E. Walker, J. C. Paulson, C.-H. Wong, J. Am. Chem. Soc. 1992, 114, 9283-9298. 59. C. Mukhopadhyay, K. E. Miller, C. A. Bush, Biopolymers 1994,34,21-29. 60. W. Jahnke, H. C. Kolb, M. J. J. Blommers, J. L. Magnani, B. Ernst, Angew. Chem., 1997,109, 27 15-271 9. 61, T. Haselhorst, T. Peters, unpublished results. 62 D. R. Bundle, N. M. Young, Curr. Opin. Struct. Biol., 1992,2, 666-673. 63 D. R. Bundle, E. Eichler, M. A. J. Gidney, M. Meldal, A. Ragauskas, B. W. Sigurskjold, B. Sinnott, C. D. Watson, M. Ydguchi, N. M. Young, Biochemistry, 1994, 33, 5172-5182.
References
1023
64. D. R. Bundle, H. Baumann, J.-R. Brisson, S. M. Gagne, A. Zdanov, M. Cygler, Biochemistry, 1994,33, 5183-5192. 65. D. R. Bundle, B. W. Sigurskjold, Meth0d.r Enzymol., 1994, 247, 288-305. 66. M. J. Milton, D. R. Bundle, J. Am. Chem. Soc., 1998,120, 10547-10548. 67. T. Sokolowski, T. Haselhorst, K. Scheffler, R. Weisemann, P. Kosma, H. Brade, L. Brade, T. Peters, J. Biomol. N M R , 1998, 12, 123-133. 68. Y. Fu, M. Baumann, P. Kosma, L. Brade, H. Brade, Infect. Immun., 1992, 60, 1314-1321. 69. L. Brade, P. Kosma, B. J. Appelmelk, H. Paulsen, H. Brade, Zrzfect. Immun., 1987, 55, 462466. 70. J. F. Espinosa, E. Montero, A. Vian, J. L. Garcia, H. Dietrich, R. R. Schmidt, M. MartinLomas, A. Imberty, J. Cafiada, J. Jimenez-Barbero, J. Am. Chem. Soc., 1998, 120, 1309-1318. 71. M. Gilleron, H. C. Siebert, H. Kaltner, C. W. von der Lieth, T. Kozar, K. M. Halkes, E. Y. Korchagina, N. V. Bovin, H. J. Gabius, J. F. G. Vliegenthart, Eur. J. Biochem., 1998, 252, 41 6- 427. 72. R. Ravishankar, A. Surolia, M. Vijayan, S. Lim, Y. Kishi, J. Am. Chem. Svc., 1998, 120, 11297-11303. 73. C. R. MacKenzie, T. Hirama, J. T. Buckley, J. Biol. Chem., 1999,274, 22604-22609. 74. K. Pervushin, R. Riek, G. Wider, K. Wiithrich, Proc. Natl Acad. Sci. USA, 1997, 94, 1236612371. 75. S. J. Glaser, T. Schulte-Herbriiggen, M. Sieveking, 0. Schedletzky, N. C. Nielsen, 0. W. S0rensen, C. Griesinger, Science, 1998, 280, 421-424. 76. N. Tjandra, A. Bax, Science, 1997, 278, 11 11-1 114. 77. B. Meyer, T. Weimar, T. Peters, Eur. J. Biochem., 1997, 246, 705-709. 78. D. Henrichsen, B. Ernst, J. L. Magnani, W.-T. Wang, B. Meyer, T. Peters, Angerv. Chem. h t . Ed., 1999, 38, 98-102. 79. M. Mayer, B. Meyer, Angrw. Chem. Int. Ed., 1999, 38, 1784--1788. 80. J. Klein, R . Meinecke, M. Mayer, B. Meyer, J. Am. Chem. Soc., 1999, 121, 5336-5337.
Carbohydrates in Chemistry and Biology Editor Beat Ernst, Gerald W. Hart, Pierre Sinay copyright OWILEY-VCH Verlag GmbH, 2000
36 Carbohydrate-Protein Interactions: Use of the Laser Photo Chemically Induced Dynamic Nuclear Polarization (C1DNP)-NMR Technique Hans-Christian Siebert and Johannes F. G. Vliegenthart
36.1 Introduction The laser photo chemically induced dynamic nuclear polarization (CIDNP) method is a sophisticated NMR technique [ 11, which can be used for the detection of surface exposed Tyr-, His- and Trp-residues of a protein. It is possible to correlate the intensity of a CIDNP signal with the degree of accessibility of the corresponding CIDNP-sensitive amino acid residue in a protein-structure [2-71. To obtain CIDNP signals it is necessary that a laser-light excited dye undergoes radical reactions with one of the three CIDNP-sensitive amino acids. Two spectra are recorded: one with and one without laser-light irradiation at each CIDNP experiment. The resulting light spectrum is subtracted from the dark spectrum, thereby establishing the CIDNP difference spectrum, containing the signals of polarized residues only. Trpand His-signals occur in positive, Tyr-signals in negative direction in the aromatic part of a CIDNP-difference spectrum [3, 81. Trp- and His-signals can be discriminated by the strong pH-dependence of the His-signals resulting in alterations of chemical shift and signal intensity. The pH-dependence of the intensities of Tyr-, His- and Trp-CIDNP-signals have been described [ 3 , 9, lo]. CIDNP-measurements are usually carried out in D20 solution, therefore one has to consider the relation: pD = pHmeterreadins0.4. In general, it is possible to distinguish highly, partly and non-accessible Tyr-, His- and Trp-residues. In case a CIDNP-reactive amino acid is located in or in the vicinity of the binding pocket of a protein, the corresponding CIDNP signals can in many cases be suppressed by the addition of a specific ligand. In the bound form this ligand hinders the excited flavin dye to undergo radical reactions with the CIDNP-reactive residues. Provided that the alternative interpretation can be excluded that ligand binding alters the general conformation of a receptor thereby changing the surface accessibilities of various amino acid residues. For this reason, CIDNP results have always to be evaluated in close correlation to a computer supported molecular modelling procedure. The CIDNP-technique is a rather quick method, but restricted to special problems, namely the detection and
+
1026
36 Carbohydrate-Protein Interactions
analysis of Tyr-, His and Trp-residues on the surface of a protein. CIDNP data provide their optimal benefit when they are combined with molecular modelling data based on X-ray and NMR structures of similar molecules [5-71. The CIDNP method sometimes also allows to detect an influence on the conformation caused by ligand interaction or by site-directed mutagenesis. The validity of CIDNP data can be checked when protein-structures are completely examined by multi-dimensional NMR experiments. In contrast to multidimensional NMR experiments the CIDNP technique is not limited by the size of the molecule [ 1 11.
36.2 The CIDNP Method The CIDNP radical reaction is initiated by the flavin I mononucleotide (N3 of the isoalloxazine ring substituted with -CHzCOOH, and N10 with -CH3) as laserresponsive dye. The laser-light used is generated by a continuous-wave argon ion laser which operates in the multi-line mode with principal emission wavelengths of 488.0 and 514.5 nm, close to the edge of the 450 nm absorption band of the dye. By an optical fibre the laser light is directed to the sample and chopped by means of a mechanical shutter controlled by the spectrometer to prevent heating of the proteincontaining solution. The irradiation leads to the generation of protein-dye radical pairs by surface-exposed Tyr-, Trp-, and His-side chains. Nuclear spin-polarization is obtained from back-reactions of the radical pairs. The irradiated dye converts from an excited singlet state to an excited triplet state where it can undergo radical reactions with the corresponding groups of the CIDNP-sensitive amino acids. Recombination and escape reactions are possible. The recombination, which only occurs in the singlet state, depends on the spin of the nuclei leading to a nuclear spin polarization which can be detected by NMRspectroscopy [3, 12-14]. The Tyr-dependent CIDNP effect corresponds to a spin-density distribution of the intermediate phenoxy radical with strong negative signals of the protons H3, H5 and less intense positive signals of the protons H2, H6. The CIDNP signals of Trp are generated by an intermediate radical with strong spin densities at the positions H2, H4, H6 of the indole unit which all yield positive CIDNP signals. The CIDNP responses of H2, H4 of His occur as positive singlets [3]. A typical experimental setting consists of a presaturation pulse of 1 s, a light pulse of 0.5 s (5 W), a resonance frequency pulse (90" flip angle) of 5 ps, an acquisition time of 1 s and a delay of 5 s. In general, an adequate signal-to-noise ratio is obtained after 16, 32 or 64 light scans [3]. The individual mixtures consisted of 0.1 mM protein, 0.1 to 2 mM ligand and 0.4 mM flavin derivative. Chemical shifts were assessed relative to acetone (2.225 ppm) and HDO (4.76 ppm at a defined temperature and pH/pD-value).
36.4 Applications
1027
36.3 CIDNP-related Molecular Modelling As mentioned before CIDNP-data should whenever possible be correlated with model structures. In case no further NMR-data are available, X-ray coordinates from the studied molecule or even from homologous structures can be used. The values of the surface accessibility of CIDNP-reactive side chains have shown to be suited parameters for the combination of experimentally and theoretically derived results. Those values can be calculated with the help of the Connolly program in Insight11 following an established method [5-7, 15-19]. The calculated values correspond to the accessible exterior part of the molecule obtained by smoothening the van der Waals surface with a test sphere that has the average radius of a water molecule (1.5 A). Additionally, the average radius of flavin (4 A) can be used without yielding different conclusions concerning the degree of accessibility of the studied residues. The average radii of water (r = 1.5A) and flavin (r = 4 A) are calculated according to a published formula [20]. The dot density of the van der Waals-like spheres representing the Connolly surfaces is generally set to a value of 1 corresponding to a distribution of calculated surface coordinate values.
36.4 Applications Beside successful studies of protein-nucleic acid interactions [19, 21, 221 and protein-folding processes [23] the method has shown to be a proper tool in glycosciences since Tyr-, His- and Trp-residues are often constituents of the carbohydrate binding domain [5-7, 15, 24, 251. Many plant and animal lectins harbour CIDNPreactive amino acid residues as an essential part of their binding pocket architecture [ 26-28]. The presence of aromatic amino acids in the binding site of various lectins raises the question why such hydrophophic amino acids are important for the binding of the hydrophilic carbohydrates. An answer may be found in Figure l a and b which documents the hydrophilic and the hydrophobic parts of a monosaccharide and its position between polar and non-polar amino acids. The CIDNP-reactive amino acid residues can form hydrophobic contacts with the unpolar parts of the carbohydrate in the binding pocket. Lectins are interesting objects to study this aspect of binding in detail. These molecules are carbohydrate binding proteins from nonimmunological origin and without enzymatic activity. They play important roles in various regulation processes in the plant as well as in the animal organism and are therefore of great interest in the field of glycosciences [25, 26, 28-30]. Since Tyr-, His- and Trp-residues are CIDNP-responsive and often involved in carbohydrate binding, alterations of the respective signal intensities of lectins can be detected after addition of a specific saccharide. It is possible to correlate such findings with the theoretical results derived by molecular modelling techniques. CIDNP
1028
36 Carbohydrate-Protein Interactions
Figure la. Hydrophobic an hydrophilic site of a monosaccharide.
36.5 Hevein-like Lectins
1029
Figure lb. Schematic representation of a monosaccharide in a binding pocket with a hydrophobic and a hydrophilic site.
data provide furthermore complementary information to structural data derived by other biochemical and biophysical methods [3I]. Molecular dynamics simulations in combination with CIDNP experiments make it possible to refine the models obtained by interpretation of the rigid X-ray coordinates alone. The success of such a study can be investigated by isoforms and/or mutants of carbohydrate-binding proteins, when available. In case X-ray data from one isoform or the wild type are known, information about important structural features can already be obtained by CIDNP-experiments.
36.5 Hevein-like Lectins The role of Tyr-, His- and Trp-residues can be exemplarily studied in the small lectins hevein, pseudo-hevein (two isolectins from the rubber tree) and the B-domain of wheat germ agglutinin (WGA) which consist of 43 amino acid residues [6, 32, 331. All hevein-like lectins have a binding specificity for D-N-acetylglucosamine oligomers (GlcNAc,). The CIDNP signals of the corresponding amino acids in the binding pocket show a significantly weakened intensity after complexation with a ligand, as demonstrated for hevein at a low pD-value of 4.4 (Figure 2). The lectin Urtica dioica agglutinin (UDA) from the stinging nettle and wheat germ agglutinin (WGA) have also an affinity to GlcNAc,. UDA consists of two and WGA of four hevein-like domains. Beside its affinity to GlcNAc, WGA also binds oligosaccharides containing sialic acids. In all mentioned examples the corresponding signals of Tyr- and Trp-residues of the binding pocket are affected by the addition of GlcNAc, indicated by CIDNP signals with lower intensity after ligand complexation. These results were confirmed by two-dimensional NMR methods
1030
36 Curbohydrute-Protein Interactions
a
b
I Tyr 30 H3,5
flavin
9.00
8.00
flavin
7.00
6.00
I
9.00
8.00
7.00
, '
6.00
Figure 2. Laser photo CIDNP difference spectra (aromatic part) of hevein and hevein-GlcNAc4 complexes at pD = 3.5. a. ligand-free hevein; b. 0.5 mmol hevein + 1 mmol GlcNAc4.
[13, 34, 351. The degree of suppression for WGA and the hevein-like monomers was significantly stronger than for UDA. Interestingly, complexation of UDA with a small amount of GlcNAc3 leads to an altered CIDNP spectrum in which a new small Trp-signal has occurred. This finding argues in favour for a small conformational change of the binding pocket during ligand binding (Figure 3 ) [6]. In order to obtain further information about the architecture of the heveindomains the CIDNP data were correlated with X-ray crystallographic coordinates from WGA [36] and completely NMR-obtained conformations from hevein and hevein-ligand complexes [ 13, 32, 33, 371. Comparison of CIDNP-derived surface accessibilities with computationally obtained values on the basis of refined X-ray or NMR-structures leads to the following results: Buried Tyr-residues have a surface accessibility below 30 A2, partly buried ones have accessibilities of 30-80 A2, whereas a value above 80A2 is calculated for Tyr-residues which are considered to be the surface exposed ones causing intense CIDNP signals. Corresponding values
36.5 Hevein-like Lectins
b
a
Trp 21 H4
H4
v 7.80
1031
7.60
7 -80
3 7.60
Trp 21 H6 Trp 23 H6
Tm 23 H4
flavin
,I I
Trp 23 H4
II
r i
II
flavin
Tyr 30 H3,5 Tyr 76 H3,5 B .'oO
7 .bO
Tyr 76 H3,5 1-
8.00
7.00
Figure 3. Laser photo CIDNP difference spectra (aromatic part) of UDA and UDA-GlcNAc4 complexes. a. ligand-free UDA; b. 0.5 mmol UDA + 1 mmol GlcNAc4.
1032
36 Carbohydrate- Protein Interactions
are estimated for Trp- and His-residues. The calculations are carried out with a dot density of 1 and a test sphere radius of 1.5A [5-7, 15-19]. These data make it possible to use CIDNP-derived results for quality control of modelled structural data [5-71. The surface accessibility values of CIDNP-reactive amino acid residues of hevein-like lectins can be compared with the intensities of the corresponding CIDNP signals. CIDNP and modelling data indicate that the architecture of the binding pockets of hevein, pseudo-hevein and the B-domain of WGAl are similar [6]. In detail: different ways in designing the models have been used. In one case the B-domain of WGAl was directly taken from the X-ray structure of WGAl . In the other case the B-domain of WGAl has been constructed by amino acid replacement in the hevein-NMR-structure. The initial structures have been energetically minimized before starting the MD (molecular dynamics) simulations. Values of the surface accessibility of CIDNP-reactive amino acid residues are obtained during the MD-simulation and listed in a table [6]. Modelled structures of pseudo-hevein and the B-domain of WGAl which are in optimal agreement with the CIDNP-results are shown in Figure 4a, b.
36.6 Galactoside-binding Lectins from Plant and Animal Origin NMR-spectra of Erythrina corallodendron lectin (EcorL) are not resolved (Figure 5) without the use of special techniques, like CIDNP. Resolved CIDNP signals from EcorL, however, can be assigned by comparison of wild-type- and mutant-EcorLCIDNP-spectra, as published [ 71. For completion, galectins, which are galactosidebinding lectins from animal origin which do not need any Ca2+-ionsfor saccharide binding and which share structural homologies with EcorL, have been included in this study. From X-ray data as well as from the results of the CIDNP experiments one can conclude that the highly accessible amino acid residues Tyrl06 in EcorL and Trp68 in galectins are involved in the binding process. However, X-ray data of EcorL and bovine galectin also indicate that the extent of surface accessibility of other CIDNPreactive moieties besides Tyrl06 and Trp68 can be examined. Since the crystallographic data sets provide the starting point for knowledge-based homology modelling, MD (molecular dynamics)-derived conformational parameters for the related proteins can likewise be correlated to the results of the CIDNP spectroscopic measurements. This comparison is also facilitated by the availability of mutants for the legume lectin, in which Tyr-residues at position 106, 108 or 229, respectively, were replaced by Thr-, Gly- or Ala-residues [38, 391. Furthermore, a mutant was studied in which Trp is replaced by Ala at position 135. The introduction of the single-site mutations in the legume protein can cause non-uniform impacts on the calculated conformational parameters beyond the immediate vicinity of the site of mutation [7, 401. These results deserve attention for the interpretation of comparative data sets of wild-type and single-site mutant proteins. The structural predictions of molecular modelling are in agreement with the CIDNP-spectra. Regarding the
36.6 Galactoside-binding Lectins,from Plant and Animal Origin
1033
a
b Figure 4a. Energy minimum conformation of pseudo-hevein, emphasising the surface exposition of Tyr21 and Trp23 in the upper part of the picture; b. energy minimum conformation of domB of WGAl, emphasising the surface exposition of Tyr64 and Tyr66 in the lower part of the picture.
1034
36 Curbohydrute-Protein Inteructions
8
v;
8
d
8 G
8 od
8
0;
36.6 Galactoside-binding Lectins from Plant and Animal Origin
1035
intensity of the Tyr-signal, the CIDNP-spectrum of the mutant Trpl35Ala (Figure 6a) does not alter remarkably from that of the wild type [7]. A single site mutation of the highly accessible Tyr 106 leads to the complete disappearance of the Tyrsignal in the CIDNP difference spectrum, Figure 6b. However, also the CIDNPspectrum of an EcorL mutant wherein Tyrl08 is exchanged against a CIDNP-inert residue does not show any Tyr-CIDNP-signal, Figure 6c. This can only be the case when Tyrl06 loses its surface accessibility due to the replacement of Tyrl08. Our experimental finding is exactly reproduced by molecular modelling calculations which are based on the X-ray structure data of EcorL [41].The derived MD data [7, 421 confirm the experimental results, as demonstrated in Table 1. Furthermore, as can also be seen from Table 1, the single site mutations of Tyrl06 or Tyrl08 have an impact on the surface accessibilities of Tyr192 and Tyr229 which are lowered significantly. On the other hand a replacement of Tyr by Ala at position 229 influences the surface presentation of Tyrl06 and Tyrl08 [7] but no impact could be detected on the binding specificity 138, 391. The conformational rearrangement in this mutant is shown in Figure 7a, b. The CIDNP method was also applied to five members of the galectin family, namely bovine heart galectin, human lung galectin, CG- 14-the galectin from chicken intestine, CG- 16-the galectin from chicken liver and the recombinant murine galectin-3. The experimental data show a significant covering of Trp68 after addition of lactose whereas the Tyr-residues of the studied galectins are unaffected by the ligand. The X-ray structure from bovine heart galectin and the CIDNP data of all studied galectins have been used as valuable information for the design of four model structures [7, 431. CIDNP-responsive amino acids are therefore important constituents in the binding site of the P-galactoside specific galectins. One galectin-domain with a bound ligand and the involvement of Trp is shown in Figure 8. The availability of well-defined X-ray structures [44-461 and sequence homology between various galectins from different species allow the prediction of structures by use of the knowledge-based protein modelling approach [47, 481. The results obtained from CIDNP experiments provide valuable information concerning the reliability of structures obtained by different modelling techniques. The occurrence of an additional Trp-CIDNP signal in a spectrum obtained from a saccharidehuman galectinl complex argues in favour of a change in orientation of the Trp-ring. Furthermore, effects on the binding strength of galectins caused by site-directed mutagenesis [49] can be analysed by a combination of CIDNP and modelling techniques. Such a protein-carbohydrate interaction study can be completed when the results from transferred nuclear Overhauser experiments (trNOE) are integrated (50). TrNOE-experiments are a proper tool in glycosciences [50-581. These kinds of experiments are used when the conformation of a small ligand in the presence of a much bigger receptor is studied. Since the mentioned biophysical methods are complementary to each other, a combination of X-ray-data, NMR methods (multidimensional experiments, trNOE, CIDNP) and molecular modelling techniques is in general the best way to analyse protein-carbohydrate complexes on an atomic scale [48, 58, 591.
I
7.00
6.00
Tyr 106 8.00
!
7.00
"
"
,
-
6.00
9.00
7.00
6.00
Figure 6. CIDNP-difference spectra (aromatic part) of three single-site mutants of recombinant EcorL. a. [WI 35AJEcorL (ligand-free); b. [Y106GIEcorL (ligand-free); c. [Y108TIEcorL (ligand-free).
8.00
1
C
36.7 Sialidase from Clostridium Perfringens (Wild Type and Mutants)
1037
Table 1. Average surface accessibilities ( 2 ) and the differences of the surface accessibility areas of CIDNP-sensitive residues derived from molecular dynamics calculations between the wild-type of EcorL ( 2 ) and the three single-site mutants [7]. All area values are given in A2. Dot density: I ; test sphere radius: 1.5 A. **** denotes the site of mutation substituting the respective tyrosine residue with CIDNP-inert alanine. X
AY106
AY108
AY229
~
Tyr 53 Tyr 82 Tyr 106 Tyr 108 Tyr 121 Tyr 172 Tyr 185 Tyr 192 Tyr 229 Trp 45 Trp 60 Trp 135 Trp 207 Trp 231 His 58 His 142 His 180 His 226
10.0 69.3 67.7 75.3 49.4 7.9 6.1 65.8 81.0 100.9 30.9 69.1 113.3 3.0 28.7 0.0 45.0 0.0
+1.2 -10.7
****
10.0 +7.0 - 14.4
-30.7 +12.2 -7.4 +3.6 -18.6 -11.9 +5.2 $2.8 -20.6 -5.5 -1.0 -10.0 0.0 +13.0 f2.1
+15.7 +7.4 -1.6 9.0 -25.4 -29.0 -17.6 123.6 -42.2 -3.0 -3.6 0.0 +9.4 0.0
-
****
+15.5 +4.4 +14.1 -32.6 $11.6 -7.9 +16.3 -30.4
****
-15.5 -26.3 -2.8 -20.6 -3.0 - 14.9 0.0 + 17.2 0.0
36.7 Sialidase from Clostridium Perfringens (Wild Type and Mutants) The presence of a number of CIDNP-sensitive amino acids on the protein surface can enable the detection of conformational alterations resulting from single site mutations. This has been demonstrated for wild-type forms and various mutants of EcorL and of the sialidase of Clostridium perfringens [5, 71. Sialidase of C. perfringens is larded with CIDNP-responsive amino acid residues. Therefore, these residues have been used as valuable sensors in NMR and modelling studies in which CIDNP-spectra of the sialidase wild type and various mutants are compared and correlated with modelled structures. Starting with the crystal structure of the bacterial sialidase of Salmonella typhimurium [60] which is used as a framework, the knowledge based homology modelling produces a set of energy-minimised conformations for the small sialidase of C.
1038
36 Carbohydrate-Protein Interactions
Figure 7. MD-derived energy minimum structures of wild-type EcorL and single-site mutants. a. wild-type EcorL; b. mutant [Y229A]EcorL.
perfringens. Although the number of CIDNP-reactive amino acids is to large to unequivocally assign signals to defined residues, as has been feasible for hevein [6], the signal intensity can be set into correlation with the model-derived expectations. In addition to the wild-type enzyme the impact of introducing amino acid substitutions by site-directed mutagenesis has been theoretically and experimentally delineated as a further test of the model structure. Changes in surface accessibilities of widely separated residues affected by a Tyr/Phe- or a Cys/Ser-substitution could be measured and calculated. This leads to the conclusion that conformational changes of mutant enzymes relative to the wild-type form should not be underestimated. However, not only CIDNP-spectra and modelling data argue in favour of an influence of single site mutations on the overall conformation. The CIDNP- and modelling-results obtained for the sialidase of C. perfringens and several of its mutants are in perfect agreement with observations concerning the enzymatic activities of these molecules determined by biochemical methods. The differences in enzymatic activities between the wild type and various mutants confirm the conclusions drawn from CIDNP experiments and molecular modelling [5].
36.8 CIDNP Analysis of Glycoproteins
1039
Figure 8. Galectin-1 monomer with a specific disaccharide in its binding pocket.
The success of knowledge-based homology modelling is critically dependent on the predictive potency of the program of structure-based calculations, which attempts to translate homologous sequences into three-dimensional structures. In order to evaluate the actual relevance of molecular-modelling-supported crystal structures for the protein topology in solution, CIDNP data providing selected parameters of the protein’s conformation can be used for quality ranking.
36.8 CIDNP Analysis of Glycoproteins When CIDNP-reactive amino acids are accessible on the surface of a glycoprotein, a possible impact of the covalently bound oligosaccharide chain on the protein part can be scrutinized [14, 15, 611 by this method. Therefore, CIDNP-experiments are also suitable to improve structural models of glycoproteins. On the basis of a X-ray structure of a glycoprotein, very often only the coordinates for the protein part are available since the flexibility of the oligosaccharide chain hampers a complete crys-
1040
36 Carbohydrate-Protein Interactions
tallographic structure. NMR measurements of the oligosaccharide chain [62-661 in combination with molecular mechanic and molecular dynamic calculations make it possible to gain the needed structural data. CIDNP experiments were applied on the glycoprotein SAP (serum amyloid P-component) from human serum [67, 681. In one series of experiments the oligosaccharide chains were intact and in another series they were desialylated [14, 151. The respective spectra of SAP and enzymatically desialylated SAP were determined. Six Trp/His signals and one Tyr signal are present in the aromatic part of the CIDNP difference spectrum of SAP. The corresponding spectrum of desialylated SAP shows remarkable alterations. The chemical shift of one Trp/His-characteristic signal is decreased by 0.1 ppm. One Trp/His-signal disappeared and a new one was formed in the CIDNP difference spectrum of desialylated SAP, while the other signals were unaffected. The Tyr signal has a clearly enhanced intensity in desialylated SAP. Therefore, the removal of sialic acid residues from the single N-glycan of each monomer apparently affects the surface presentation of distinct CIDNP-reactive amino acids of SAP [ 14, 151. A conformational change of the protein part of SAP in relation with a different orientation of the desialylated oligosaccharide chain in comparison to the sialylated chains or a covering of CIDNP-responsive amino acids by the oligosaccharide chain itself arc possible explanations of the CIDNP results. In order to prove these experimental data molecular dynamics simulations of SAP with an intact and an asialo-saccharide chain were carried out. The structural data of the protein part of SAP were obtained by using X-ray crystallographic coordinates of SAP [69, 701 as a start structure for the molecular dynamics simulations. The computationally constructed oligosaccharide chains were energetically minimised with the SWEET program [71]and linked to the protein part (Figure 9) before the MD-run. The MD-simulations address the question in which way an interaction between the oligosaccharide and protein part is possible. Small long range interactions within oligosaccharide chains of 0-acetylated and non-0-acetylated gangliosides (90Ac-GDla and GDla) have already been identified with help of NMRsupported MD-simulations [72].
36.9 Conclusions In summary, the CIDNP method is a potent addition to the arsenal of biophysical methods which arc successfully used in the field of structural glycosciences. The combination of X-ray data, molecular modelling, trNOE-methods and laser-photo CIDNP provides valuable structural information about protein-carbohydrate complexes. This is of particular importance, when multidimensional NMR-data can not be recorded due to the size of the molecule. Furthermore, the analysis of similar carbohydrate-binding proteins sharing a sufficient sequence homology is possible when complete structural data are available only for a few of them and CIDNPand molecular modelling data are present for all of them. The successful role, which CIDNP-experiments can play for the solution of important structural glyco-
36.9 Conclusions
1041
Figure 9. X-ray structure of human serum amyloid P component with modelled oligosaccharide chains.
biochemical problems, is based on the important function of Tyr-, His- and Trpresidues in constituting the hydrophobic interactions between the corresponding combining sites of a carbohydrate and a protein. Acknowledgments This work was supported by the Human Capital and Mobility Program of the European Community, the Netherlands Foundation for Chemical Research (SON) and the Netherlands Organisation for Scientific Research (NWO).
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36 Carbohydrate-Protein Interactions
References 1. D. Neuhaus, M. P. Williamsen, The Nuclear Overhauser EfSect, 1989, VCH, Weinheim-New York. 2. R. Kaptein, K. Dijkstra, K. Nicolay, Nature, 1978, 274, 293-294. 3. R. Kaptein in Biological Magnetic Resonance, 1982,4, (L. J. Berliner, ed.) pp. 145-191, Plenum Press, New York. 4. P. J. Hore, R. W. Broadhurst, Progr. N M R Spectrosc., 1993,25, 345-402. 5. H.-C. Siebert, E. Tajkhorshid, C.-W. von der Lieth, R. G. Kleineidam, S. Kruse, R. Schauer, R. Kaptein, J. F. G. Vliegenthart, H.-J. Gabius, J. Mol. Model., 1996, 2, 446-455. 6. H.-C. Siebert, C.-W. von der Lieth, R. Kaptein, J. J. Beintema, K. Dijkstra, N. van Nuland, U.M.S. Soedjanaatmadja, A. Rice, J.F.G. Vliegenthart, C.S. Wright, H.-J. Gabius, Proteins, 1997,28, 268-284. 7. H.-C. Siebert, R. Adar, R. Arango, M. Burchert, H. Kaltner, G. Kayser, E. Tajkhorshid, C.-W. von der Lieth, R. Kaptein, N. Sharon, J. F. G. Vliegenthart, H.-J. Gabius, Eur. J. Biochem., 1997,249, 27-38. 8. S. Stob, R. Kaptein, Photochem. Photobiol., 1989, 49, 565-577. 9. P. F. Heelis, B. J. Parsons, G. 0. Phillips, Biochim. Biophys. Acta, 1979, 587, 455-462. 10. K. A. Muszkat, T. Wismontski-Knittel, Biochemistry, 1985, 24, 5416-5421. 11. R. M. Scheek, R. Kaptein, J. W. Verhoven, FEBS Lett., 1979, 107, 288-290. 12. Y. N . Molin (ed.), Spin Polarization and Magnetic Effects in Radical Reactions, 1984, Elsevier, Amsterdam. 13. H.-C. Siebert, R. Kaptein, J. J. Beintema, U. M. S. Soedjanaatmadja, C. S. Wright, A. Rice, R. G. Kleineidam, S. Kruse, R. Schauer, P. J. W. Pouwels, J. P. Kamerling, H.-J. Gabius, J. F. G. Vliegenthart, Glycoconj. J., 1997, 14, 53 1-534. 14. H.-C. Siebert, S. Andre, G. Reuter, R. Kaptein, J. F. G. Vliegenthart, H.-J. Gabius, Glycoconj. J., 1997, 14, 945-949. 15. H.-C. Siebert, S. Andre, G. Reuter, H.-J. Gabius, FEBS Lett., 1995, 371, 13 -16. 16. B. Lee, F. M. Richards, J. Mol. Biol., 1971, 55, 379-400. 17. M. L. Connolly, J. Appl. Cryst., 1983, 16, 548-558. 18. M. L. Connolly, Science, 1983, 221, 709-713. 19. E. Kellenbach, T. Hiird, R. Boelens, K. Dahlman, J. Carlstedt-Duke, J.-A. Gustafsson, G. A. van der Marel, J. H. van Boom, B. Maler, K. R. Yamamoto, R. Kaptein, J. Biomol. N M R , 1991, I , 105-110. 20. J. T. Edward, J. Chem. Educ., 1970, 47, 261-270. 21. F. Buck, H. Riiterjans, R. Kaptein, K. Beyreuter, Proc. Natl Acad. Sci. USA, 1980, 77, 51455148. 22. S. Stob, R. M. Scheek, R. Boelens, R. Kaptein, FEBS Lett., 1988, 239, 99-104. 23. R. W. Broadhurst, C. M. Dobson, P. J. Hore, S. E. Radford, M. L. Rees, Biochemistry, 1991, 30, 405-412. 24. F. Quiocho, Pure Appl. Chem.; 1989, 61: 1293-1306. 25. N. Sharon, H. Lis, H., Science, 1989,246, 227-246. 26. N. Sharon, Trends Biochem. Sci., 1993, 18, 221-226. 27. J. M. Rini, Annu. Rev. Biophys. Biomol. Struct., 1995, 24, 551-577. 28. H.-J. Gabius, S. Gabius, Glycosciences: Status und Perspectives, 1997, Chapman & Hall, Weinheim-London. 29. R. A. Dwek, Chem. Rev., 1996, 96, 683-720. 30. H.-J. Gabius, Eur. J. Biochem., 1997, 243, 543-576. 31. T. Diaz-Mauriiio, D. Solis, J. JimCnez-Barbero, M. Martin-Lomas, H.-C. Siebert, J. F. G. Vliegenthart, Carbohydr. Eur., 1998, in press. 32. J. L. Asensio, F. J. Caiiada, M. Bruix, A. Rodriguez-Romero, J. Jimenez-Barbero, Eur. J. Biochem., 1994,230, 621-633. 33. J. L. Asensio, F. J. Caiiada, M. Bruix, C . Gonzales, N. Khiar, A. Rodriguez-Romero, J. Jimenez-Barbero, Glycobiology, 1998,8, 569-577. 34. K. Hom, M. Gochin, W. J. Peumans, N. Shine, FEBS Lett., 1995,361, 157-161.
References
1043
35. H.-C. Siebert, R. Kaptein, J. F. G. Vliegenthart, in Lectins und Glycobiology (H.-J. Gabius, S. Gabius, eds), 1993, pp. 105-1 16, Springer Verlag, Heidelberg, New York. 36. C. S. Wright, J. Mol. B i d , 1989,20Y, 475-487. 37. N. H. Andersen, B. Cao, A. Rodriguez-Romero, B. Arreguin, Biochemistry, 1993, 32, 14071422. 38. R . Arango, E. Rodriguez-Arango, R. Adar, D. Belenky, F. G. Loontiens, S. Rozenblatt, N. Sharon, FEBS Lett., 1993, 330, 133-136. 39. R. Adar, N. Sharon, N., Eur. J. Biochem., 1996,239, 668-674. 40. T. P. Kogan, B. M. Revelle, S. Tapp, D. Scott, P. J. Beck, J. Biol. Chem., 1995, 270, 1404714055. 41. B. Shaanan, H. Lis, N. Sharon, Science, 1991,254, 862-866. 42. E. Moreno, S. Teneberg, R. Adar, N. Sharon, K.-A. Karlsson, J. Angstrom, Biochemistry, 1997,36,4429-4437. 43. E. Tajkhorshid, H.-C. Siebert, M. Burchert, H. Kaltner, G . Kayser, C.-W. von der Lieth, R . Kaptein, J. F. G. Vliegenthart, H.-J. Gabius, J. Mol. Model., 1997, 3, 325-331. 44. Y. D. Lobsanov, M . A. Gitt, H. Leffler, S. H. Barondes, J. M. Rini, J. Biol. Chen?.,1993, 26, 27034-27038. 45. Y. Bourne, B. Bolgiano, D. Liao, G. Strecker, P. Cantau, 0. Herzberg, T. Feizi, C. Cambillau, Nature Struct. Biol., 1994, I , 863-870. 46. D. Liao, G. Kapadia, H. Ahmed, G. R. Vasta, 0. Herzberg, Proc. Natl Acad. Sci. U S A , 1994, 91, 1428-1432. 41. M. S. Johnson, N. Srinivasan, R. Sowdhamini, T. L. Blundell, Crit. Rev. Biochem. Mol. B i d , 1994,29, 1-68. 48. M. W. MacArthur, R. A. Laskowski, J . M. Thornton, J. M., Curr. Opin. Struct. Biol., 1994, 4, 731-737. 49. J. Hirabayashi, in Glycosciences: Status und Perspectives (H.-J. Gabius, S. Gabius, eds.), 1997, pp. 355-368, Chapman & Hall, London-Weinheim. 50. H.-C. Siebert, M. Gilleron, H. Kaltner, C.-W. von der Lieth, T. Kozar, N. V. Bovin, E. Y. Korchagina, J. F. G. Vliegenthart, H.-J. Gabius, Biochem. Biophys. Rex Commun., 1996, 219, 205-212. 51. V. L. Bevilacqua, D. S. Thomson, J. H. Prestegard, Biochemistry, 1990, 29, 5529-5537. 52. V. L. Bevilacqua, Y. Kim, J. H. Prestegard, Bioeliemistry, 1992, 31, 9339-9349. 53. J. L. Asensio, F. J. Cafiada, J. Jimenez-Barbero, Eur. J. Biochem., 1995, 233, 618-630. 54. T. Weimar, T. Peters, Anyew,. Chem. Int. Ed Enyl., 1994, 33, 88-91. 5 5 . L. Poppe, G . S. Brown, J. S. Philo, P. V. Nikrad, B. H. Shah, J. Am. Chem. Soc., 1997, I I Y , 1727- 1736. 56. M. Gilleron, H.-C. Siebert, H. Kaltner, C.-W. von der Lieth, T. Kozar, N . V. Bovin, E. Y. Korchagina, H.-J. Gabius, J. F. G. Vliegenthart, Eur. J. Biochem., 1998, 252, 416-427. 57. H.-C. Siebert, C.-W. von der Lieth, M. Gilleron, G. Reuter, J. Wittmann, J. F. G. Vliegenthart, H.-J. Gabius, in Glycosciences: Status und Perspectives (H.-J. Gabius, S . Gabius, eds.), 1997, pp. 291-310, Chapman & Hall, London-Weinheim. 58. A. Poveda, J. Jimenez-Barbero, <'hem. Soc. Rev., 1998, 27, 133-143. 59. G. Wagner, S. G. Hyberts, T. F. Havel, Annu. Rec. Biophys. Biomol. Struct., 1992,21, 167-198. 60. S. J. Crennel, E. F. Garman, W. Graeme Laver, E. R. Vimr, G. L. Taylor, Proc. Nut/ Acud. Sci. U S A , 1993, YO, 9852-9856. 61. K. Hird, J. P. Kamerling, J. F. G . Vliegenthart, Carbohydr. Rex, 1992, 236, 315-320. 62. K. HBrd, J. F. G . Vliegenthart, in Glycobiology. A Prcicticul Approach (M. Fukuda, A. Kobata, eds.), 1993, pp. 223-~242,Oxford University Press, Oxford, New York, Tokyo. 63. T. de Beer, C. W. E. M. van Zuylen, K. HBrd, R . Boelens, R. Kaptein, J. P. Kamerling, J. F. G . Vliegenthart, FEBS Lett., 1994, 348, 1-6. 64. T. de Beer, C. W. E. M. van Zuylen, B. R. Leeflang, K. HBrd, R. Boelens, R. Kaptein, J. P. Kamerling, J. F. G. Vliegenthart, Eur. J. Biochem., 1996, 241, 229-242. 65. J. P. M. Lommerse, L. M. J. Kroon-Batenburg, J. Kroon, J. P. Kamerling, J. F. G. Vliegenthart, J. Biomol. N M R , 1995, 5, 79-94. 66. J. P. M. Lommerse, L. M. J. Kroon-Batenburg, J. P. Kamerling, J. F. G. Vliegenthart, Biochemistry, 1995, 34, 8196-8206.
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67. G. A. Tennent, M. B. Pepys, Biochem. Soc. Trans., 1994,22, 74-79 68. M. B. Pepys, T. W. Rademacher, S. Amatayakul-Chantler, P. Williams, G. E. Noble, W. L. Hutchinson, P. N. Hawkins, S. R. Nelson, J. R. Gallimore, J. Herbert, T. Hutton. R. A. Dwek, Proc. Nut1 Acad. Sci. USA, 1994, 91, 5602-5606. 69. J. Emsley, H. E. White, B.P. O’Hara, G. Oliva, N. Srinivasan, I.J. Tickle, T. L. Blundell, M. B. Pepys, S. P. Wood, Nature, 1994,367, 338-345. 70. A. K. Shrive, G. M. T. Cheetham, D. Holden, D. A. A. Myles, W. G. Turnell, J. E. Volanakis, M. B. Pepys, A. C. Bloomer, T. J. Greenhough, Nuture Struct. Biol.,1996, 3, 346-354. 71. A. Bohne, E. Lang, C.-W. von der Lieth, J. Mol. Model., 1997, 3, 1-5. 72. H.-C. Siebert, C.-W. von der Lieth, X. Dong, G. Reuter, R. Schauer, H.-J. Gabius, J. F. G. Vliegenthart, Glycohiology, 1996, 6, 561-572.
Carbohydrates in Chemistry and Biology Editor Beat Ernst, Gerald W. Hart, Pierre Sinay copyright OWILEY-VCH Verlag GmbH, 2000
37 Biacore Wolfgang Jager
37.1 Introduction Since the first commercially available Biacore system (Biacore AB, Uppsala, Sweden) was introduced back in 1990, the technology has advanced to become a standard method in studying biomolecular interactions. This Chapter describes the function and practical utilization of this technology, discusses typical user questions and offers suggestions for problem solving. Studies of oligosaccharide interactions are presented, while the whole application range includes all kinds of biomolecules. Since applications are continually optimized and new reagents and consumables are being developed, detailed protocols will not be given. The reader is referred to updated protocols provided by the supplier, to the literature cited in the text, and to the current reference list on the Internet under http://www.biacore.com. 37.1.1 Real-time Analysis by Surface Plasmon Resonance The detection principal of Biacore relies on the physical phenomenon of surface plasmon resonance (SPR: Figure 1). Specifically, it is based on an optical unit which measures the refractive index located on the matrix side of the flat sensor chips (Figure 2). One of the interacting partners is immobilized on the chip matrix. Substance accumulation by molecule binding to the immobilized partner directly influences the refractive index, which is detected in real-time and indicated immediately [ 11. Inside the instrument, the chip matrix forms a roof of small flow cells. In current instrument configurations, each sensor chip serves two or four of these flow channels, giving two or four separate measuring surfaces. The integrated flow system provides a continuous flow of buffer or samples over the chip surface. Sample injections and measurements run automatically: only specimen loading to a sample loop can be
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37 Biacore
A) srslscrr chip masrix
B) Mctlecule immobilisation
C ) Interaction analysis
D) Regeneration and reuse
Figure 1. Different steps for applying the biosensor are displayed. A) A suitable sensor chip is chosen. B) Immobilization of biomolecules in the chip matrix generates a specific measuring surface. C) Interaction is analyzed by injecting binding partners along the surface. D) After the regeneration the surface is used for the next analysis.
optical
Figure 2. The sensor chip is composed of a glass carrier coated with a thin gold layer on which a biocompatible matrix is attached. An evanescent field is generated when light under the condition of total internal reflection is directed to the glass side. This field extends about 300 nm into the solution on the other side (the matrix side) and interacts with the refractive index of the solution close to the gold surface. The electromagnetic phenomenon of surface plasmon resonance (SPR) arises in the gold film, resulting in extinction of the reflected light at a specific angle. The angle of minimum reflected intensity (the resonance angle) varies with the refractive index on the matrix side. Changes in the resonance angle are directly proportional to changes in mass concentration due to binding or dissociation of biomolecules. The signal is presented in resonance units (RU), where 1 RU corresponds approximately to 0.8 pg carbohydrate or 1 pg protein bound per mm2.
37.1 Introduction
1047
either automatic or manual, depending on the system used. Signal detection occurs simultaneously in all channels and a serial flow along different surfaces allows monitoring of separate interactions with just one sample injection, or measuring the in-line control signal at a reference surface simultaneously. Because detection directly reacts to substance accumulation, the signal depends both on the molecular weight and concentration of the injected partner, and on the number of binding sites present on the matrix. However, detectable limits are picomolar concentrations or a minimal mass of about 180 Da [2]. 37.1.2 Information in a Sensorgram In the sensorgram the actual signal corresponding to mass concentration at the sensor surface is plotted against time (Figure 3 ) . Interaction with the immobilized partner during sample injection causes an increase in signal. At the end of the injection, the system automatically switches to buffer flow and a decrease in signal now reflects the dissociation of interactants. The matrix is regenerated and reused.
Signal (kKli)
P
16
14
12
J
AAMAAAA
I
I
I
I
I
100
20
300
400
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Figure 3. Interaction progress is monitored in a sensorgram. Binding of molecules during sample injection and dissociation during buffer flow are indicated. The height or the initial slope of the signal are directly correlated to sample concentration, while the shape of the curves reflect association and dissociation kinetics. After each measurement, remaining material is washed away by injecting an appropriate regeneration solution leaving only the immobilized partner at the surface, and a new analysis cycle can be done.
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37 Biacore
Multiple usage of one surface greatly enhances reproducibility of results and, therefore, is especially valuable for comparative studies. Analysis can be purely qualitative (yes or no), in order to determine the presence or absence of binding activity in a particular sample, or under selected conditions. Whole complexes can be built up on the matrix, step by step, and simultaneously viewed on the screen. The concentration of biologically active molecules can be determined by use of a standard curve. Association and dissociation kinetics as well as the affinity of an interaction are calculated by the fitting routines of the evaluation software [3].
37.2 Experimental Procedures This biosensor allows experiments to be organized in different ways. The choice can be made as to which interacting partner to immobilize on the chip matrix and which to inject in solution. Different immobilization strategies may be applied, and both purified samples or extracts can be used for the measurements. Interaction analyses are either performed by direct binding to the immobilized molecule, or as an inhibition assay with both partners free in solution. With this flexibility, the easy handling of the instruments, and a wide diversity of standardized protocols, each user has the opportunity to immediately start their analysis. On the other hand, many assays can be carried out in different ways. Therefore, it is recommended to obtain a general view of the technology and to clearly define the goal for each measurement, in order to structure an analysis in the best way.
37.2.1 Immobilization of Biomolecules at the Sensor Surface Before the actual measurement, a sensor chip is specifically prepared by immobilizing the biomolecule of interest in the matrix. There is a variety of sensor chips available with different surface properties, and there are standardized methods for coupling just about every biomolecule. A summary of the currently available sensor surfaces and the corresponding immobilization strategies is presented in Table 1. The immobilization level can be controlled on the screen and varied to fit any particular application. It is recommended to use purified samples for the coupling in order to create a homogeneous measuring surface, while the total amount of material consumed is usually only 1-5 pg. Covalent coupling via amine groups is the standard immobilization procedure [21], which includes three steps. First, the carboxymethyl (CM)-groups on the sensor chip matrix are activated by a mixture of EDC (N-ethyl-N’-(3-dimethylaminopropy1)-carbodiimide) and NHS (N-hydroxysuccinimide), then covalently bound to free amine groups of the biomolecules, and finally all remaining activated
37.2 Experimental Procedures
1049
Table 1. Sensor surfaces. Sensor chip matrix
Coupling via*
Immobilized molecules/comment
Standard CM-dextran
Proteins or small compounds
Streptavidin-dextran
NH2-, SH-, CHO- or COOH-groups Biotin
NTA-dextran** Flat hydrophobic surface**
Histidin-6mer Hydrophobic adsorption
Biotinylated molecules like Oligosaccharides or nucleic acids His-tag fusion proteins Lipid-monolayers
New sensor chips (continually developed, surfaces might change) Low carboxylated dextran Shortened dextran Flat CM-surface Lipid-anchor-dextran** Pure gold surface
As standard CMdextran As standard CMdextran As standard CMdextran Liposome capture User-defined
Reduced non-specific binding of culture medium or cell homogenate Reduced non-specific binding of serum, injection of phages or cells When dextran is not required, injection of phages or cells Liposomes (lipid bilayer) Customized surfaces, spin-coating, selfassembled monolayers
* for detailed protocols please refer to information supplied by the manufacturer ** surface can be completely regenerated
groups are deactivated by an excess of ethanolamine. In the same manner, CMsurfaces can be chemically modified, in order to bind proteins or other molecules via thiol-, aldehyde-, or carboxyl-functions [4]. In some cases, covalent coupling can result in loss of biological activity. Then, the immobilization strategy has to be changed, for instance from amine to thiol coupling or to indirect binding by high affinity capture. Covalent immobilization ensures a stable baseline, which is important whenever low signals are expected in the measurements. Molecule capture is an indirect immobilization, carried out for instance by covalent coupling of an antibody for capturing the corresponding antigen. This method allows removal of the captured interaction partner after measurement, and the surface can be re-loaded with the same or a new molecule. Moreover, specifically captured molecules need not be purified, but can be present in extracts, so that the danger of protein inactivation by a purification protocol is eliminated [ S ] . Carbohydrates are usually biotinylated and bound to a streptavidin matrix. A simple procedure has been published for the preparation of 4-(biotinamid0)phenylacetylhydrazine and the employment of this substance to biotinylate oligosaccharides at the reducing end [6]. An additional incubation step at 4°C at pH 3.5 greatly reduces the mixture of stereoisomers resulting in an excess of the cyclic P-glycoside form, which also exists in natural N-linked oligosaccharides. This aspect is important in that the type of modification can influence the activity of bio-
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37 Biucore
Signal (mu)
Lipsome capture
Toxin binding
Conditioning
1 1
Regeneration
18 16
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molecules. Heparin, for instance, showed slightly changed binding characteristics depending on whether amino or oxidized cis-diol groups were biotinylated [7]. Glycolipids can be incorporated in liposomes, directly captured on the matrix of the sensor chip (Figure 4),and completely removed after a measurement [22]. In a similar approach using immobilized antibodies for liposome capture, bacterial toxin affinity and binding specificity for glycolipid receptors were determined [ 81. 37.2.2 Surface Regeneration
After immobilization, surface activity and regeneration conditions are tested. Repeated injections with the same sample and regeneration of the surface must lead to identical binding signals and to a stable baseline. Regeneration is needed, whenever the injected partner does not dissociate in buffer flow within an acceptable time. Care should be taken while testing the conditions, in order not to inactivate the surface-bound molecules. Injections of 1 min pulses are recommended, using successively harsher solutions, until all complexes have dissociated and the signal is back on the original baseline level. Acidic solutions down to pH 1 (100 mM HCl), basic solutions up to pH 13 (100 mM NaOH), hydrophobic solutions (up to 50% ethylene glycol), chaotropic ions, detergents, nonpolar solvents, and also chelating agents may be applied. If the inactivation of the matrix-bound molecule by the regeneration procedure cannot be avoided, then a capturing strategy should be applied, by completely regenerating the surface after each analysis and re-loading it with an active interaction partner.
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37.2 Experimental Procedures
37.2.3 Interaction Analysis and Controls Sample injections are typically performed at flow rates of 5-30 yl min-' using sample concentrations corresponding to the affinity of the interaction (K D ,affinity dissociation constant). A measurement will immediately give an answer as to whether binding occurs or not. Nevertheless, each response should be verified through a surface and a sample control. A second trace on the same sensor chip, chemically modified in the same manner as the active surface (e.g. activated/ deactivated), usually serves as a control channel. However, an optimized reference surface carries an inactive molecule, which is similar to the active molecule and immobilized at the same amount. Negative samples like non-specific proteins or carbohydrates, or inactive extracts, serve as a control in solution. The interaction signal during sample injection is divided into two main parts, the so-called bulk response at the beginning and the end of the injection and the actual binding signal (Figure 5). Since the detector reacts to changes in the refractive index, every solution which is optically denser or thinner than the running buffer causes this signal jump. Using the sample buffer as running buffer greatly minimizes this effect and improves the data quality. Possible background binding to the chip surface can be minimized by choosing a different sensor chip (see Table 1). If background is based on ionic interaction with the CM-groups, then the negative charge at the matrix should be reduced by expanding activation and deactivation times to about 15 min each. Increasing the ionic strength in the injected sample up to 500 mM sodium chloride can also help.
Si,
I
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I
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i*
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Time (sec)
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37 Biucove
For the analysis of crude samples, like extracts or serum, a 1 : 10 dilution with running buffer is often sufficient to decrease any background to an acceptable level. Non-specifically bound material or aggregated proteins can usually be removed from the surface by using detergent or 5-50 mM sodium hydroxide.
37.2.4 Determination of Kinetic Rate Constants The dynamics of an interaction are characterized by the speed of complex building and decay and described by the rate constants of association and dissociation. Quantitative kinetic analysis is performed by using a set of curves obtained by injecting samples in concentrations of about 0.1-100 times KD. Based on the mathematical rate equation for the assumed interaction model, a global fitting routine directly evaluates the measured curves. 1 : 1 binding and reactions, like heterogeneity, conformational changes, or bivalence, are predefined in the software, and customized models can be created by the model editor. Published dissociation rate constants range from 6 x lop7 sec-' [9] to 10 sec-' [lo]. Association rate constants, which can be determined by global fitting, are shown in Figure 6. The quality of a fit is assessed by means of the corresponding residual plot. Differences between measured and calculated curves should be near the noise level and regularly dispersed. Any trend in the residuals might indicate use of a wrong interaction model or inaccuracy during the analysis. The choice of the correct model is important as well as proof that the binding partners react in the way the model assumes. For example, a monovalent molecule will bind multivalently if it ag-
-
.---
Very little kinetic information
_.--
Quantitative kinetic information
lo'
-
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37.2 Experimental Procedures
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gregates in solution. This was in fact found during the kinetic analysis of a single chain Fv antibody raised against Salmonella 0-polysaccharide [ 111. Deviations from a 1 : 1 interaction are indicated by altered dissociation curves following injections with different contact times [3]. Mass transport to the surface can affect binding signals of interactions with fast association rates, since the last distance to the immobilized partner must be overcome by diffusion. Nevertheless, due to the defined laminar flow mass transport can be mathematically described and, if necessary, taken into account during the data evaluation [ 121. Transport has no influence on the interaction, when the initial slopes of binding curves obtained at flow rates of 15 and 75 pl min-' do not deviate more than 10% from each other. In general, kinetic analyses should be carried out at flow rates of 2 3 0 p1 min-' on a surface with a low immobilization level corresponding to a maximum surface binding capacity (Rmax)of 10-100 RU. The experimental arrangement should be as simple as possible, in order to ensure robust and secure data evaluation. It can be achieved, for instance, by coupling a bivalent molecule to the surface and injecting the monovalent partner. This results in a 1 : 1 binding scheme, whereas in an opposite construction a bivalent interaction would occur. 37.2.5 Affinity Determination
Affinity can be directly determined from the equilibrium response, which is reached during an injection, when as many complexes are formed as decay and the signal no longer shows any change. The sample concentration leading to 50% surface saturation directly corresponds to the affinity dissociation constant ( K D ) The . data can be evaluated by the existing software program or a standard Scatchard plot [13]. Affinity determination with both partners free in solution can be performed by an inhibition assay [ 14, 151. Interaction partners are mixed and injected after equilibrium binding has been reached in solution. Biacore specifically detects free molecules of one of the interactants. This concentration is inversely proportional to the amount of formed complexes. Low molecular weight carbohydrates often show weak and rapid interactions, reaching equilibrium within seconds after injection and dissociating rapidly during buffer flow. The corresponding response looks like a square pulse, similar to bulk signals. Nevertheless, even transient binding can be quantitatively analyzed by realtime detection (Figure 7). When working with small sugars, active and reference surfaces should be immobilized at the same density, in order to avoid differences in the bulk signal [ 161, or a correction factor should be included [ 2 3 ] . A comparison of affinity and kinetic data ensures consistency of the analysis. For a 1 : 1 interaction, affinity is equal to the quotient of dissociation and association rate constants ( K D = kdiss/k,,,). However, some reactions do not follow a 1 : 1 interaction, and it must be noted that the apparent affinity of bi- or multivalent reactions can be directly affected by the density of immobilized interaction partners. Nevertheless, the chip surface resembles the situation on a cell surface and possibly reflects the in vivo environment better than an assay free in solution [ 171.
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37 Biacore
Req vs Conc plot ,
1500rn
t7 I/
Figure 7. Maltose binding to immobilized anti-maltose antibody is shown. The concentration of maltose ranges over 5.8-1500 1 M with duplicate injections of each concentration. Depicted are the binding signals after subtraction of the background response measured on a surface, which was coated with an unspecific antibody. Affinity determination was done by fitting data from the Plot of equilibrium response (Req)against concentration to the Langmuir 1 : 1 binding isotherm using BIA evaluation software 3.0.
37.3 Application Areas The application range of Biacore for studying biomolecular binding is quite diverse. Some of the references describing analyses of oligosaccharide interactions have already been cited in the text, while three publications are presented below in more detail. 37.3.1 Selectin Binding to a Glycoprotein Ligand Selectins are transmembrane proteins with membrane-distal Ca2+-dependent lectin domains. This family of cell adhesion molecules is involved in the tethering and rolling of leukocytes on the blood vessel endothelium. Nicholson and colleagues [ 101 published the analysis of leukocyte selectin, CD62L, binding to the mucin-like glycoprotein, Gly-CAM-1. The extracellular portion of CD62L was expressed as a fusion protein with two domains of CD4. The resulting soluble monomeric form of CD62L was used to measure the monovalent affinity and kinetics of its interaction with the native GlyCAM-1. Injection of CD62L-CD4 along a matrix coated with GlyCAM-1 resulted in an immediate increase in signal, attaining equilibrium within seconds. Different concentrations of CD62L-CD4 were applied and subtracting the signals on the control surface carrying no GlyCAM-1 from the signals on the active surface resulted in the actual binding response. Fitting the graph of equilibrium binding level against concentration to a 1 : 1 binding model showed a low affinity of K D NN 108 pM. No binding occurred when CD62L-CD4 was applied in the presence of EDTA, or when the control chimera sCD48-CD4 was injected. The binding response dropped with a half time of 0.07 sec after switching to buffer, corresponding to a dissociation rate constant of kdlss NN 10 sec-'. But, since it
37.3 Application Areas
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also takes this time to wash the sample out of the flow cell, dissociation of the complex may even be faster and was therefore given as kdi,, 2 10 sec-'. Direct measurement of the association rate constant was not possible because the reaction reached equilibrium within one sec. However, with the values for affinity, 108 pM, and dissociation rate, 210 sec-', the association rate could be calculated as k,,, 2 lo6 M-' sec-'. A higher affinity was found when the analysis arrangement was changed by immobilizing CD62L-CD4 and injecting GlyCAM-1 along the surface. Binding was apparently detectable at concentrations as low as 30 nM GlyCAM-1, and the dissociation rate constant was reduced approximately by a factor of 10,000 when compared to the data obtained in the above 1 : 1 binding assay. This strongly suggested that GlyCAM-1 interacts in a multivalent manner to immobilized CD62L-CD4. The data supported the hypothesis that the highly dynamic cell adhesion process is based on the low affinity and fast kinetics of selectin interaction. GlyCAM-1 was shown to bind multivalently to immobilized CD62L at concentrations just above its mean serum level, indicating a physiological role by interacting with CD62L embedded in leukocyte membranes. 37.3.2 Oligosaccharide Characterization
An analytical method was described by Blikstad et al. [ 181 to detect unlabelled oligosaccharides or glycopeptides in column effluents. Sumbucus nigru agglutinin (SNA) specific for sialic acid, and Ricinus comrnunis agglutinin (RCA) specific for terminal galactose were covalently immobilized and used to identify different biand triantennary N-linked oligosaccharides. After each measurement, the lectin surfaces were regenerated with a 3 min pulse of 100 mM HCl. Surface stability was found for at least 300 measurements over a period of 2 weeks. For 25 consecutive analysis cycles the coefficient of variation was calculated to be as low as 1.21%. Detection was highly specific, since Sialylated oligosaccharide bound to SNA but not to RCA, whereas desialylated oligosaccharide with a terminal galactose interacted only with RCA. The sensitivity of the analysis was determined by injecting a concentration series of trisialyated triantennary N-linked oligosaccharide (A3) and disialylated biantennary N-linked oligosaccharide (A2) over the SNA surface. Both oligosaccharides could be detected at concentrations of less than 1 nM, while above 200 nM no further increase in signal occurred, indicating surface saturation. Similarly, asialo-galacto biantennary N-linked oligosaccharide (NA2) was detectable at a concentration of 10 nM, using immobilized RCA as detector. During the measurements only 35 pl of sample were consumed for each injection. The feasibility of this assay was demonstrated by using a pronase digest of transferrin. After a chromatographic separation, individual fractions from the column were diluted and injected over the SNA surface. While measuring binding activity to SNA, one major peak showed the presence of responding glycopeptides. These fractions showed practically no UV absorbance. By using other lectins, the method can be extended to detect oligosaccharides containing other structures.
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Combining gel filtration, different glycosidases and different lectins for an analysis, the overall structures of oligosaccharides could be deduced.
37.3.3 In situ Modification of Immobilized Carbohydrates Beside qualitative detection of oligosaccharides and quantitative affinity or kinetic analyses, the in situ modification of carbohydrates immobilized at a sensor chip surface has been published [6, 191. Van der Menve et al. [20] used this approach to analyze the binding of B-lymphocyte antigen, CD22, to the highly glycosylated leukocyte surface protein CD45. Native CD45 (CD45-thy) purified from rat thymus was covalently coupled to sensor chip CM5. CD22Fc (domains 1-3 of mouse CD22 fused to the Fc portion of human IgG) interacted with immobilized CD45-thy. The in situ modification of the sialoglycoconjugates present on CD45-thy by desialylation and resialylation provided the basis for a detailed characterization of this interaction. Muuckiu umurensis agglutinin (MAA) and Sumbucus nigru agglutinin (SNA) lectins, which are specific for a2-3- and 1x2-6-linkedsialic acids, respectively, bound to CD45-thy, indicating the presence of the corresponding sialic acids. Treatment of immobilized CD45-thy by a 30 min injection of Vibrio cholerae sialidase abolished CD22Fc interaction and substantially decreased both MAA and SNA binding. Resialylation of CD45 with GalPl-4GlcNAc a2-6-sialyltransferase using NeuAc as substrate increased SNA binding, while CD22Fc did not bind. CD22Fc interaction was fully restored after 1x2-6-resialylatingCD45-thy using NeuGc as a substrate. Binding of SNA and the lack of MAA binding confirmed the specificity of the a2-6resialylation. The results showed that CD22Fc binds to NeuGca2-6Gal~1-4GlcNAc, which are carried on CD45-thy N-glycans. Moreover, the experiments demonstrated the potential for analyzing binding specificities to selectively modified carbohydrates immobilized at a sensor chip.
References 1. Jonsson U. and Malmqvist M. (1992) Real time biospecific interaction analysis. The integration of surface plasmon resonance. Detection, general biospecific interface chemistry and microfluidics into one analytical system, Adv. Biosensors 2:29 1-336. 2. Karlsson R. and Stihlberg R. (1995) Surface plasmon resonance detection and multi-sensing for direct monitoring of interactions involving low molecular weight analytes and for determination of low affinities, Anal. Biochem. 228:274-280. 3. Karlsson R. and Falt A. (1997) Experimental design for kinetic analysis of protein protein interactions with surface plasmon resonance biosensors, J. Immunol. Methods 200:121-133. 4. Johnsson B., L o f h S., Lindquist G., Edstrom A,, Miiller-Hilgren R.-M. and Hansson A. (1995) Comparison of methods for immobilization to carboxymethyl dextran sensor surfaces by analysis of the specific activity of monoclonal antibodies, J. Mol. Recognit. 8: 125-131. 5. Nath D., van der Menve P. A,, Kelm S., Bradfield P. and Crocker P. R. (1995) The aminoterminal immunoglobulin-like domain of sialoadhesin contains the sialic acid binding site. Comparison with CD22, J. Biol. Chem. 270:26184-26191.
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6. Shinohara Y., Sota H., Gotoh M., Hasebe M., Tosu M., Nakao J. and Hasegawa Y. (1996) Bifunctional labeling reagent for oligosaccharides to incorporate both chromophore and biotin groups, Anal. Chem. 68:2573-2579. 7. Mach H., Volkin D. B., Burke C. J., Middaugh C. R., Lindhardt R. J., Fromm J. R., Loganathan D. and Mattsson L. (1993) Nature of the interaction of heparin with acidic fibroblast growth factor, Biochemistry 325480-5489. 8. MacKenzie C. R., Hirama T., Deng S., Bundle D. R., Narang S. A. and Young N. M. (1997) Quantitative analysis of bacterial toxin affinity and specificity for glycolipid receptors by surface plasmon resonance, J. Biol. Chem. 2725533-5538. 9. Deka J., Kuhlmann J. and Muller 0. (1998) A domain within the tumor suppressor protein APC shows very similar biochemical properties as the microtubule-associated protein tau, Eur. J. Biochem. 253591-597. 10. Nicholson M. W., Barclay A. N., Singer M. S., Rosen S. D. and van der Merwe P. A. (1998) Affinity and kinetic analysis of L-selectin (CD62L) binding to glycosylation-dependent celladhesion molecule-1, J. Biol. Chem. 273:763-770. 11. MacKenzie C. R., Hirama T., Lee K. K., Altmann E. and Young N. M. (1996) Analysis by surface plasmon resonance of the influence of valence on the ligand binding affinity and kinetics of an anti-carbohydrate antibody, J. Biol. Chem. 271:1527-1533. 12. Myszka D. G., He X., Dembo M., Morton T. A. and Goldstein B. (1998) Extending tha range of rate constants available from BIACORE: Interpreting mass transport-influenced binding data, Biophys. J. 75583-594. 13. Karlsson R., Fagerstam L., Nilshans H. and Persson B. (1993) Analysis of active antibody concentration. Separation of affinity and concentration parameters, J. Immunol. Methods 166:75-84. 14. Karlsson R. (1994) Real-time competitive kinetic analysis of interactions between low-molecular-weight ligands in solution and surface-immobilized receptors, Anal. Biochem. 221:142-1 5 1 . 15. Nieba L., Krebber A. and Pluckthun A. (1996) Competition BIAcore for measuring true affinities: Large differences from values determined from binding kinetics, Anal. Biochem. 234: 155-165. 16. Ohlson S., Strandh M. and Nilshans H. (1997) Detection and characterization of weak affinity antibody antigen recognition with biomolecular interaction analysis, J. Mol. Recognit. 10:135138. 17. Shinohara Y., Hasegawa Y., Kaku H. and Shibuya N. (1997) Elucidation of the mechanism enhancing the avidity of lectin with oligosaccharides on the solid phase surface, Glycobiology 7: 1201-1208. 18. Blikstad I., Fagerstam L. G., Bhikhabhai R. and Lindblom H. (1996) Detection and characterization of oligosaccharides in column effluents using surface plasmon resonance, Anal. Biochem. 233:42-49. 19. Hutchinson A. M. (1994) Characterization of glycoprotein oligosaccharides using surface plasmon resonance, Anal. Biochem. 220:303-307. 20. van der Merwe P. A,, Crocker P. R., Vinson M., Barclay A. N., Schauer R. and Kelm S. (1996) Localization of the putative sialic acid-binding site on the immunoglobulin superfamily cellsurface molecule CD22, J. Biol. Chem. 271:9273-9280. 21. Johnson B. and Lofis S. (1991) Immobilization of proteins to a carboxymethyldextran modified gold surface for biospecific interaction analysis in surface plasmon resonance, Anal. Biochem. 198:268-277. 22. Cooper, M. A,, Hansson, A,, Lofas, S. and Williams, D. H. (2000) A vesicle capture sensor chip for kinetic analysis of interactions with membrane-bound receptors, Anal. Biochem. 277r196-205. 23. Karlsson, R., Kullmann-Magnusson, M., Hamalainen, M., Remaeus, A,, Anderson, K., Borg, P., Gyzander, E. and Deinum, J. (2000) Biosensor analysis of drug target interactions, Anal. Biochem. 278:l-13.
Part I Volume 2
V Carbohydrate-Carbohydrate Interactions
Carbohydrates in Chemistry and Biology Editor Beat Ernst, Gerald W. Hart, Pierre Sinay copyright OWILEY-VCH Verlag GmbH, 2000
38 Carbohydrate-Carbohydrate Interactions Dorothe Spillmann and Max M. Burger
38.1 Introduction Cell recognition and adhesion are important events in the formation and maintenance of functional tissues. These processes must be selective and flexible in order to guarantee proper development as in embryogenesis, during the development of the nervous system, for continuous activity of the lymphoid system, but can also be misused by parasitic intruders or aberrant cells like tumor cells. How are glycans involved in these recognition and adhesion phenomena so important for all living organisms? Carbohydrates are the most ubiquitous and prominently exposed molecules on the surface of living cells. The combination of a few building blocks in a seemingly countless array of structures is a principle characteristic of carbohydrate chains that distinguishes carbohydrate sequences from all other biomolecules whether nucleic acids or peptides. Such, these chains offer simultaneously flexible, ordered and easily modulatable motives for cellular interactions. The specific arrangement of individual carbohydrate sequences on surfaces and within matrices creates yet another dimension of diversity: Well defined structures or patches of carbohydrate motives can be created within linear stretches of glycans and glycosaminoglycans (GAG), attached to core protein backbones or located on freely movable lipid anchors for interaction with other molecules [ 1, 21. Glycans are therefore predestined to serve as crucial molecules for recognition and attachment at the moment of cell encounter, Carbohydrates have been recognized as interaction sites in many different instances. Many of these processes are mediated by lectin- or lectin-like molecules that recognise specific carbohydrate motifs. Lymphocyte homing is a multistep process with increasing interaction strengths. A first rolling stage of circulating lymphocytes is dependent on recognition of glycan ligands by protein receptors, the selectins, on the endothelium as well as on the lymphocytes [2, 31. Neuronal development depends on reversible cell extensions and connections followed by disruption which requires the proper interaction with cells, extracellular matrix and solu-
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ble factors like growth factors. Many of these interactions have glycans implicated [4-61. Plant and mammalian lectins have defined roles in tissue maintenance and defense. Glycan ligands are also used as primary receptors by microbial pathogens [7, 81. Many of these interactions are characterized by fairly weak forces which, however, are easily potentiated by orders of magnitude through multimerization of ligand and receptor [2, 9, lo]. Further along this conceptual line is the idea of carbohydrate-carbohydrate interactions mediated by specific carbohydrate sequences in an ordered polyvalent array [ l l ] . Instead of the protein receptor for a glycan ligand, a second glycan is receptor for the first. Such interactions could be both specific and easily controlled by external influences like ionic conditions, shear forces, availability of substrates and biosynthetic activity of genes involved [ 121 and would therefore be ideal for early steps in cell interactions where links should have modest affinities in order to guarantee a quick and flexible reversion. The property of potentially weak interactions, on the other hand, raises one major difficulty, namely the question how to measure such interactions. In cases of structural components as found for instance in plants where direct carbohydrate-carbohydrate interactions have been postulated first, the number of binding sites is extremely large and therefore the avidity reasonably high to be measurable by classic methods of biophysics and biochemistry. In more dynamic systems, like in mammalian tissues that undergo continuous restructuring or underlay shear forces from body fluids the possibilities of carbohydrate-carbohydrate interactions are obvious, but hard to measure. The number of publications dealing with this special aspect of glycobiology is therefore limited. However, the advent and continuous refinement of modern methods like atomic force microscopy, nuclear magnetic resonance, surface plasmon resonance or the increasing availability of tools like anti-
Figure 1. Schematic overview of carbohydrate-carbohydrate interactions. Binding of carbohydrate sequences to one another can be envisaged both in cis and trans mode at the cell surface between different or identical glycoconjugates and between identical or different types of sequences. Also cell-matrix interactions might be stabilized in this way. I protein core, 1 lipid core, I carbohydrate chains
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bodies and homogeneous, synthetic ligands should allow it to study such phenomena equally well as other types of interactions where carbohydrates are involved. This Chapter is divided into a first general part with an overview of the biological background for the different models and is followed by a discussion of the molecular details assumed to play a role in such interactions. Finally, methods in use for assessing carbohydrate-carbohydrate interactions are described. An extensive description of “cook-book’’ recipes is omitted as the use of most methods is discussed in detail in other parts of this series and does not differ for different applications whether carbohydrate-carbohydrate or carbohydrate-protein interactions are analyzed.
38.2 From Structural Components to Cell Recognition 38.2.1 Carbohydrate-Carbohydrate Interactions as Part of Structural Components Extracellular Matrix of Seaweeds-Agarose, Carrageenan and Alginate
The classical view on carbohydrates is the one of the energy store and the structural component, whether extracellular space filler and cushion or as part of a cell wall. In this latter form, carbohydrate-carbohydrate interactions have been proposed for the first time. Agarose, carrageenan and alginate, are examples of carbohydrate networks which are produced by different species of algae as intercellular matrix. They are examples for structural networks created from regular and random stretches of carbohydrate chains which form hydrated, elastic gels, that are stabilized by interchain hydrogen-bonding and ionic complexing thereby providing a very basic form of carbohydrate-carbohydrate interaction. Agarose and carrageenan are each built of two alternating sugar residues that form a repeating unit ([3,6-anhydrofor agarose ~-Galal-3o-Galpl-4]and [3,6 anhydro-~-Galwl-3~-Ga1(4-OS0~)/31-4] and K-carrageenan, respectively). The chains can form double helices which are interrupted at sites where the chains kink due to further modification of the basic units by sulfation or dehydration. The extent of modification, which is under biosynthetic control, determines the structural behaviour of the chains and thereby affects the functional properties of the gelling polysaccharides [ 131. Alginates are another group of glycan networks. They are composed of homo- and heteropolymers of p-D-mannuronic acid and its C5 epimer a-L-guluronic acid, and are produced by brown algae and certain strains of Pseudomonas and Azotobacter bacteria [ 141. The special characteristics of alginates are determined by the sequence of building blocks in the single chain. Blocks of homogeneous mannuronate or guluronate polymers alternate with heteropolymers containing both units in variable order of sequence. Due to the conformation of the two uronic acids, the different types of sequences adopt different three-dimensional conformations that in turn determine the extent of chain-interaction. While the mannuronic acid is in a preferred 4C1 chair conformation and the mannuronan-homopolymer therefore adopts
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an extended structure of either a ribbon in its acid form or a threefold helix in its salt form, the guluronic acid conformation of Cq drives the guluronan-homopolymer to adopt a twofold buckled ribbon, that is more condensed [ 141. In presence of Ca2+ ions these so called G-blocks complex to egg-box like structures, that lead to intra- and interchain cross-linking, for the first time suggested by Rees 1131. A high content of guluronic acid is consequently resulting in much stiffer gels due to carbohydrate-carbohydrate binding while high mannuronic acid contents provide more loosely, flexible gels that have a low degree of chain contacts. The extent of interaction can be modulated not only by the ratio and distribution of guluronic and mannuronic acid along the chain, but also by post-polymerization modification. Some of the bacterial alginates for instance, though identical in basic structure to the algae structures, are 0-acetylated on mannuronate which changes their Ca2+uptake and complexing characteristics [ 151. Cell Walls Many cell walls from bacteria to plants are predominantly composed of carbohydrates. Especially capsular polysaccharides of bacteria have attracted the interest because of their properties being similar to other polysaccharide structures found in mammalians and as virulence factors. In the case of Bucteroides fragilis the identity of a virulent capsular polysaccharide has been identified and reveal an unusual dimeric carbohydrate complex of a polysaccharide chains A and B. The two polysaccharides are linked by non-covalent electrostatic interactions between negatively and positively charged groups within both polysaccharide chains providing a carbohydrate-carbohydrate interaction [ 161. Considering findings with isolated polysaccharides as bacterial colominic acid 1171 or the behavior of GAGS (18, 191, one might anticipate similar phenomena to occur within capsular structures in more than the Bucteroides example. Another form of very simple carbohydrate-carbohydrate interactions can be found in plant cell walls. These walls contain a high content of carbohydrates of which approximately 20% are cellulose microfibrils, some 70-80% are non-cellulose polysaccharides, hemicelluloses and pectins, and up to 10% of the mass is glycoproteins. While some of these structures are very conserved over a wide range of organisms (like cellulose), others are not, as detected by specific antibodies 1201. Cellulose, the homogeneous polymer of P-4-linked D-glucose and the polymer with the largest biomass on earth, forms the basic frame of plant cell walls with rigid microfibrils. Hydrogen-bonds and hydrophobic interaction between the individual, parallel chains stabilize the assembly to a near crystalline form [21, 221. These homogeneous fibrillar structures are cross-linked by an uncounted number of different heteropolysaccharides. Hemicelluloses (xylans, gluco- and galactoglucomannans, xyloglucans and branched glucans), on the one hand, and pectins (galacturonans, rhamnogalacturonans, arabinans, galactans and arabinogalactans), on the other hand, create a fine network between different cellulose fibres. The non-cellulose chains are all more heterogeneous and linear and branched elements create even more versatile structural properties. In this way a three-dimensional network is created that guarantees the physical properties of the plant cell wall. Covalent ester
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b
0
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Figure 3. Segment of a plant cell wall (schematic drawing). Bundles of cellulose microfibers in near crystalline packing (thick bundles) are crosslinked by hemicelluloses (drawn as double-lined intercalated network) and pectins (fine lines with egg-box interaction sites). Carbohydrate-carbohydrate interaction occurs between individual cellulose chains, between cellulose and xyloglucan (a hemicellulose) and between different hemicelluloses and pectins. Besides different forms of non-covalent interactions (Ca2+ mediated “egg-box” structures as well as ion-independent forms of molecular interactions) also covalent ester linkages between carbohydrate chains are found (adapted from ~31).
linkages between individual polysaccharides are found as well as ion-complexed “egg-box” structures and non-covalent carbohydrate-carbohydrate hydrogenbonding [21, 23, 241. Xyloglucan for instance associates in carbohydrate--carbohydrate manner to cellulose, both within the cell wall as well as extracted in in vitro assays [21, 2.51. Still there are plenty of questions open concerning the threedimensional structure of different cell walls especially considering the needs of the plant during development and growth, when the cells should overcome the rather rigid network and be able to extend themselves [20]. Mammalian Extracellular Matrix Components
Although the number of different molecules present in matrices of higher organisms is clearly larger than in more simple organisms similar concepts of structural organization of carbohydrate networks might still serve as a basic fundament. The extracellular matrix and cell surfaces of higher eukaryotic organisms consist of a rich assembly of highly glycosylated molecules [26-281. Among those, proteoglycans with their long GAG chains provide motifs that could serve not only for binding proteins [ l , 291 but might also show a higher order of structural organisation. Matrix constituents are glycoproteins, proteoglycans and the only type of free carbohydrate chains, hyaluronan (HA). This last one is not only an important component of extracellular matrices, but also essential component of the vitreous body of the eye and of synovial fluid. HA chains are the most simple type of GAG chains with the basic structure of an alternating glucuronic acid and glucosamine unit to HA chains in form long, uniform chains of the structure [GlcA~l-3GlcNAc~l-4].. solution are highly hydrated randomly kinked coils, which are entangled at concentrations of less than 1 mg/ml as seen by sedimentation analysis and viscosity
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measurements, function as lubricator and as controller of liquid and macromolecular flow (for review see [30]). Although they interact with proteins in the matrix and on the cell surface their gel forming properties are also occurring in protein-free preparations [311, by direct interaction of chains with one another. Fibrillarization of HA chains with one another has been observed by electron microscopy [ 19, 321 as well as other techniques like spectroscopic methods [33] and viscosity measurements [34]. This purely carbohydrate mediated network might well serve as basic cushion into which other molecules are integrated. In the crystal an antiparallel helix has been described [35, 361. Driving force for the complexing could be the exposure of hydrophobic stretches stabilized by intramolecular hydrogen bonding [37] which would lead to hydrophobic association of the chains. The protein-bound forms of GAG chains, namely the heparan sulfates (HS) and chondroitin sulfates (CS) that are parts of the ubiquitous proteoglycans have been tested for direct carbohydrate-carbohydrate interaction in vitro. Isolated GAG chains free from their core proteins were found to form double-helical structures with other GAGS [38]. GAG chains immobilised on beads to mimic their natural, polyvalent appearance on protein cores, showed GAG specific interactions [ 39, 401. HA modified beads aggregate with chondroitin-6-sulfate (CSC) beads and less so with chondroitin-4-sulfate (CSA) but only very modestly with heparin or dermatan sulfate (DS) modified beads. Neither of the beads showed homotypic aggregation. These interactions were independent of Ca2+ ions and required intact, full-length chains, as neither oxidized nor fragmented chains were mediating bead aggregation, thus indicating a strong need for polyvalent interaction [39]. Similarly, rotary shadowing and electron microscopy showed aggregation to meshes of CSC with itself or HS, but not with CSA, and CSA did not form networks with itself [ 181. A potential explanation could be the interaction of hydrophobic sites within CSC to aline with hydrophobic stretches within HA, or in another molecule of CSC, whereas the 4-sulfation of N-acetyl-galactosamine in CSA would disturb these hydrophobic patches explaining the reduced self-complexing of CSA compared to CSC [18]. HS and DS chains, on the other hand, were proposed to bind in a homotypical way to the respective type of GAG either as free chains or multimerized on the core protein [41,42]when tested by affinity chromatography on HS-modified matrices. This affinity interaction was dependent on intact sulfation and carboxylation of the chains, desulfation or reduction of the carboxyl groups did abolish the binding [43], and on the cooperative interactions of several sites within an intact chain [44]. Such GAG-GAG interactions as measured in vitro could be of value at the interface of cells and matrix in tissues with a high content of GAGS (i.e. cartilage) while they are probably of rather negligible importance in others considering the large number of potentially GAG-binding proteins present in the tissue. Of relevance they might be in particular pathogenic situations where GAG-chains become accumulated [45] as for instance in the deposition of plaques in amyloidoses. Glycolipids have been postulated to assume structural purposes via carbohydratecarbohydrate mediated interactions for instance by stabilizing myelin layers around axons. Galactosylceramide (GalCer) and cerebroside sulfate (CBS) are the predominant glycolipids found in myelin in higher vertebrates and it has been suggested that the
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Table 1. Different classes of glycans suggested in carbohydrate-carbohydrate interactions. Structure”
Type of interactionb
Organism/System’
K-Carrageenan Agarose Alginate Cellulose Cellulose/Hemicellulose Neutral glycans Glycosaminoglycans Acidic glycans Zwitterionic glycans Glycolipids
homo homo homo homo hetero hetero homo/hetero homo/hetero? hetero homo/hetero
Algae Algae Algae, bacteria Plants, bacteria Plants In vitro In vitro Marine sponge, In vitro Bucteroides frugilis Different cell types
Reference
a Classes of glycan structures associated with carbohydrate-carbohydrate binding hIndicates whether identical or different sequences of the same class of glycans are interacting with one another “Orgdnism/model in which these interactions have been described
amount of these ceramides correlates with the stability and compactness of the myelin sheet [46]. In vitro liposome assays have demonstrated a preferred heterogeneous interaction of GalCer-liposomes with CBS-liposomes in presence of Ca2+ over a homogeneous interaction of either glycolipid type with itself [46, 471. 38.2.2 Carbohydrate-Carbohydrate Interactions as Part of Recognition Keys? In all of the examples described so far, often homogeneous types of carbohydrate sequences interact with neighboring chains. Stable structures are created by cooperative action of a multitude of low affinity binding sites that accumulate to an avidity keeping for instance cell walls together. In none of these examples, however, is there flexibility tolerated unless on the expense of stability. A hyaluronan-pad can be compacted or extended by exchange of H 2 0 and ions, but it is stable unless it becomes degraded by enzymatic or chemical means. Cell walls, once created, are rigid and flexibility must be “bought” by the action of reconstructive enzymes, sofisticated hydrolysis processes and biosynthetic adaptation to new requirements [23].A totally different situation arises, if carbohydrate-carbohydrate are considered in recognition and adhesion process of tissues that are dependent on flexibility. Here, advantage could be taken from the fact that carbohydrate-carbohydrate interactions are rather low affinity interactions and be used as primary, reversible contact glues that allow the organism to quickly release or reinforce adhesion between two cells. Mainly two lines of research have been followed testing these possibilities: The properties of an extracellular aggregation molecule that mediates aggregation of marine sponges, on the one hand, and cell surface glycoconjugates that participate in the adhesion of embryonic and tumor cells, on the other hand.
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Carbohydrate Interactions in Invertebrates-The Marine Sponge Microciona prolifera as a Model System Sponges are the most simple multicellular organisms. They do not have any defined tissues or organs instead their pluripotent cells remain motile within the animal and maintain different functions. For this simplicity and for the fact that mechanically isolated cells have the property to reaggregate and reform animals [48, 491 the marine sponges have been model systems for studying recognition and adhesion in multicellular organisms since the beginning of the century. Early it was recognized that cell suspensions from two different sponges do sort out when mixed and reaggregate in a species-specific fashion. As the sponge is feeding on filtration basis, the animal is continuously in contact with foreign particles a fact which probably has helped to let the animal evolve a modus for recognising self u s foreign. Equally well has this sponge model been one of the first examples where carbohydratecarbohydrate interactions have been postulated in context of a recognition phenomenon [50].The extracellular matrix of the sponge is similarly composed as higher eukaryotic tissues, containing both proteoglycan-like complexes as well as collagens and other glycoproteins. A major adhesion mediating factor of the sponge tissue has been isolated in form of a proteoglycan-like complex from Microciona prolifera [51].This large complex, the Microciona aggregation factor (MAF), with a carbohydrate content of more than half the molecular mass of the macromolecule has been shown to mediate species-specific aggregation of dissociated sponge cells [52-54] in a Ca2+-rich environment (-10 mM, as in sea water). In the electron microscope, the macromolecule appears as a sunburst [55]with a ring of -200 nm diameter to which up to 20 arms are attached, a feature confirmed by more physiological assessments of the structure by atomic force microscopy (AFM) in its hydrated form [56]. EDTA-treatment of the complex disrupts the arms from the central ring core, indicating that the cation is important for the structural integrity of the complex [54]. Several hundred Ca2+-binding sites are also important for the Ca2+-dependent self-interaction [ 571. Two functionally distinct binding sites have been identified for the complex, one of which is a Ca2+-independent binding site to cellular receptors and the second, the Ca2+-dependent self-association site which is providing the intercellular adhesion force [57]. Cellular receptors may include membrane-associated glycoproteins of 210 kDa and 68 kDa [58] and have a high affinity to both, the cells as well as the aggregation factor [59, 601. The carbohydrates are essential for the adhesion and recognition process as either glycosidase treatment [61, 621 or periodate oxidation abolish the aggregation of isolated sponge cells [57].A carbohydrate-carbohydrate interaction of the glycans by themselves was suggested by experiments with protein-free glycan preparations from MAF, where a Ca2+-dependent aggregation of beads could be demonstrated with multimerized glycans [I I]. Species-specific binding could be achieved by either the intact aggregation factor or the polymerized glycans to create de novo polyvalence [50, 631. Ca2+ is essential for aggregation of the glycans and cannot bereplaced by other divalent cations [64]. At much lower charge concentrations, polycations as polybrene or polylysine are able to replace Ca2+ indicating a cooperative effect of the polycations with the polyanionic carbohydrate chains. The quantitative
1070
38 Carbohydrate- Carbohydrate Interactions
Figure 4. Model of polyvalent interaction between two Microciona prolifera cells. The high molecular weight proteoglycan-like complex of MAF is composed of -20 arms attached to a central ring each composed both of protein and carbohydrate components. The small, N-linked g6 glycans are predominantly localized in the arms of the molecule [55] and supposed to interact with the cell surface [67]. The large glycans associated with the self-association property of the complex [I 11have not been unambiguously localized but might correspond to the larger glycans identified in the central ring ([55] and J. Jarchow personal communication) and are recognised by anti-MAF-antibodies blocking the self-aggregation [ 11, 68, 691, adapted from [ 121.
differences in interactive strength of various polycations could reflect the variations around an optimal fit of the polycations to the polyanionic glycan chains of MAF as demonstrated by phase-partition assays [65]. Whereas many different polycations are able to precipitate the intact factor glycans, the self-interaction of glycans under physiological conditions, i.e. in the presence of Ca2+ ions, is restricted to glycan chains of the same species [50, 631. These findings indicate that the self-interaction
38.2 From Structuvul Components to Cell Recognition
1071
of the glycan chains is not due to unspecific charge interaction of polyionic chains. Rather, the glycans contain a species-specific arrangement of residues in a proper spacing of interactive sites, whether these are single-charged residues in a defined distance [65] or sequences of several residues 1661. Monoclonal antibodies which inhibit the glycan mediated self-aggregation of factor molecules [ 11, 671 are directed against distinct carbohydrate motives [68, 691 favoring the sequence model. Observation of cross-reactivity of some of the monoclonal antibodies with glycans from different species [ I I ] and the observation that different sponge type cells may first aggregate randomly before sorting out [88] could easily be explained by the different arrangement of otherwise identical or practically identical motives. Carbohydrate Interactions in Vertebrates-Embryonal and Tumor Cells Eukaryotic cells express, besides glycoproteins, high amounts of different glycosphingolipids (GSL) on their plasma membranes and many of them are subjected to developmental regulation. Not only embryonic cells but also tumor cells undergo changes in the expression pattern of GSL and therefore these glycoconjugates became classified as onco-developmental antigens [47, 701. Early mouse embryos at the 8- to 16-cell stage when expression of the stage specific embryonic antigen 1 (SSEA-1) is highest, are susceptible to decompaction by the analog free hapten, namely the blood group antigen LewisX (Le": Gal01 +4[Fucul-+3]GlcNAc~l+ 3Gal/31+4Glcp) [71]. The embryonal carcinoma cell line F9, which shows a selfaggregation capacity, expresses also a large number of LeX epitopes, whereas the more differentiated embryonal cell line PYS-2 does not self-aggregate and does not express LeXepitopes [72]. The correlation of self-aggregation capacity with Le" expression level was corroborated by binding properties of these cells to Le" coated culture dishes indicating that F9 cells depend on a Le" based binding mechanism. F9 cells were therefore used to analyze the mechanism behind the LeX mediated decompaction of either morula stage embryos or aggregated tumor cells. Indeed, also F9 cell aggregation turned out to be sensitive for competition by Le" containing oligosaccharides. In isolates from F9 cell membranes both Le"-containing glycoproteins [72, 731 as well as GSL having this hapten were found to bind in a Ca2+-dependent fashion to Le"-modified matrices [73]. The glycan nature of the aggregation mediator was further supported by the observation that protein-free embryoglycan chains from the F9 cells precipitated in presence of Ca2+, but not EDTA and were sensitive towards removal of fucose residues, suggesting that the LeXepitopes can mediate a Ca2+-dependent carbohydrate-carbohydrate interaction [72]. Not only Le" epitopes can mediate cell interaction but also diverse other glycans. Undifferentiated human embryonal carcinoma 2 102 cells express high amounts of the LeXprecursor nLc4 and SSEA-3 (with the major epitope GalGb4) and Gb4 and these cells bind well to Gg3 (GalNAc~1+4Gal~1+4Glc~l+lCer) (GalNAc~1+3Galu1+4Gal~l+4Glc~l+lCer) coated dishes while they do not adhere to several other tested surfaces. The binding is dependent on the differentiation state of the cells as retinoic acid or bromodeoxyuridine induced differentiation alters both, the expression level of these GSL on the cell surface and the adhesion phenotype of the cells [74]. Furthermore, binding to Gb4 was reported to induce
1072
38 Curbohydrute~CurhohydrateInteractions
activation of the transcription factors AP1 and CREB, while binding to Gg3 did not result in any cell activation [74] indicating that there is also some qualitative difference in binding to different GSL layers. A similar observation was made for B16 melanoma cells either plated on non-coated, G M (NeuAca2+3Galpl+ ~ 4Glcpl+lCer) or Gg3 coated surfaces. Only on the preferential coating, the Gg3 surface, adhesion went along with enhanced tyrosine phosphorylation of FAK 1751. That direct binding between G& on the cell surface to Gg3 on the culture dish is the activating mechanism, is suggested by the ability to activate FAK also with ~ antibodies against G M1751. When cells interact with other cells or with the extracellular matrix, not only glycolipids may be involved in the interaction with the surroundings but equally well glycoproteins. In what context do these different molecules act? What is the role of other adhesion molecules that are well known mediators of cell interaction and what forces could be tolerated by the different combinations? Work with the mouse melanoma cell line B16 addressed these questions. As seen before with the embryonal carcinoma cell lines, also the melanoma cells could adhere in a GSLdependent manner to either other cells or glycolipid coated culture dishes [76]. The adhesion and spreading on coated culture dishes was most obvious at early stages of cell plating indicating that GSL mediated interactions are very early phenomena in cell interactions and will be overtaken by protein-mediated binding 1771. This fact was also confirmed by plating cells on dishes coated both with GSL and extracellular matrix proteins. There, melanoma cells could adhere and spread faster on glycolipid coated plates while the adhesion and spreading on laminin or fibronectin coated plates were slower [78]. Different clones of the melanoma line B16 with different expression levels of the predominant GSL were tested for their binding behavior to non-activated endothelial cells. Binding of melanoma cells was de~ level in the static system and faster, but weaker pendent on their G Mexpression than binding to laminin or fibronectin. Under shear forces, binding strength of the , also stronmutant B16 cells was still correlated to the expression level of G M ~but ger than the binding via the proteins. From these findings the hypothesis was raised that metastatic tumor cells make use of the high expression rates of certain glycolipids to attach to the unstimulated endothelium, before the next steps in cell activation and transmigration are mediated by protein-protein interactions [79]. Repulsive Carbohydrate-Carbohydrate Interactions
Where there are adhesive carbohydrate-carbohydrate interactions, there must also exist anti-adhesive or repulsive ones. Aggregation factors and glycans from different sponge species show only little or no cross-reactivity [ I l l or similarity in glycan composition [80], and beads coated with aggregation factors from different species sort out [SO, 811 as cells have been known to do since long ago. These properties might suggest that there are sequence compositions that are not promoting adhesion as explained by the zipper model [ 121 and therefore participate in the self us non-self recognition phenomenon of sponge cells. Examples of non-adhesive carbohydrate-carbohydrate interactions are also seen between different GSLs. GSL-GSL interactions have been found to promote
38.2 From Structural Components to Cell Recognition
1073
Table 2. Carbohydrate motifs suggested to interact in carbohydrate-carbohydrate interactions. MotiP -
-
Organism/System"
Reference
Algae, bacteria In vitro Early mouse embryo, teratocarincoma cells Melanoma/lymphoma cells Melanoma/endothelial cells Teratocarcinoma cells Teratocarcinoma cells In vitro liposome assay In vitro liposome assay In vitro liposome assay
~ 4 1 [171 [71, 1221 [761 [791
~
[GulA],-(GulA], PSA-PSA Lex-LeX G~3-Gg3 GM3-lacCer Gb4-nLc4 Gb4-GalGb4 GalCer-Gal(S03)Cer H-H H-Ley
PI
(741 [461 ~901 [901
Identified structure or epitope suggested to interact in a carbohydrate-carbohydrate interaction bDescribes in what context these observations were made
a
binding, to be neutral or to be anti-adhesive [82]. An example of an anti-adhesive GSL interaction has been observed for B 16 melanoma cells coated on GM3-plates. While cells coated on Gg3 were nicely spreading, less than 1% of all cells did so on G M ~Sialidase . treatment of the dish could revert the effect, so that cells did spread to control levels, indicating that a G~3-Gh.13interaction is of repulsive nature [77]. Control of GSL expression could therefore easily allow the switch from a nonadhesive to an adhesive phenotype or vice versa by the activation/inactivation of a sialyltransferase. A fascinating oligosaccharide modulating neuronal plasticity by repulsive carbohydrate-carbohydrate interactions is polysialic acid (PSA). PSA is a homogeneous polymer of a2-8 linked sialic acid residues [83], which in neuronal tissue is expressed almost exclusively on the neuronal cell adhesion molecule (N-CAM) attached to a N-linked core glycan [84]. The role of PSA on embryonal forms of N-CAM, but also on adult forms as on axons that undergo plasticity or synaptic remodelling, is to prevent axon fasciculation and thereby guaranteeing easier axon migration towards the target, and higher accessibility of the axon for signalling molecules from the target. The injection of endo-neuraminidase during embryonal development leads for instance to serious pathfinding problems for the motorneurons as they should brake away from their neighbors in the plexus once they have emerged from the neural tube. In adult tissue, where the amount of PSA on N-CAM is drastically reduced this repulsive effect is lost. The mechanism behind this anti-adhesive carbohydrate-carbohydrate interaction could be pure ionic repulsion or physical hindrance due to the large hydration volume of the polymer. In either way, cell contacts can be affected in trans or cis fashion, hindering either receptor-receptor interaction between two cells or affecting receptor mobility and interaction with other molecules within the cell membrane which ultimately also leads to altered cell-cell contacts [84]. An unexpected finding with isolated PSA from either bacterial or mammalian source, however, demonstrates an adhesive capacity of the
1074
38 Carhohydrate-Carbohydrate Inteructions
Table 3. Carbohydrate motifs suggested to function in anti-adhesive mode. Motif
Organism/System
Reference ~-
PSA>
LeY>
Embryonal brain Melanoma cells In vitro liposome assay Marine sponges
[61 [771 [901
P O , 811 ~~
"A carbohydrate-carbohydrate interaction is only indirectly suggested as the sponge glycans are postulated to bind in a homophilic way [ 11, 671. By bead-aggregation and AFM, however, sorting out of MAF and HAF modified beads could be demonstrated, whereas a direct proof of MAFglycan-HAF-glycan interaction has not yet been established.
chains as observed by atomic force microscopy. Similar to HA, PSA forms bundles in the presence of Ca2+ beyond a chain length of 12 units and longer fragments have a tendency to form branched bundles [17]. These findings refresh the discussion about the mechanism and functioning of PSA in the developing brain and in bacterial cell walls [ 171.
38.3 Molecular Aspects of Carbohydrate Interactions 38.3.1 Polyvalence to Inforce Weak Interactions Molecular interactions where carbohydrates are involved are usually weak interactions. Proteins usually recognize and bind to just a few carbohydrate residues within an oligosaccharide, whether it is an antibody or a lectin. Nature adopts to the needs of higher affinity requirements by the multimerization of either protein or carbohydrate or both units in order to reach avidities that can hold molecules and cells under physiological conditions [2, 661. Even more true is the need for polyvalence in the case of carbohydratecarbohydrate interactions. Association of glycoconjugates as small as a mono- or disaccharide with another sugar molecule can be observed by methods as mass spectrometry [85, 861 or crystallography [87]. The affinities are, however, so low that the stability of the complexes would not even survive the measuring time in NMR [72] not to think of a situation in vivo. Polyvalence, i.e. the repetition of a binding motif in either of different modes, allows these molecules to bind together and gives the scientist the chance to measure them by different methods. Sponge cell aggregation mediated by isolated glycans needs the de nouo polymerization of the isolated chains into longer, multivalent complexes to achieve approximately the size of the native aggregation factor [50, 631. Polyvalence could also be achieved by attachment of glycans to beads which could aggregate in a species-specific manner [ 11, 501 confirming earlier proposals and data [65].
38.3 Molecular Aspects of Carbohydrate Interactions
1075
The same needs hold true in the GSL-mediated interactions of tumour cell lines. The adhesion capacity of cells as well as liposome aggregation and binding are dependent on the concentration of glycolipids on the receptor side. Correlation of binding strength was both seen for the amount of a specific GSL expressed on a certain cell line as well as to the concentration of the glycolipid on the substrate side 174, 771. Also the length and degree of hydroxylation of the fatty acid portion has an influence on the interaction of glycolipids with one another, suggesting that developmental control or pathological changes can affect carbohydrate-carbohydrate mediated cell interactions [46]. In the carbohydrate-carbohydrate model of cell interaction advantage is taken from single low affinity sites. Control of the polyvalence by various means (surface density of presented structures, ionic strength to modulate attractive vs repulsive forces, subtle changes in biosynthesis of the carbohydrate sequences, etc.) to change the affinity of the interactive molecules provides an adaptable system beyond a structural scaffold. Such recognition systems are created which allow different cells to test surrounding surfaces and release or reinforce interactions [SS]. Speciesspecific sorting is one example of the value for moderate interaction strengths which achieve biological relevance through polyvalence. Moderate strength is also required within one species, e.g. within an individual sponge, otherwise the cells could not migrate past each other. In an experiment where factor-mediated aggregation was induced by polybrene instead of Ca2+, secondary migration of cells was inhibited. This observation demonstrates the drastic effect of “locking” the molecules in a permanent tight interaction mode 1651. 38.3.2 Arrangement of Motifs and the Possibility to Control Specificity
A Velcro pad or a zipper can be used as simple models to highlight the value and simplicity by which nature may create specific and lasting binding sites by such carbohydrate-carbohydrate interactions from compositionally rather similar structures [66]. The creation of repetitive, interacting glycan sequences is feasible in different modes. Oligosaccharide motives can be repeated along the primary glycan sequence as seen in the examples of plant cell wall carbohydrates. Glycans can also be arranged in a repetitive pattern along a backbone structure which is not directly participating in binding, as illustrated by mucin structures on protein backbones [2] or by branching carbohydrates on a glycan scaffold as for instance blood group antigens on poly-N-acetyllactosamine type glycan backbones [70, 891. Finally, proteoglycans, glycoproteins or glycolipids can be presented in clusters or surface superstructures partly due to mobile anchors. Only in this last version advantage is taken of the fluidity of the biomembrane for control of the surface density of these glycans and therefore their avidity [2, 66, 901. There are indications that GSL form clusters within membranes and are not randomly distributed 1751. Indeed, GSL accumulate on apical surfaces of epithelial cells [91, 921, are observed as patches in erythrocyte membranes [93] and peripheral lymphocytes 1941 or as morphological clusters in vitro in liposomes as detected by electron microscopy of freeze fractured samples 1951.
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38 Carbohydrate-Carbohydrate Interactions
Primary glycan sequences determine whether there is the possibility of interaction, but how these sequences will be arranged in a three-dimensional context will define the specificity of interaction. Even though sequences could be similar, different spacing in the three-dimensional architecture could create a “non-binding” phenotype as illustrated with two half-chains of a zipper being of different dimensions. These architectural differences could even be gradual, allowing for instance two different sponges to first slightly complex before sorting out again [88]. Or a tumor cell with a high density of a certain GSL be an effective metastatic colonizer, whereas the one with a lower concentration of the same GSL be less efficient due to reduced primary binding capacity [96]. The next step in binding via carbohydrates is the question how for instance a certain combination of GSL would trigger the cells to activate signalling molecules while another combination, although still mediating binding would not activate the cell [75].
38.3.3 Molecular Basis of Carbohydrate-Carbohydrate Interactions The forces playing between carbohydrates are no different from those acting between other biomolecules. Most carbohydrates are neutral or negatively charged due to carboxy- and sulfate groups, although positively charged glycans also occur [16, 971 and ionic interactions can be anticipated. Except for rare ionic interactions between oppositely charged glycans [ 161, direct carbohydrate-carbohydrate interaction between two anionic carbohydrate rather leads to repulsive forces as nicely illustrated for PSA [84].With the aid of divalent cations, this effect can be overcome as demonstrated for sponge cell aggregation, where both carboxyl and sulfate groups as well as Ca2+-ionsare important for proper species-specific aggregation of cells [57, 981. However, not just any kind of acidic sponge glycan is interacting in the presence of Ca2+ indicating that the effect of Ca2+ is not merely a charge effect. Another possibility of the action of Ca2+ is the property to complex carbohydrates via a suitably arranged combination of sugar hydroxyl groups. Single hydroxyl groups in sugars are too weak to coordinate cations in the presence of water molecules. However, in the combination of two to three well positioned hydroxyls on one sugar residue or over two adjacent residues, cations can become coordinated to the carbohydrate which can force the chain into a specific conformation or lock it there as seen for instance with guluronic acid sequences in alginate [ 141 or pectins [23].The preference of Ca2$ over other cations lies in the molecular dimensions and complexing properties: Complexing strength raises from mono- to trivalent cations, while the ionic radii determine how well the ions fit into molecular dimensions of the molecules which are optimal around 100-1 10 pm as seen for Na+, Ca2+,La3+ [99].Some, but not all, of the GSL interactions have been shown to depend on Ca2+ and it is not clear what is the difference between those. For the interactions of G M ~ with Gg3 and Le”-Le” an association of the molecules via their hydrophobic sides that complement each other followed by a locking through Ca2+ has been proposed based on molecular modelling [ 72, 761. However, a similar molecular modelling approach has led to the suggestion that Ca2+ is the interlinking force between the
38.3 Molecular Aspects of Carbohydrate Interactions
1017
two Le" molecules [85].No Ca2+ was present in the Le" crystal [87], why further studies are required to resolve the exact role of Ca2+ in this interaction. Carbohydrates offer a rich source for hydrogen-bonds due to hydroxyl-, amineand carboxy-groups. Hydrogen bonding can be seen intramolecular as well as intermolecular or in combination with the solvent. An extremely high number of hydrogen bonds could be seen in the crystal of Le" [87] with bonds between the trisaccharide and water and between the carbohydrates themselves. An indirect consequence of intramolecular hydrogen bonding has been suggested for HA. As with the locking properties of Ca2+ a similar fixing the conformation has been proposed for neighbouring residues in HA, which would lead to exposure of a larger hydrophobic patch in the chain, which in turn would favor hydrophobic interactions between different chains explaining chain interactions [ 371. An opposite interpretation of similar data from molecular dynamics models for short saccharide sequences would suggest the rapid exchange of different intramolecular H-bonds in favor of a prolonged solubility of even high concentrations of HA [ 1001. Improved measuring methods must be awaited for deciding which ones are the driving forces in vivo. Also hydrophobic interactions and Van der Waals forces are likely to occur as not only HA-chains present hydrophobic patches [ 18, 1011. Hydrophobicity of the COO'
Figure 5. Schematic drawing of forces that might act between two carbohydrate chains in sponge glycans. Possible forces are sketched as ionic interaction via Ca2+ ions between two carboxyl groups (Ca2+ between arrows), hydrogen-bonds (arrows with dashed lines) might occur within the chain, between chains and between a chain and the solvent, and hydrophobic interactions (broad interaction band) between apolar surfaces of the sugar rings (adapted from [ 121).
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38 Curbohydrate- Carbohydrate Inteructions
carbohydrates depends on the conformation of the rings, the epimer conformation and the glycosidic linkages and therefore not all carbohydrate chains are equally amphiphilic [ 1021.
38.4 Experimental Approaches 38.4.1 General Considerations Low affinity interactions are difficult to measure and the intrinsic problem is to distinguish specific from non-specific interaction. In this context, it is important to realise that not all biological interactions are equally “specific”, in other words, to understand specificity not merely as a term of high affinity, unique binding site, but rather as an adaptation to the physiological possibilities and needs. The coexistence in space and time of potential partners in an interaction scheme is therefore a more important requirement than the existence of unique structural motifs recognized by individual molecules. A measure for the strength of two molecules interacting with one another is the affinity between them. This can be expressed in terms of an association constant which can differ for many orders of magnitude depending on the kind of interaction. Association strengths between two sugar molecules can be below lo2 M-l, whereas low affinity interactions have a range of approximately 102-104 M-’ and high affinity interactions more than lo5 M-’, already indicating that the possibilities of detection and choices to measure such interactions can be limited by the inherent technical possibilities of different approaches. As measurements of interactions often happen in dissected systems where molecules are tested in other than the original context, it should be crucial to test a model in conceptually different assay set-ups to validate findings and try to eliminate experimental artefacts. These requirements can be difficult to achieve for the carbohydrate-carbohydrate interaction; even more difficult than to find the natural components of protein-carbohydrate interaction which is already a difficult task as demonstrated by the hunt for the “real” selectin ligands [103]. Much of older literature on carbohydrate-carbohydrate interaction does not necessarily hold the requirements for unambiguous testing in different systems, but due to lack of possible alternatives at the time they were performed they have been included in this summary. Improvement of methods and technology should give the chance to test these concepts in future. The collection of methods presented below is chosen on the basis of experiments described in the literature used for studies of carbohydrate-carbohydrate interactions. Rather than being a collection of ready to use recipes the different methods are discussed considering important points as most of the techniques are discussed in full extent in other parts of these volumes and do not differ conceptually whether measuring carbohydrate-carbohydrate interactions or any other type of interaction.
38.4 Experimental Approaches
1079
38.4.2 Affinity Interactions
In principle all studies to measure interaction between two molecules are affinity interactions. What is described under this heading could also be described as traditional cell biological or biochemical approaches to measure binding between two systems. It was historically started with the observation of whole cells interacting, before more sophisticated methods of cell fractionation and artificial reconstruction have progressed to the point that almost any molecule can be isolated and packed into a de nozio assembly to test for its role in the intact system. Cell Binding Studies The advantage to use intact cells for binding studies is that the molecular assembly is in its original form and effects are less likely due to altered exposure of molecules, artificial combinations, lack or changed arrangement of components. Obviously that creates also a disadvantage in itself, as designing which of the components does what is less ambiguous in an intact system. Cell binding studies can be performed in several different approaches. Single-cell suspensions are allowed to aggregate with themselves or another cell type, to bind to a supporting cell layer or to an artificial substrate. Cell aggregation
Cell aggregation is a classical type of experimental set-up and has been used extensively, hemagglutination just being the most prominent one. Mechanically dissociated and well washed cells are allowed to aggregate in the presence of a potential adhesion mediator or inhibitor. Cells can be left untouched or exposed to shear forces for instance on a gyratory shaker. To exemplify this approach, sponge cells can be mechanically dissociated from the sponge matrix, washed extensively and allowed to reaggregate in the presence of Ca2+ and an aggregation factor [51]. This approach can be further extended to test the role of different components of the adhesion molecule. Chemical treatment of the factor or enzymatic removal of certain sugars [57, 61, 621, fragmentation into smaller subunits [63] or chemical cross-linking of factor glycans to a multimeric complex [ I l l aid to substantiate the mode of interaction between two cells. The reversed scenario can also be applied: A cell complex is broken up by adding competitors from which one assumes that they have the capacity to inhibit the interaction. This can be antibody molecules against different surface molecules or glycan motifs to be tested for their effect as done with early embryos [71]. Cell adhesion to substrate
The binding capacity of cells to different substrates is a first step in dissecting the system and testing individual components for binding. While the cell is still an intact entity and serves as a tool to screen with, the complementary adhesion site is modified and theoretically can be built from almost any kind of combination of
1080
38 Carbohydrate-Carbohydrate Interactions
Table 4. Cell lines applied to GSL-mediated binding studies.
Cell line"
Expressionb
Binding"
Substrated
B 16 melanoma
( 3 ~ 3high
Strong
B16 melanoma
( 3 ~ 3high
Strong
B 16 melanoma
G M high ~
Weak
B16-FI0 clone
G Mhigh ~
Strong
B16-F1 clone
G M medium ~
Medium
B16-WA4 clone
G M lOW ~
Weak
F9 teratocarcinoma PYS-2 teratocarcinoma 2102 embryonal carcinoma
LeXhigh Le" low
Yes No
LacCer-coated plate, non-stimulated endothelial cells (LacCer expression) Gg3 coated plate, Tcell lymphoma L5178 AA12 (high Gg3 expression) T-cell lymphoma L5178 AV27 (low Gg3 expression) Non-stimulated endothelial cells (LacCer expression) Non-stimulated endothelial cells (LacCer expression) Non-stimulated endothelial cells (LacCer expression) Le" coated plate Le" coated plate
High nLc4 and GalGb4, moderate Gb4, no Gg3 High nLc4 and GalGb4, moderate Gb4, no Gg3
Yes
Gg3 and Gb4-coated plate
No
Gb3, Lc3, Lea, Le", G M ~GM3-coated , plate
2 102 embryonal carcinoma
Reference
Cell lines and clones used for studying GSL-mediated binding Expression levels are described with low, moderate or high according to the original description 'The binding strength is graded as described in the original publication a
molecules. The advantage lies in the simplicity of the test and the better control of components, whether concerning amounts or combinations. The caveat arises from the same point as mentioned earlier, namely the artificial combination of components and the uncertainty as to the steric properties of the molecules when immobilized on a surface. Nevertheless this type of approach has been used extensively in the study of GSL mediated cell binding. Individual GSL alone or in combination with different glycoproteins have been immobilized to culture dishes and testing occurred with different cell lines under static or flow conditions. Conclusions drawn from this kind of assays are that different GSL have different adhesion promoting/ preventing potentials for a given cell line, an effect which is clearly dependent on
38.4 Experimental Approaches
1081
both the concentration of GSL expressed on the cell surface and that coated on the culture dish [74, 75, 771. Combination of GSL with proteins and testing cell adhesion under both static and flow conditions indicate that GSL-GSL interactions are primarily mediating cell adhesion in the early phases of cell coating and are replaced by protein-carbohydrate and protein-protein interactions after that, but are more prominent under shear forces than under static conditions [78, 791. Aggregation of de novo Complexes
Once a cell is dissected into single components and these should be tested individually, different options can be tested. Aggregation or complexing are easily observed and do not need too much of technical equipment. Macromolecules that allow such observations have been favored due to simplicity and easiness to create such polyvalent assemblies. Modification of inert beads and integration of molecules into liposomes have been two different approaches used extensively in the study of carbohydrate-carbohydrate interaction. Aggregation of beads can be followed macroscopically or with a normal light microscope, or, when using fluorescent beads, by fluorescent microscopy. Liposome aggregation can be followed simply by measuring absorption with a spectrometer. ModiJication of inert beads The choice for beads is merely empirical. Two important points must be considered before an experiment: First, how shall the molecules be coupled to the beads in order to exert a minimal effect of the immobilization method on the assay. Is noncovalent adsorption possible and stable enough or does it need a covalent-linking scheme. The chemistry of linking molecules to beads depends on molecular characteristics (which chemical groups are available for linking, which ones should not be used to conserve the biological activity) and can affect the properties of the linked molecule in terms of its binding properties, i.e. its functional integrity and its way of presentation (steric factor, architecture on the bead). Integrity and access of the molecule on the bead should therefore be tested with a suitable probe as could be an antibody, a lectin or another molecule for which the binding behavior is known. Second, the chemical groups on the beads used for covalent coupling of the sample must not affect the binding properties of the beads. It must be possible to neutralize them without changing the binding characteristics of the bead. This problem is usually rather easily circumvented as control beads can be modified and blocked in parallel without any ligand or an alternative ligand and be used as negative (or positive) control. Beads have been used to demonstrate that either intact aggregation factor or immobilized factor glycans can mediate the sponge cell aggregation [57, 671, that GAGScan bind to one another [39,44]and to demonstrate GSL-GSL interaction [72]. Integration of glycans into liposomes Glycolipids are highly suited to be integrated into liposomes for testing glycandependent liposome aggregation and liposome binding to surfaces. No chemical
1082
38 Carbohydrate- Carbohydrate Interactions
modification is needed, as the quality of liposomes be controlled by the choice of lipids used to create the liposomes. The integration of radioactively marked glycolipids can help to control the level of incorporation. Different glycan motifs can be integrated and tested in parallel. The possibility to create neoglycolipids from short glycans also opens the possibility to test glycans that originally are not isolated as glycolipids but may originate from a glycoprotein and could be tested independent of the protein core [ 1041. Hypotheses of GSL-mediated cell interactions have been tested by demonstration of the aggregability of GSL-liposomes and adhesion of such liposomes to glycolipid coated surfaces [46, 82, 901. Affinity Chromatography Alternative to using immobilized glycans in aggregation assays, cell or liposome binding and similar experiments, they can also be used in chromatography. Whereas high affinity interactions are easily seen by affinity chromatography, low affinity interactions might be overlooked due to insufficient retention. For larger glycans that are inherently polyvalent, retention on a glycan modified column could be manifested on the basis of carbohydrate-carbohydrate [42].As holds true for the bead coating, the inclusion of an identically modified column matrix without the ligate of choice attached should be considered as a control or precolumn to avoid looking at non-specific matrix-ligand interactions. At least the theoretical possibilities of weak affinity chromatography are impressive if one succeeds to immobilize the ligate at high enough densities to cope with the low affinity of carbohydratecarbohydrate interactions [ 1051. Distribution between Compartments Boyden chamber
The distribution of molecules between two phases or two liquid chambers separated by a membrane has been occasionally used for the demonstration of carbohydratecarbohydrate interaction. In the Boyden chamber, two compartments with identical buffer content are separated by a membrane with suitable molecular weight cut-off. To one chamber the molecules to be tested, for instance a soluble oligosaccharide and a potential macromolecular ligand are added, and the diffusion of the oligosaccharide into the other chamber is followed. If it does not interact with the macromolecule, the distribution of the oligosaccharide should be under free diffusion and an equilibrium must be reached. Similarly, two freely diffusible species can be observed, if their interaction creates complexes that become too big for free diffusion (by correct choice of the membrane pore size), and will remain in the chamber they have originally been placed in. Binding of radiolabelled oligosaccharides to stationary liposomes containing different glycolipid species was observed in the Boyden chamber. Oligosaccharides that bound to the GSL in the liposomes did not pass the membrane, whereas oligosaccharides that could not bind to the GSL were recovered also from the other compartment [73].
38.4 Experimental Approaches
1083
Phase partitioning In a similar type of approach, phase partitioning, the distribution of molecules between two different liquid phases, is analyzed without any separating membrane. In this assay, the solubility and interaction with the solvent in the two different phases is driving force for the separation behavior of the analytes. The distribution of 1251-MAFbetween a lower, dextran-rich and an upper, polyethylene glycol phase had been analyzed as a function of cations added. While the non-complexed MAF distributed to the dextran-rich phase due to its polyanionic character, aggregation of MAF in the presence of cations reduced the net charge of the aggregate and resulted in a shift to the polyethylene glycol phase [65]. The limitation of this assay system is, however, that the effects of pure charge neutralization by the addition of cations to the polyanion cannot be distinguished from the effect of cation-mediated complexing of the polyanion to a less charged macrocomplex. The change of distribution properties of liposomes modified with neutral glycans like dextran, pullulan or mannan between a carbohydrate-rich and a carbohydrate-poor phase, might rather been taken as a sign of direct carbohydrate-carbohydrate interaction between the carbohydrate on the liposome and the bulk carbohydrate [ 1061.
38.4.3 Microscopy Electron Microscopy Tertiary structures beyond the cellular level can be observed by electron microscopy (EM). Both, macromolecular complexes of proteoglycan-like aggregation factors [ 551 and isolated glycosaminoglycan chains forming fibrillar structures have been observed by EM [19, 321. Many of the observations have been made on replicas of rotary shadowed structures that had been dried on the carrier. These dehydrated samples have most obviously lost some of their physiological properties beside the fact that the preparation as such could be deleterious to the sample and result in artefacts. The development of atomic force microscopy (AFM) in the 1980s is therefore a valuable improvement of technology towards more physiological sample application possibilities. Atomic Force Microscopy AFM is an attractive development which has resulted from scanning tunneling microscope technology. In AFM a sensor is raster scanned in nanometer distance over a surface with the immobilized sample giving a picture of the specimen. In this imaging mode, measurements can be achieved for single molecules as well as whole cells allowing the observation of biological phenomena in a more physiological context. In contrast to EM, samples do not need to be fixed or shadowed and they can be observed in hydrated form. What was anticipated a few years ago to be a general method for imaging from molecules to cells [ 1071 has already been successfully used for imaging cells [108], proteins [109], ribonucleic acid [lo81 and carbo-
1084
38 Carhohydrute-Carbohydrate Interactions
hydrates [56, 1101 but even more to measure forces acting between molecules [56, 1101. In the force-distance approach, the tip with an immobilized molecule is moved in an approach-and-retract cycle towards the sample on the substrate surface. The forces acting on the sensor are recorded under all the approach-and-retract cycle and give a picture of singular binding events, the distance at which the binding ruptures, the adhesion probability and, at least theoretically, also the energy dissipated during the process [ 11 11. In order to really be able to measure forces acting between the two samples and not those between the samples and their respective immobilization surface, it is important that the molecules are immobilized so well as to resist forces larger than the ones acting between the molecules. As discussed with other immobilization techniques, it is also important with AFM, that the immobilization procedure has to conserve the right orientation and binding activity of the molecules to be analyzed. And equally well is it important to discriminate against any unspecific interaction with the surface and sensor [ 1111. Applying AFM technology, imaging demonstrated the same contours of the aggregation complex from sponges as had earlier been observed by EM [56]. In the force mode, an average molecular contour length of -220 nm and arm lengths of -150 nm for MAF could be determined [ 11 11. Furthermore, the average forces of individual interaction sites between two MAF molecules were estimated to -40-50 pN [56, 1111. As multiple jump-off events of -40 pN f 15 pN were seen in a singular approach-and-retract cycle, but the average forces measured between the molecules amounted to -125 pN with maxima up to -400 pN, this result seems to favor multivalent binding of 3-10 binding pairs [56]. As it is, however, still unresolved how the single glycan species are integrated in the whole aggregation factor complex a conclusive picture of this interaction awaits further experimentation. An unexpected finding by AFM is the observation of PSA filaments in the presence of Ca2+-ions [ 171. 38.4.4 Crystallography Crystallography is one method of choice to receive detailed conformational information about molecules. The flexibility and heterogeneity of oligosaccharides, however, sets serious limitations to the analysis of either glycoconjugates or carbohydrate ligands bound to other macromolecules [ 1 121. Crystallization of carbohydrates might be more difficult and even impossible for certain structures, but still has a considerable value in cases where the crystallization succeeds. Not surprisingly HA, which is very homogeneous in sequence, has been described as a crystal and assumes a double helical form [35, 361. More recently, crystallization of a blood group antigen succeeded for the first time ever [87]. This Le" crystal is characterized by a high degree of hydration with water molecules filling all spaces in the crystal and participating in the intermolecular hydrogen bonding pattern. Hydrophobic interaction within a single trisaccharide can be seen between fucose and galactose rings, but no intramolecular hydrogen bonding. The conformation of the trisaccharides in the crystal fit to the conformations predicted from NMR and molecular modelling data [ 1131, and the order of the trisaccharide in a head-to-head arrange-
38.4 Experimental Approaches
1085
ment in the crystal would suggest that a tight packing of glycolipids could occur in a similar fashion [87].
38.4.5 Mass Spectrometry Mass spectrometry (MS) has been used since its beginnings for assisting the characterization of carbohydrate structures. More recently it has gained much of value due to improved ionization tools that provide milder treatment of the sample without fragmentation risk. Whereas the advent of matrix assisted laser desorption ionization (MALDI) MS has improved the characterization of even larger compounds, electrospray ionization (ESI) MS has opened the field to studies of interactions between molecules. In ESI-MS highly charged liquid droplets in a strong electric field are dispersed and evaporated into the mass analyzer [ 1141. Due to this soft ionization technique even non-covalent interactions can therefore be observed. By ESI-MS homodimers could be observed for both LeX and Le"-LacCer in presence of divalent, but not monovalent cations. A heterospecific interaction in presence of divalent cations could also be observed for Lex-LacCer in interaction with LacCer, GalCer and even Cer with a decreasing affinity in this order [85] confirming findings realized by other methods. Another example for the value of ESI-MS is provided by the study of GalCer with CBS, that shows a Ca2+ dependent oligomerization and confirms results from liposome assays. A qualitative picture of the affinity between different oligomers can be gained by measuring the stability at different declustering potentials. Under increasing declustering potentials GalCerCa2+ was the most stabile complex which contrasts to findings with the liposome assay. The divergence might, however, be explained by the choice of the solvent used for the ESI-MS measurements [86]. Collision induced decomposition of the complexes, on the other hand, showed that the non-covalent interactions between the carbohydrate portions and Ca2+ are more stable than covalent linkages, as the ceramide portion could be split off before the complex broke. Combining the results from changing declustering potentials and performing collision induced decomposition suggests therefore, that the GalCer-CBS-Ca2+ complex is the most stabile form [ 861 which corresponds to the liposome assays. Similar observations were also made with the Lewis antigens where ceramide and fucose were split off before the Ca2+-stabilized carbohydrate-carbohydrate interaction was disintegrated [85].
38.4.6 Nuclear Magnetic Resonance The advantage of nuclear magnetic resonance (NMR) lies in the fact that measurements occur in aqueous solutions, i.e. the most natural form if one considers biological materials. NMR is widely used for structural characterizations of carbohydrates even though the requirements for homogeneous probes and relative insensitivity can pose limitations. The flexibility of carbohydrates sets another factor of limitation and therefore structural and dynamic properties must be determined in a combination of different methods [ 1121. Conformations of bound carbohydrates
1086
38 Carbohydrate-Curbohydrute Intermtiom
us non-bound carbohydrates can be measured by transfer nuclear Overhauser effect
(trNOE) experiments. These trNOEs can only be determined for a carbohydrate complexed to a large molecular weight molecule, for instance a protein, when the relaxation is governed by the tumbling time of the protein. The only information gained in this way is the difference between non-bound and bound carbohydrate. To elucidate the structure of the entire complex needs also the parallel analysis of the protein structure by either crystallography or NMR and the combination of the data acquired in the different experiments with molecular modelling techniques [ 112, 1131. As carbohydrate-carbohydrate interactions are usually weak and require polymerization of both binding partners resulting in larger complexes, trNOEs are not feasible for measuring carbohydrate-carbohydrate interactions. The only possibility for analyzing characteristics of two carbohydrate molecules against one another is by modelling interactions with data obtained for the individual components from either NMR or crystallographic approaches. 38.4.7 Molecular Modelling
Due to the nature of carbohydrates, appropriate force-fields must be used to describe the conformation and dynamic properties of these structures. A number of different programs have been adapted or created especially for modelling carbohydrates (for review see [ 113, 1151). Due to the limited number of data for larger carbohydrates, models only exist for a few carbohydrate-carbohydrate interactions mediated by small oligosaccharides. The interactions of the GSL headgroups Gg3 ~ been modelled by creating minimum energy conformation models of and G Mhave the two head groups based on hard sphere exoanomeric calculations, suggesting the interaction via an exposed hydrophobic patch in the two molecules [76]. Similarly, Le"-LeX interaction was modelled and proposed to assume a head-to-head alignment of the molecules [72] as also suggested by crystal data [87]. The increasing potential of calculation power and development of enhanced measuring capacity with NMR equipment will probably add information to the field of carbohydratecarbohydrate interactions.
38.4.8 Tools Important tools for the study of carbohydrate interactions have been antibodies directed against carbohydrate epitopes. Even more important is the synthesis of carbohydrate sequences to create homogeneous, well defined sequences which allow the study of interactions without problems created by heterogeneous populations of glycans in functional and structural tests. Synthetic Oligosaccharides
The synthesis of well defined and homogeneous structures does allow the study of interaction phenomena in different ways. For obvious reasons oligosaccharide syn-
1087
38.4 Experimental Approaches
thesis involves more effort than peptide- or oligonucleotide-synthesis, but nevertheless the number of structures synthesized for different purposes is increasing. Sponge epitopes [ 1161, GSL-epitopes [ 1171 and, the most prominent examples, Lewis blood group antigens have been synthesized and applied for direct measurement of interaction by NMR, crystallography 1871, ESI-MS [85] and other methods [118]. Antibodies against Carbohydrate Motifs Antibodies against carbohydrate determinants have been widely used to study the functional aspects of carbohydrate interactions. They are applied to control the expression level of a given carbohydrate epitope as GSLs 1771, to block binding sites [ 111 or to affinity purify and detect oligosaccharide structures containing the epitope [68]. One important aspect with antibodies is the fact that their reactivity can depend both on the conformation of the epitope due to different surroundings and on the degree of polyvalence due to clustering [ 1 191. The recognition of the same antenna structure on glycopeptides and glycolipids has been shown to differ for various antibodies as a consequence of different conformation of these epitopes depending on the respective anchoring sequences [ 1 191. Two monoclonal antibodies inhibiting Microciona aggregation are directed against the carbohydrate portion of the aggregation factor (Block 1 and Block 2), while a third one does not block the aggregation although recognizing the carbohydrate portion [ 111. All three have been characterized by a combination of chemical degradation and characterization of the recovered structures by enzyme susceptibility, NMR and MS [68, 691. Several antibodies directed against glycolipid structures have been used in the characterization of GSL-GSL interaction to Table 5. Monoclonal antibodies applied for studying carbohydrate-carbohydrate mediated interaction.
Antibody
Epitope recognized
Assay systema
Reference
Block 1
Microciona aggregation
[681
Block 2 DH2
Pyr-4,6Galp 14GlcNAcp1-3F~c GlcNAc(30S03)p1-3Fuc Anti-GM3
~ 9 1 ~761
2D4
Anti-Gg3
T5A7 9G7 1B2 MC63 1
Anti-LacCer Anti-Gb4 Anti-nLc4 Anti-SSEA-3 (GalGb4 main epitope) Anti-SLex Anti-Lex Anti-H
Microciona aggregation Melanoma adhesion to T-cell lymphoma Melanoma adhesion to T-cell lymphoma B16 melanoma adhesion 2 102 lymphoma cell adhesion 2 102 lymphoma cell adhesion 2 102 lymphoma cell adhesion B16 melanoma adhesion GSL-GSL in vitro B16 melanoma adhesion
[791
SNH3 SH I BE2
“Assay system indicates in which context the antibodies were used for binding studies
[761 1791 [ 741
P I P I
PI
[791
1088
38 Curbohydrute-Curbohydrute Interactions
quantitate the GSL-expression on cell lines and to interfere with the binding of cells and liposomes to one another [76, 77, 79, 82, 117, 1201. Cells
What started in the beginning of the century with the observation of cells taken from different sponges to allow the study of recognition processes [48, 491 has developed into a major research tool in biology with the use of both, primary and immortalized cells from many different sources. The selection of different clones from the same parent line has further stimulated the application and direct comparison of cells with similar but not identical surface composition. Examples of such cell lines that have been used for the study of carbohydrate-carbohydrate mediated phenomena are variants of the mouse melanoma cell line B16 expressing different levels of GM3-ceramides [79]or T-cell lymphoma clones with different levels of Gg3ceramides on the cell surface [76] (see also Table 4).
References D. Spillmann and U. Lindahl, Curr. Opin. Struct. B i d , 1994, 4, 677-682 A. Varki, Proc. Natl Acad. Sci. USA, 1994, 91, 7390-7397 T. A. Springer, Cell, 1994, 76, 301-314 H. Rauvala and H. B. Peng, Proy. Neurobiol., 1997, 52, 127-144 M. Schachner, Curr. Opin. Cell Biol., 1997, 9, 627-634 U. Rutishauser, J. Cell. Biochem., 1998, 70, 304-312 K.-A. Karlsson, Curr. Opin. Struct. Biol., 1995, 5, 622-635 K. S. Rostand and J. D. Esko, Infect. Immun., 1997, 65, 1-8 N. Sharon and H. Lis, Science, 1989,246, 227-234 10. T. Feizi, Trends Biochem. Sci., 1994, 19, 233-234 11. G. N. Misevic, J. Finne and M. M. Burger, J. Bid. Chem., 1987,262, 5870-5877 12. D. Spillmann and M. M. Burger, J. Cell. Biochem., 1996,61, 562-568 13. D. A. Rees, Biochem. J., 1972, 126, 257-273 14. P. Gacesa, Carbohydr. Pol., 1988, 8, 161-182 15. F. Clementi, Crit. Rev. Biotech., 1997, 17, 327-361 16. A. 0. Tzianabos, A. Pantosti, H. Baumann, J.-R. Brisson, H. J. Jennings and D. L. Kasper, J. Biol. Chem., 1992,267, 18230-18235 17. J. Toikka, J. Aalto, J. Hayrinen, L. J. Pelliniemi and J. Finne, J. Biol. Chem., 1998, 273, 28557-28559 18. J. E. Scott, Y. Chen and A. Brass, Eur. J. Biochem., 1992,209, 675-680 19. J. E. Scott, C. Cummings, A. Brass and Y. Chen, Biochem. J . , 1991,274, 699-705 20. P. Albersheim, J. An, G. Frshour, M. S. Fuller, R. Guillen, K.-S. Ham, M. G. Hahn, J . Huang, M. O’Neill, A. Withcombe, M. V. Williams, W. S. York and A. Darvill, Biochem. Soc. Trans., 1994,22, 374-378 21. M. McNeil, A. G. Darvill, S. C. Fry and P. Albersheim, Annu. Rev. Biochem., 1984,53, 625663 22. L. M. J. Kroon-Batenburg and J. Kroon, Glycoconj. J . , 1997, 14, 677-690 23. N. C. Carpita and D. M. Gibeaut, Plant Cell, 1993,3, 1-30 24. N. Carpita, M. McCann and L. R. Griffing, Plant Cell, 1996,8, 1451-1463 25. G. 0. Aspinall In The Biochemistry of Plants; Academic Press: New York, 1980; pp 473-500. 26. T. N. Wight, M. G. Kinsella and E. E. Qwarnstrom, Curr. Opin. Cell Biol., 1992, 4, 793-801 27. H. Lis and N. Sharon, Eur. J. Biochem., 1993,218, 1-27 28. A. Varki, Glycobiology, 1993, 3, 97-130 1. 2. 3. 4. 5. 6. 7. 8. 9.
References
1089
29. L. KjellCn and U. Lindahl, Annu. Rev. Biochem., 1991, 60, 443-475 30. T. C. Laurent, U. B. G. Laurent and J. R. E. Fraser, Imm. Cell. Biol., 1996, 74, A3-A7 31. L. A. Sellers and A. Allen In Mucus and Related Topics; University Press Corporation: 1989; pp 65-71. 32. M. Morgelin, M. Paulsson, T. E. Hardingham, D. Heinegdrd and J. Engel, Biochem. J., 1988, 253, 175-185 33. R. E. Turner, P. Lin and M. K. Cowman, Arch. Biochem. Biophys., 1988,265, 484-495 34. E. R. Morris, R. D. A. and E. J. Welsh, J. Mol. Biol., 1980, 138, 383-400 35. J. K. Sheehan, K. H. Gardner and E. D. T. Atkins, J. Mol. Biol., 1977, 117, 113-135 36. S. Arnott, A. K. Mitra and S. Raghunathan, J. Mol. Biol., 1983, 169, 861-872 37. J. Ed Scott, Ciba Found. Symp., 1989, 143, 6-20 38. L.-A. Fransson and L. Coster, Biochim. Biophys. Acta, 1979, 582, 132-144 39. E. 4. Turley and S. Roth, Nature, 1980,283, 268-271 40. L.-& Fransson, I. Sjoberg and V. P. Chiarugi, J. Biol. Chem., 1981,256, 13044-13047 41. L.-A. Fransson, L. Coster, A. Malmstrom and J. K. Sheehan, J. Biol. Chem., 1982, 257, 6333-6338 42. L.-A. Fransson, I. Carlstedt, L. Coster and A. Malmstrom, J. Biol. Chem., 1983, 258, 1434214345 43. L-4.Fransson and B. Havsmark, Carbohyd. Res., 1982, 110, 135-144 44. L.-A. Fransson, B. Havsmark and J. K. Sheehan, J. Biol. Chem., 1981,256, 13039-43 45. A. D. Snow, H. Mar, D. Nochlin, R. T. Sekiguchi, K. Kimata, Y . Koike and T. N. Wight, Am. J. Pathol., 1990, 137, 1253-1270 46. R. J. Stewart and J. M. Boggs, Biochemistry, 1993, 32, 10666-10674 47. S.4. Hakomori, Pure Appl. Chem., 1991, 63, 473-482 48. H. V. Wilson, J. Exp. Zool., 1907, 5, 245-258 49. P. S. Galtsoff, J. Exp. Zool., 1925, 42, 223-251 50. G. N. Misevic and M. M. Burger, J. Biol. Chem., 1986,261, 2853-2859 51. T. Humphreys, Dev. Biol., 1963, 8, 27-47 52. T. Humphreys In The Speczjicity of Cell Surfaces; Englewood Cliffs: Prentice Hall, NJ, 1967; pp 195-210. 53. P. Henkart, S. Humphreys and T. Humphreys, Biochemistry, 1973. 12, 3045-3050 54. C. B. Cauldwell, P. Henkart and T. Humphreys, Biochemistry, 1973, 12, 3051-3055 55. S. Humphreys, T. Humphreys and J. Sano, J. Supramol. Struct., 1977, 7, 339-351 56. U. Dammer, 0. Popescu, P. Wagner, D. Anselmetti, H.-J. Giintherodt and G. N. Misevic, Science, 1995,267, 1173-1 175 57. J. E. Jumblatt, V. Schlup and M. M. Burger, Biochemistry, 1980, 19, 1038-1042 58. J. A. Varner, M. M. Burger and J. F. Kaufman, J. Biol. Chem., 1988,263, 8498-8508 59. J. A. Varner, J. Cell Sci., 1995, 108, 3119-3126 60. J. A. Varner, J. Biol. Chem., 1996,271, 16119-16125 61. S. R. Turner and M. M. Burger, Nature, 1973,244, 509-510 62. G. N. Misevic and M. M. Burger, J. Cell. Biochem., 1990, 43, 1-8 63. G. N. Misevic, J. E. Jumblatt and M. M. Burger, J. Biol. Chem., 1982,257, 6931L6936 64. D. J. Rice and T. Humphreys, J. Biol. Chem., 1983,258, 6394-6399 65. W. Burkart and M. M. Burger, J. Supramol. Struct. Cell. Biochem., 1981, 16, 179-192 66. D. Spillmann, G/ycoconj. J., 1994, I I , 169- 171 67. G. N. Misevic and M. M. Burger, J. Biol. Chem., 1993,268, 4922-4929 68. D. Spillmann, K. Hgrd, J. Thomas-Oates, J. F. G. Vliegenthart, G. Misevic, M. M. Burger and J. Finne, J. Biol. Chem., 1993,268, 13378-13387 69. D. Spillmann, J. E. Thomas-Oates, J. A. van Kuik, J. F. G. Vliegenthart, G. Misevic, M. M. Burger and J. Finne, J. Biol. Chem., 1995,270, 5089-5097 70. T. Feizi, Nature, 1985, 314, 53-57 71. B. A. Fenderson, U. Zehavi and S.4. Hakomori, J. Exp. Med., 1984,160, 1591-1596 72. N. Kojima, B. A. Fenderson, M. R. Stroud, R. I. Goldberg, R. Habermann, T. Toyokuni and S.4. Hakomori, Glycoconj. J., 1994, 11, 238-248 73. I. Eggens, B. Fenderson, T. Toyokuni, B. Dean, M. Stroud and S.4. Hakomori, J. Biol. Chem., 1989, 264, 9476-9484
1090
38 Carbohydrate- Carbohydrute Interactions
74. Y. Song, D. A. Withers and S.4. Hakomori, J. Biol. Chem., 1998, 273, 2517-2525 75. K. Iwabuchi, S. Yamamura, A. Prinetti, K. Handa and S.-i. Hakomori, J. Bid. Chem., 1998, 273, 9130-9138 76. N. Kojima and S.4. Hakomori, J. Biol. Chem., 1989,264,20159-20162 77. N. Kojima and S.4. Hakomori, J. Biol. Chem., 1991,266, 17552-17558 78. N. Kojima and S.4. Hakomori, Glycobiology, 1991, 1, 623-630 79. N. Kojima, M. Shiota, Y. Sadahira and S.-i. Hakomori, J. Biol. Chem., 1992, 267, 1726217270 80. J. Jarchow and M. M. Burger, Cell Adhes. Comm., 1998. 81. 0. Popescu and G. N. Misevic, Nuture, 1997, 386, 231-232 82. S.4. Hakomori, Biochem. Soc. Trans., 1993, 21, 583-595 83. J. Finne, J. Bid. Chem., 1982,257, 11966-11970 84. U. Rutishauser and L. Landmesser, Trends Neurosci., 1996, 19, 422-427 85. G. Siuzdak, Y. Ichikawa, T. J. Caulfiedl, B. Munoz, C.-H. Wong and K. C. Nicolaou, J. Am. Chem. Soc., 1993,115, 2877-2881 86. K. M. Koshy and J. M. Boggs, J. Biol. Chem., 1996,271, 3496-~3499 87. S. PCrez, N. Mouhous-Riou, N. E. Nifant’ev, Y. E. Tsvetkov, B. Bachet and A. Imberty, Glycobiology, 1996, 6, 537-542 88. M. M. Burger In The Role of Intercellulur Signals: Nuvigution, encounter, outcome; Verlag Chemie: Berlin, 1979; pp 119-134. 89. M. Fukuda, Biochim. Biophys. Actu, 1985, 780, 119-150 90. N. Kojima, Trends Glycosci. Glycotechnol., 1992, 4 , 491-503 91. K. Simons and G. van Meer, Biochemistry, 1988, 27, 6197-6702 92. D. A. Brown and J. K. Rose, Cell, 1992, 68, 533-544 93. T. W. Tillack, M. Allietta, R. E. Moran and W. W. J. Young, Biochim. Biophys. Actu, 1983, 733, 15-24 94. M. Sorice, I. Parolini, T. Sansolini, T. Garofalo, V. Dolo, M. Sargiacomo, T. Tai, C. Peschle, M. R. Torrisi and A. Pavan, J. Lipid Res., 1997, 38, 969-980 95. P. Rock, M. Allietta, W. W. j. Young, T. E. Thompson and T. W. Tillack, Biochemistry, 1990,29, 8484- 8490 96. S.-i. Hakomori, Cancer Rex, 1996, 56, 5309-5318 97. M. Salmivirta, K. Lidholt and U. Lindahl, FASEB J., 1996, 10, 1270-1279 98. W. J. Kuhns, G. Misevic and M. M. Burger, Biol. Bull., 1990, 179, 358-365 99. S. J. Angydl, Adv. Curhohydr. Chem. Biochem., 1989, 47, 1-43 100. A. Almond, A. Brass and J. K. Sheehan, Glycobiology, 1998, 8, 973-980 101. R. U. Lemieux, Chem. Soc. Rev., 1989,119, 347-374 102. D. Balasubramanian, B. Raman and C. S. Sundari, J. Am. Chem. Soc., 1993,115,74-77 103. A. Varki, J. Clin. Invest., 1997, 99, 158-162 104. T. Feizi, M. S. Stoll, C. T. Yuen, W. Chai and A. M. Lawson, Methods Enzymol., 1994, 230, 484-519 105. D. Zopf and S. Ohlson, Nature, 1990, 346, 87-88 106. E.-C. Kang, K. Akiyoshi and J. Sunamoto, Int. J. Biol. Mucromol., 1994, 16, 348-353 107. M. Radmacher, R. W. Tillmann, M. Fritz and H. E. Gaub, Science, 1992,257, 1900-1905 108. H. G. Hansma, K. J. Kim, D. E. Laney, R. A. Garcia, M. Argaman, M. J. Allen and S. M. Parsons, J. Struct. Biol., 1997, 119, 99-108 109. D. J. Muller, C.-A. Schoenenberger, F. Schabert and A. Engel, J. Struct. Biol., 1997, 119, 149-157 110. M. Rief, F. Oesterhelt, B. Heymann and H. E. Gaub, Science, 1997, 275, 1295-1297 111. J. Fritz, D. Anselmetti, J. Jarchow and X. Fernandez-Busquets, J. Struct. Biol., 1997, 119, 165-1 71 112. T. Peters and B. M. Pinto, Curr. Opin. Struct. Biol., 1996, 6, 710-720 113. A. Imberty, Curr. Opin. Struct. Biol., 1997, 7, 617~-623 114. G. Siuzdak In Muss Spectrometry f o r Biotechnology; Academic Press, Inc.: San Diego, New York, Boston, London, Sydney, Tokyo, Toronto, 1996; pp 1-161. 115. R. J. Woods, Glycoconj. J., 1998, 15, 209-216 116. T. Ziegler, Curbohyd. Rex, 1994, 262, 195-212
References
1091
1 1 7. M. R. Stroud, S. B. Levery, S. Mgrtensson, M. E. K. Salyan, H. Clausen and S.4. Hakomori,
Biochemistry, 1994, 33, 10672- 10680 118. T. Le Bouar, F. Pincet, J.-M. Mallet, J. Esnault, Y. M. Zhang, E. Perez and P. Sinay, Glycoconj. J., 1997, 14, Sl2-73 119. S. Saito, S. B. Levery, M. E. K. Salyan, R. I. Goldberg and S.4. Hakomori. J. Biol. Chem., 1994,269, 5644-5652 120. M. R. Stroud, S. B. Levery, E. D. Nudelman, M. E. K. Salyan, J. A. Towell, C. E. Roberts, M. Watanabe and S.4. Hakomori, J. Biol. Chem., 1991,266, 8439-8446 121. D. Gupta, R. Arango, N. Sharon and C. F. Brewer, Biochemistry, 1994,33, 2503-2508 122. I. Eggens, B. A. Fenderson, T. Toyokuni and S.4. Hakomori, Biochem. Biophys. Rex Commun., 1989, 158, 913-920
Part I
Volume 2
VI Carb0hydrate-Nucleic Acid Interactions
Carbohydrates in Chemistry and Biology Editor Beat Ernst, Gerald W. Hart, Pierre Sinay copyright OWILEY-VCH Verlag GmbH, 2000
39 Carbohydrate-Nucleic Acid Interactions Heinz E. Moser
39.1 Introduction Among the various classes of biomolecules, nucleic acids play a central role not only as carrier of genetic information but also in the form of RNA as important link for the production of proteins. Specific interactions both on DNA and RNA are essential to retrieve the information required at various stages of life cycles. In general this is largely achieved by a variety of DNA or RNA binding proteins to regulate accessibility and transcription of DNA as well as modification, transport and translational processes of messenger RNA. Molecular interference with these processes can result in more or less specific disturbance of normal cell functions and evolution produced a variety of relatively small ligands that bind with high affinity to DNA or RNA, thereby disrupting crucial functions for life. This often translates in cytostatic or cytotoxic activity and depending on specificity, quite a few of these molecules found a therapeutic application as antiinfective or antitumor agents. Only a limited number of these compounds can be considered as pure carbohydrates. The binding to highly charged nucleic acids required structural properties that are usually not common for carbohydrates. Features like reduction of hydrophilicity and incorporation of positive charges are often found in carbohydrate containing molecules that tightly bind nucleic acids and they indicate how nature managed to optimally benefit from the richness of structural information in carbohydrates by adopting required physicochemical properties. Only a few structures of pure carbohydrate nucleic acid complexes are known thereby limiting our understanding of these interactions. However, many of the DNA ligands known to date carry one or more carbohydrate residues (recognition elements [ 11). The increasing availability of x-ray and NMR structures helps to understand their influence on interaction with either DNA or RNA at the molecular level. A small number of review articles have previously addressed carbohydrate nucleic acid interactions [2-51. The focus of this overview is the discussion of
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39 Carbohydrate-Nucleic Acid Interactions
selected examples from recently published structures with the aim to better understand common rules that govern interactions between nucleic acids and modified carbohydrates.
39.2 Carbohydrates Binding to DNA 39.2.1 Ene-Diyne Antibiotics and Antitumor Agents A family of extremely potent antitumor antibiotics were discovered over the past decades with many common features [ 3 , 61. These molecules all bind double stranded DNA in the minor groove and consist of a bicyclic core containing a strained 9- or 10-membered ring with an ene-diyne functionality and at least one sp2-like bridgehead carbon atom. Either an intra- or intermolecular nucleophilic attack at this central ring triggers a conformational change allowing a spontaneous cyclization to take place. The resulting, highly reactive aryl diradical was demonstrated to initiate single or double strand DNA cleavage based on H-abstraction from the deoxyribose backbone. In addition, most compounds carry one or more carbohydrate side chains and/or an intercalating group, adding to structural complexity and tuning their biological function. Selected members of this family include neocarzinostatin (l),esperamicin A1 (2), calicheamicin y1 (3), and dynemicin A (4) (Scheme 1). Recent publications of high resolution NMR structures of such ligands to short DNA duplexes [7, 81 allow a much better understanding of DNA interaction and modification by this complex class of molecules, including the important function contributed by carbohydrate moieties. The incredibly potent family of calicheamicins were discovered independently almost 20 years ago at Lederle Laboatories and Brystol Myers [9-121 based on a focused effort to identify natural products with the ability to inhibit tumour growth. Esperamicin A1 (2) and calicheamicin y1 (3) were among the most potent members isolated (approximately 1000-fold more potent than adriamycin (8b) against murine tumors, Scheme 3), and they both share the same aglycon but vary in their side chains influencing their specificity to interact with and damage DNA. Their biological activity triggered extensive research efforts to understand structure and function of aglycon and side chains to gain better insight at the molecular level of this fascinating and complex class of compounds. A key component to unravel this puzzle was the synthetic accessibility of the core ene-diyne structure that was successfully tackled by various groups [ 13-20]. Mastering the synthetic accessibility to these complex molecules paved the way to explore structure activity relationship of analogs. Esperamicins
Esperamicin A1 (2) and related members of this family, originating mainly from chemical hydrolysis or nuclephilic attack inducing the cycloaromatization, were
39.2 Cuvbohydrutes Binding to D N A
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39 Curbohydrate-Nucleic Acid Interactions
studied extensively to understand their mechanism of action in relation to biological activity [21-241. Esperamicin A1 (2) was shown to be highly cytotoxic in various eukaryotic cell lines with ICSOSof 0.3-4.5 ng/ml. Removal of parts D and E (esperamicin C) reduced the cytotoxic effect by roughly 200-fold. Additional removal of carbohydrate moiety B (esperamicin D) further reduced the potency by approximately five-fold. Interestingly, this trend was also found for their ability to induce DNA breaks in HCT116 cells; however, the increase of concentration needed to trigger a similar amount of strand breaks varied 5000-fold for removal of D and E and additional 30-fold for esperamicin D, indicating that additional functions might be involved in the observed cytotoxicity [21]. Surprisingly examination of DNA scission in vitro showed equipotent behaviour of both esperamicin A1 and C, indicating a role of the deoxyfucose-anthranilate moiety D and E for drug accumulation in cells. However, esperamicin A1 predominantly produced single strand breaks at low concentration, pointing out the importance of the deoxyfucoseanthranilate moiety to position the ene-diyne aglycon and ultimately the aryl diradical in the minor groove of DNA, facilitating H-abstraction mainly from one of the two sugar phosphate backbones. These findings as well as sequence preferences and the proposed intercalation of the methoxyacrylyl-anthranilate E [24, 251 were experimentally supported by the NMR solution structure of esperamicin A1 (2) bound to the self complementary DNA duplex d(CGGATCCG) [S] (Figure la). Upon closer examination of these published structures some of the previously observed properties of the various esperamicins can be much better understood and indicate the role of the carbohydrate side chains. The ene-diyne aglycon R and the methoxyacrylyl-anthranilate E are linked by the deoxyfucose D which serves as ideal spacer accommodating the necessary 90" turn and distance between these residues. In addition, a hydrogen bond between the axial hydroxyl group and the carbonyl group of cytosine (C7', see Figure lb) contributes to the overall stability of the complex. Remarkably, this hydrogen bond could hypothetically be formed also with the three other bases replacing cytosine as they all contain a hydrogen bond acceptor at this position (thymine: 0-2; adenine: N-3; guanosine: N-3), thereby supporting the lack of a clear sequence preference for this particular interaction. The trisaccharide A-B-C on the opposite side of aglycon R has a key role to help fixing the ene-diyne moiety deep in the minor groove (Figure lb). All sugar residues are deoxygenated and carry rather unusual groups optimizing favorable interactions (hydrophobic and/or opposite charges) with the DNA duplex. Carbohydrate moiety A serves as connection point between the aglycon R and residues B and C, facing the relatively wide minor groove with the 6-membered ring and interacting with the phosphodiester backbone via its equatorial hydroxyl group. Residue B is placed through the unusual NH-0 linkage deep into the minor groove with the ring parallel to the rims of the groove, establishing extensive hydrophobic contacts in addition to a hydrogen bond with N-3 of adenine via its only axial hydroxyl group. As mentioned above, this specific interaction could in principle take place with the other three nucleic acid bases as well. An additional interaction was pointed out between the large polarizable sulfur atom on ring B and the exposed exocyclic amino proton of the proximal guanine base, detectable by the large upfield shift for this proton. This observation would indicate a sequence pref-
39.2 Carbohydrates Binding to DNA
U
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U
b Figure 1. Stereo representation of esperamicin A1 bound to the self complementary oligodeoxyribonucleotide duplex d(CGGATCCG)Z ([8], structures were retrieved from the Brookhaven database: lpik; to simplify the discussion only structure #2 is represented, with the hydrogen atoms omitted). a) Different parts of esperamicin A1 are colored as follows: aglycon R and methoxyacrylyl-anthranilate E (light grey), carbohydrate residues A , B, C, and D (dark grey). b) Same structure with one base pair omitted at each end. Specific heteroatoms intermolecularly interacting between esperamicin A1 and the DNA via hydrogen bonds are highlighted as small balls. Oxygen atoms are represented in light grey and both, nitrogen and sulfur, are shown in dark grey; all the other elements are black.
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39 Carbohydrate-Nucleic Acid Interactions
erence for a guanine at this position; even though such sites are found upon close examination of esperamicin A1 induced cleavage patterns, overall this interaction does not seem to play a dominant role. Residue C is placed over the sugar phosphate backbone of the DNA duplex forming a hydrogen bond between its charged isopropyl ammonium group and one of the phosphodiester oxygens. All the interactions discussed above are in excellent agreement with the observed, relatively weak sequence preferences for DNA cleavage by esperamicin AJ. The reported structure provides a plausible explanation for the preferred break of only one strand. The ene-diyne aglycon R is tightly placed deep in the minor groove fixed on both sides by the anthranilate-deoxyfucose D-E and the trisaccharide A-B-C sidechains, respectively, allowing only minimal conformational changes relative to the duplex DNA. Whereas one of the potential radical positions will be ideally placed to abstract HI’ from the bottom strand (Figure lb), the other position on the opposite site is not only further away from Hs’ of the other strand but also not ideally directed at this position. It is important to note that these differences have to be read with caution as in particular the position of Hs’ is not as well defined and the interpretation of this structure in terms of cleavage prediction is limited. Nevertheless, the hypothesis that esperamicin A J predominantly cleaves one strand based on conformational fixation is supported by the cleavage behavior of esperamicin C: removal of the intercalating part E and linking sugar D yields an esperamicin analog that exclusively produces double strand scissions [211. Calichearnicins
Calicheamicin y, (3) shares the same ene-diyne aglycon R and highly similar trisaccharide A-B-C (for 2) or A-B-E (for 3) with esperamicin Al (2) but lacks the intercalating methoxyacrylyl-anthranilate deoxyfucose part and contains an additional extension on the trisaccharide [6].Two major differences were found between these antibiotics: calicheamicin y1 (3) shows a clear sequence preference for TCCT (found as well for the oligosaccharide moiety alone [26-281) and produces mainly double strand cleavages even at low concentrations with a typical 3 base 3‘-shift of the cleavage sites on opposite strands, commonly observed for DNA cleavers bound to the minor groove of DNA [6, 291. Further insight was gained from the cleavage behaviour of calicheamicins either lacking rings D or E:Similar to esperamicin A1 , omission of the 4-ethylamino sugar E did not cause a change in the observed specificity patterns but dropped the efficiency by 2-3 orders of magnitude indicating a function for binding affinity but not specificity. The calicheamicin lacking the terminal rhamnose D exhibited a similar cleavage specificity with a roughly 100-fold lower efficacy [30]. Many groups initiated various studies to better understand the molecular interaction between DNA and calicheamicin y1 (3); in particular NMR proved to be extremely valuable [31-341. Finally, a high resolution structure of calicheamicin y1 (3) bound to the 23-mer hairpin duplex d(CACTCCTGGTTTTTCCAGJgG19AGTG) was obtained and published that is used here as basis for the discussion of specific DNA carbohydrate interactions [7]. Calicheamicin y1 (3) binds in an extended conformation to the minor groove of DNA, placing the terminal ene-diyne aglycon deep into the groove (Figure 2a).
39.2 Carbohydrates Binding to D N A
1101
h
Figure 2. Stereo representation of calicheamicin y, (3) bound to the 23-mer hairpin duplex d(CACTCCTCGTTTTTCCAGGAGTG) ([7], the structure was retrieved from the Brookhaven database as 2pik and due to simplicity only the first structure is represented omitting hydrogen atoms and the T5 hairpin loop. a) Different parts of calicheamicin y, (3) are coloured as follows: aglycon R and thiobenzoate ring C (light grey), carbohydrate residues A , B, D, and E (dark grey). b) Same structure with one base-pair omitted at the top end. Specific heteroatoms intermolecularly interacting between calicheamicin y, (3) and the DNA via hydrogen bonds are highlighted as small balls. Oxygen and iodine atoms are represented in light grey and both nitrogen and sulfur, are shown in dark grey; all the other elements are black.
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39 CarbohydrateeNucleic Acid Interactions
Determination of the structure without target DNA under various conditions indicated a high degree of conformational preorganization [ 351, allowing this rather big molecule to bind its target site in an extended conformation with high affinity. The interactions for sugar rings A , B, and E closely resemble those discussed for the similar counterparts in esperamicin Al (2) discussed previously (see Figure 2b). It is worthwhile to point out that the required orientation between rings A and B is maintained with the unusual N - 0 bond, ideally adopting an eclipsed conformation with a torsion angle of close to 120" [36, 371 (-128.4 2.6" [7]) providing a relatively rigid, gentle curvature in the middle of the molecule to optimally achieve the overall shape to bind the minor groove. A change of the natural configuration at the anomeric center on ring A completely disrupts the binding affinity of the corresponding aryltetrasaccharide to the target DNA site [28], further supporting the importance of structural preorganization. As mentioned for esperamicin A*, the sulfur atom is in proximity with the exocyclic amino group of guanine 19 causing a large difference in the chemical shift of the corresponding proton. However, replacing this guanine by inosine lacking the exocyclic amino group caused only a modest change in the binding affinity of the aryltetrasaccharide and therefore indicates this interaction to be of minor importance [ 381. The thiobenzoate ring C is buried similar to sugar B deep in the minor groove with its methyl and iodine substituents facing the bottom of the groove and establishing stacking interactions with the deoxyribose rings of T7 and A20 on opposite strands. The large polarizable iodine atom is in proximity to the exocyclic amino group of guanine 18 favorably contributing to the sequence preference of calicheamicin y1 (3) [ 11. Substitution of the iodine atom by bromine, chlorine, fluorine, methyl, or hydrogen [38] indicated the importance of iodine in this position for binding of the corresponding aryltetrasaccharide as these substitutions progressively reduced the binding affinity as experimentally determined by a competition cleavage experiment. In agreement with these findings, substitution of the critical guanine 18 by inosine greatly diminishes calicheamicin y1 (3) binding and subsequent DNA cleavage [38]. Rings B and D both showed the highest resolution with the least standard deviations of the four structures published [7] giving a high degree of confidence in this central part of the molecule. The terminal rhamnose sugar D is twisted relative to the rims of the minor groove and interacts via its 2-hydroxyl group with the proximal phosphodiester group on the purine strand of the recognition site. In addition, the second hydroxyl group is within distance to interact with a phosphodiester oxygen on the opposite strand. Finally, the ene-diyne aglycon R lacking further substitution on the opposite side of its aryltetrasaccharide substituent, interacts via its unsubstituted hydroxy group with the proximal oxygen atom on the terminal phosphodiester of the complex. The calicheamicin oligosaccharide 5 (Scheme 2) was shown to bind with low micromolar affinity to its target site with similar selectivity as compared to calicheamicin y1 (3) [38] (see also references in [39]). This unique property could be used to compete off specific transcripton factors from their target sequence at the expected concentrations [39]. The limited selectivity and potency, however, were not sufficient for a meaningful biological application and the recent progress in the synthesis of complex oligosaccharides allowed this approach to be explored further.
39.2 Carbohydrates Binding to D N A
1103
1 104
39 CurbohydruteeNucleic Acid Interactions
Based on structural knowledge of the calicheamicin oligosaccharide bound to its target DNA tetramer site, head to head and head to tail dimers 6 and 7 were designed and synthesized [40-421. Examination of the inhibitory activity on calicheamicin y, (3) induced DNA cleavage revealed an estimated 100-fold higher affinity of the head to head dimer 6 and more than 1000-fold improved binding of dimer 7 for the target sites as compared to monomer 5 [43]. Both dimers bound with >100-fold selectivity to the corresponding target site but dimer 6 interacted differently with two distinct target sites indicating the influence of neighboring sequences on the overall activity. This dimer was subjected to structure elucidation by NMR as complex with the self complementary oligonucleotide duplex d(CGTAGGATATCCTACG)2 [34]. Close examination of the head to head dimer 6 interaction with the DNA duplex essentially revealed a highly similar binding mode of each carbohydrate unit as compared to the monomer 5, reproducing all the individual interactions discussed previously (see above). The recently published structure of the head to tail dimer 7 confirmed the previous findings and added an interesting insight on sequence selectivity [44]: the structure was resolved with the target duplex d(GCACCTTCCTGC) x d(GCAGGAAGGTGC) containing two slightly different affinity sites. As discussed with calicheamicin y, (3) itself, the 3-OH group of ring B hydrogen bonds with N-3 of A20 [7] (see Figure 2b). Flipping this base pair in the duplex positions the 0 - 2 carbonyl function in almost exactly the same place allowing the hydrogen bond to be formed as evident from the NMR structure. The calicheamicin y1 (3) and calicheamicin oligosaccharide structures 6 and 7 discussed above [7, 34, 441 revealed an almost perfect fit between duplex DNA and bound carbohydrate residues. Structural analysis of the DNA binding sites revealed no major conformational changes at the sites of carbohydrate interactions whereas the accommodation of the en-diyne moiety required a widening of the minor groove. These findings are in contrast to an earlier hypothesis [31, 331 that assumed the conformational flexibility of the DNA binding site to be of crucial importance for the selective binding event. The head to head dimer 6 was also tested for its ability to interfere with transcriptional events [45]. At low micromolar concentrations oligosaccharide 6 was able to disrupt binding of transcription factors (AP-1, STAT-3, NFI, and PU.l) to the corresponding target sites containing a overlapping binding site as shown by gel mobility assays and in vitro transcription by polymerase 11. These experiments demonstrate the validity of modified carbohydrate units as minor groove recognition elements, not limiting their function as originally believed to tune the pharmacokinetic behavior. The required degree of conformational preorganization with an evolutionary adaption of the individual units by decreasing hydrophobicity and adding positive charges make the functionally rich carbohydrates a truly unique scaffold for minor groove recognition of DNA. Whereas the work described above was mainly based on further elaboration of oligosaccharides from natural sources, the increasing structural understanding on molecular level paired with powerful technologies like combinatorial (carbohydrate) chemistry, improved synthetic methodologies, and experimental binding optimization by NMR [46-481 might lead to new horizons for DNA-carbohydrate interactions. In particular, probing preformed DNA-carbohydrate complexes by NMR for adjacent
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39.2 Cuvhohydrates Binding to DNA
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39 Curhohydrute-Nucleic Acid Interactions
binding events of structurally diverse carbohydrate residues (or other low molecular weight compounds) could lead after appropriate linkage to new classes of high affinity DNA ligands that will go beyond the scope of currently available molecules from natural sources.
39.2.2 Anthracyclins Anthracycline antibiotics or antitumor agents are a class of biologically active compounds isolated from various Streptornyces strains. They have indicator-like properties and consist of a core tetracyclic anthraquinone chromophore carrying one or multiple carbohydrate substituents. The first members of this family were isolated roughly half a century ago and shown to display potent antibacterial activities (for recent reviews see 149-521). The high toxicity in mice, however, precluded their further application in man and this changed in the sixties with the discovery of the structurally closely related daunorubicin (or daunomycin, 8a) 153, 541 and doxorubicin (or adriamycin, 8b) [55], both currently used in the clinical treatment of leukaemias and solid tumors, respectively. They interfere with DNA replication and transcription by tightly binding to double stranded DNA via intercalation, preferably at alternating pyrimidine-purine tracts, placing the carbohydrate moiety in the minor groove. In addition, they interfere with topoisomerase I1 activity 1561. Due to an intense interest in their biological behaviour and shortcomings in the clinical application (cardiotoxic side effects and development of resistance) efforts were initiated to understand the interaction with DNA on the molecular level. To date well over 40 high resolution structures are available from the Brookhaven database, providing insight in the role of the carbohydrate-DNA interaction that might lead to the design of derivatives with improved properties. Daunorubicin (8a) was the first anthracycline antibiotic for which structural information at high resolution became ayailable of its DNA-bound state 1571. This structure was refined to remarkable 1.2 A 1581 and, later on, was followed by additional structures using various DNA templates 159-611. In the highly refined 2: 1 complex with d(CGTACG)2 [58],the tetracyclic aglycon is intercalated between the terminal CG base pairs, oriented almost at a right angles to the long axis between the base pairs (Figure 3a). In this complexed form the conformation of ring A varies from the x-ray structure obtained from daunorubicin (8a) alone [62]: only C-9 lies outside the plane defined by the fused aromatic rings, positioning 0-9 almost perfectly perpendicular to this plane and wtthin hydrogen bonding distance to N-3 and N-2 of G2 (O-N distances: 2.6 and 2.9 A, respectively; see Figure 3b). Interestingly, this hydroxy group at C-9 is essential for the biological activity of daunorubicin, supporting the importance of this intermolecular interaction. Daunorubicin (Sa) was reported to preferentially bind to 5’-A/T-CG-3’ or 5’-A/T-GC-3’ triplets, albeit with only modest preference over other sequences 163, 641. This is in agreement with the hydrogen bonding interaction discussed above, as the more important interaction with N-3 of GZcan take place with the other three bases as well (N-3 of adenosine or 0 - 2 of either thymine or cytosine). The second main difference between the two x-ray structures is the torsion angle of the glycosyl linkage (C7-07-C1’-C2’)
1107
39.2 Curbohydrutes Binding to D N A
a
Figure 3. Stereo pictures of daunorubicin (8a) bound to d(CGTACG)* [58]. The co-ordinates have been retrieved from the Brookhaven database (1 l d l ) and for clarity, hydrogens are omitted. a) Representation of the DNA duplex with two daunorubicin molecules intercalated; the tetracyclic anthraquinone aglycon is represented as CPK model with carbon atoms in dark grey and oxygen/ nitrogen atoms in light grey. b) Part of the same structure including selected interacting water molecules (small balls in light grey) and one sodium atom (ball in dark grey). Heteroatoms involved in hydrogen bonding are drawn as small balls. Carbon and phosphorous atoms are shown in black, oxygen in light grey and nitrogen in dark grey.
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39 Carbohydrute-Nucleic Acid Interactions
which is smaller in the DNA-bound molecule. This small change allows the aminosugar (daunosamine) to adopt the correct orientation for an optimal fit in the minor groove of the right-handed DNA duplex. In order to optimally accommodate the carbohydrate in the minor groove the proximal G2-Cll base pair needs to be dislocated by roughly 1.3 A towards the major groove. Opposite to interactions seen in other complexes, the amino group does not directly bind to the negatively charged phosphodiester backbone but hydrogen bonds to 0-2 of C-5 and two water molecules. The axial hydroxy group at C-4’ points away from the DNA duplex into the solvent region where it might play an important role for the interaction with topoisomerase 11. As shown previously by footprinting studies of various anthracyclines with DNA, the attached carbohydrate residues do not largely influence the sequence selectivity for DNA binding [65], even though they greatly influence both the binding affinity [66] and biological activity [49]. These observations are supported by the high resolution structure of the complex: the binding affinity is increased by co-operative van der Waals contacts of the carbohydrate moiety in the minor groove. However, specific hydrogen bond interactions between carbohydrate and bases are lacking. The wealth of detailed information of the complex discussed above served as starting point for structure-based design of new bis-intercalating anthracycline antibiotics [67]. In all structures reported to date, a 2: 1 ratio of daunorubicin (8a) to target DNA was observed. Both drug molecules intercalate at the end of the duplex positioning their carbohydrate groups in the minor groove pointing at each other (see Figure 3a). In this bound form, the two amino groups of both daunorubicins are roughly 7 apart and therefore could easily be bridged with an appropriate linker to give the bis-intercalator 9. Binding studies confirmed the bis-intercalating binding mode and revealed a picomolar binding affinity (27 pM) to herring sperm DNA at 20”C, roughly four orders of magnitude higher than daunorubicin @a). The hypothesized binding mode of dimer 9 was later confirmed by structure determination using x-ray crystallography [68] and high resolution NMR [69]. The structural information on daunorubicin (8a) and calicheamicin y1 (3) bound to duplex DNA guided the design of a hybrid molecule between the daunorubicin aglycon and the calicheamicin y1 tetrasaccharide [70]. The main goal of this approach was the creation of an intercalating daunorubicin analog with improved sequence selectivity by replacing the carbohydrate moiety. Examination of models indicated the requirement of a P-glycosidic linkage and a five-atom spacer (CH2CH20CH2CH2) between aglycon and tetrasaccharide. The corresponding “calichearubicin” was indeed shown to intercalate double stranded DNA, whereas an analog lacking the five atom spacer failed to intercalate. Footprinting studies on a 155 base pair fragment of pBR322 with methidium propyl [71] indicated a gain in binding specificity compared to daunorubicin. Due to the difficult interpretation of these results and the lack of discrete binding affinities for this hybrid molecule, a clear picture on sequence preference could not be obtained. During the late sixties the structurally more complex nogalamycin (10) [72] was isolated and characterized. This anthracycline is unique as two carbohydrate groups (nogalose and aminoglucose) are linked to the opposite ends of the tetracyclic aglycon, yielding a dumbbell shaped molecule. As a consequence, one of the bulky
A
39.2 Curbohydrutes Binding to D N A
1109
sugar residues needs to move through the helix to reach the opposite side, most likely requiring a local melt of base pairs. This special feature is reflected in the slow on- and off-rates for binding. The publication of various high resolution structures of nogalamycin (9) to short DNA duplexes [73-811 confirmed the proposed binding mode (Figure 4a) which is discussed in more detail below. In all the structures reported to date nogalamycin 10 is intercalating DNA, preferentially between purine-pyrimidine tracts, with the doubly connected aminoglucose on ring D positioned in the major groove. The conformation of the DNA bound ligand [77] closely resembles the ligand itself [82], indicating a well preorganized structure. A subtle difference is the slightly closer proximity between nogalose and aminoglucose in the bound state that allows nogalamycin (10) to optimize its interactions with the DNA in the complex (see below). The aglycon intercalates almost perpendicular to both flanking GC base pairs, similar to the orientation reported for daunorubicip (8a). The main difference, however, is the shift towards the minor groove by -2 A, caused by interactions of the aminoglucose in the major groove and the large nogalose in the minor groove. The aminoglucose faces the GC base pair with its flat surface of the six-membered ring, positioning both hydroxy groups (OzG and 04G) in hydrogen bonding distance to N-7 of GZ and N-4 of m5Cll, respectively (Figure 4b). The dimethylamino group is not directly interacting with the DNA or its negatively charged phosphodiester backbone but is bridged through a water molecule to N-6 of A,". Ring A of the aglycon adopts a similar conformction as reported for daunorubicin with CS dislocated from the mean plane by 0.53 A, placing the hydroxy group in an equatorial position pointing away from the minor groove. The axial methyl ester at C-10 points in direction of the minor groove and interacts with N-2 of GI2 through a hydrogen bond. The large hydrophobic nogalose inserts sideways in the minor groove and primarily interacts by Van der Waals contacts, thereby widening the groove by roughly 3 A. Only one direct interaction was proposed between the glycosidic 0-7 between nogalose and ring A and the hydrogen donating N-2 of G2. The distance between these two and the lower electron density of the involved oxygen lone pair due to atoms (3.5 the stereoelectronic conjugation with the neighboring C-0 bond, however, suggest that this interaction is only weak. As seen previously for the calicheamicin antibiotics, the anthracyclins represent another family of DNA binding ligands carrying modified carbohydrates. To gain energetic benefits from binding to DNA, carbohydrates have to be less hydrophilic and/or contribute to the overall charge neutralization. This allows optimal interaction at the bottom and sidewalls of the lipophilic minor groove allowing excellent shape recognition through van der Waals contacts. In addition, the conformationally rigid six-membered ring is an excellent scaffold to place hydroxy or amino groups such that they can specifically interact with the functional groups of nucleotide bases on the floor of both grooves. Nature chose a variety of approaches to make sugar residues successful DNA or RNA ligands: often the carbohydrate is deoxygenated and some or all of the remaining hydroxy groups are methylated as seen in nogalose. The aminoglucose found in nogalamycin (10) is deoxygenated as well and, in addition, contains a dimethylamino functionality that energetically contributes to the interaction with DNA by charge neutralization. Both the remain-
A)
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39 Carbohydrate-Nucleic Acid Interactions
a
b Figure 4. Stereo representation of the x-ray structure of nogalamycin (9) (Brookhaven: ld21; [77]) bound to d[m5CGT(pS)Am5CG]2.The hydrogen atoms are omitted to simplify the representation of the complex. a) 2 nogalamycin molecules are shown in space filling models intercalated in the modified duplex with water molecules removed, carbon atoms are shown in dark grey and oxygen/ nitrogen atoms in light grey. b) Part of the same structure including selected interacting water molecules (small balls in light grey). Heteroatoms involved in hydrogen bonding are drawn as small balls. Carbon and phosphorous atoms are shown in black, oxygen in light grey and nitrogen in dark grey.
39.2 Carbohydrates Binding to DNA
1 11 1
ing hydroxyl groups are perfectly positioned for direct interaction with hydrogen bond donor or acceptor sites at the binding site. 39.2.3 Pluramycins and Aureolic Acids
Pluramycins are closely related to nogalamycin (10): they consist of a central, tetracyclic chromophore with up to three substituents (altromycins) at different positions. Besides carbohydrates they contain epoxy functionalities that provide them with alkylating properties in the bound state. This class of antibiotics was recently reviewed [83] and will not be discussed further in this review. Chromomycin A3 (11) is an antitumor antibiotic belonging to the group of aureolic acids along with related analogs like mithramycin and olivomycin. Chromomycin is believed to bind DNA, thereby inhibiting RNA transcription and regulation [84, 851. It was demonstrated, that it binds in the presence of Mg2+ to double-stranded DNA [86] with a sequence specificity to GC rich sites [87-901. Even though the central, tricyclic aglycon indicated an intercalative binding mode, NMR structure elucidation revealed a unique binding mode of the magnesium bound dimer in the minor groove of DNA [91, 921. The complex is bound symmetrically around the central (GC)2 sequence of the target duplex d(AAGGCCTT)Z (Figure 5, [91]) with its unligated hydroxy group of the aglycon directly interacting with the exocyclic amino group of the central guanosines. The binding of the dimer requires a widening of the minor groove to accommodate both chromophores and
Figure 5. Stereo picture of a Mg2+ chelated chromomycin A3 (11) dimer bound to d(AAGGCCTT)z [91] as retrieved from the Brookhaven database (ld83). The hydrogen atoms are omitted to simplify the representation of the complex. The DNA duplex is represented as a CPK model (grey) and both the chromomycin dimer and magnesium ion are shown in black.
1112
39 CurbohydruteeNucleic Acid Interactions
sugar moieties C and D side by side in a parallel orientation to the walls of the groove. Carbohydrates E extend on both sides in the minor groove, facing the bottom of the groove. The trisaccharide C-D-E along with mainly with the sugar moiety A , primarily stabilize the complex by van der Waals intermolecular interactions within the minor groove of the target duplex. As seen with the previous antibiotics, the hydrophilic nature of all carbohydrate moieties has been altered by deoxygenation (ABCDE), methylation ( B E ) and acetylation ( A E ) to improve the overall binding characteristics. This complex reveals a different molecular recognition approach for DNA binding molecules that is related to the recent finding of alternate binding modes for distamycin A or netropsin related compounds [93, 941. However, the metal ion plays a crucial role indicating new possibilities in the design of specific ligands that achieve higher molecular weight by chelating to physiologically present metal ions. A different but nevertheless related approach was described for protease inhibitors that bind the target enzyme as dimers chelated to Zn2+ [95]. In summary, the properly functionalized, six-membered carbohydrates can fit into the minor groove of DNA, depending on the groove width either facing the floor or squeezed between the phosphodiester backbone facing the walls. Besides changing the overall properties like solubility and pharmacokinetic behavior, these carbohydrates usually improve the overall binding affinity either in a selective or non-selective manner. As discussed previously, these carbohydrates are modified to increase their lipophilic properties and in some cases carry cationic functionalities to contribute to the overall charge neutralization. Such scaffolds seem to be ideally suited as DNA recognition elements due to size, conformational preorganization, and the high degree of potential functionalization. The synthetic difficulties, however, to fully explore this potential explain why there is currently only a limited understanding on energetic contributions of individual groups to overall DNA binding. The combination of combinatorial techniques with improvements in carbohydrate chemistry and biosynthesis might not only contribute to our understanding of interactions at the molecular level but could also provide compounds with improved therapeutic potential.
39.3 Carbohydrates Binding to RNA Quite a few antibiotics containing modified carbohydrates interfere with protein biosynthesis rather than transcriptional processes by binding to DNA. Examples are erythromycin, streptomycin (12), tetracycline, spectinomycin, hygromycin, edeine and the neomycin family of aminoglycosides. Even though the observed biological effects have been correlated with perturbation of particular ribosomal events, the understanding of specific interactions on molecular level between such antibiotics and ribosomal components remained unknown for a long time. In analogy to enzyme inhibitors, it was assumed for these molecules to directly and specifically interact with functional sites of ribosomal components but until quite recently, no convincing evidence was found. The hypothesis that rRNA is the
39.3 Carbohydrates Binding to RNA
11 13
essential determinant of ribosome function lead to close examination of the interaction between a variety of antibiotics and rRNA by chemical footprinting [96, 971. Over the past decade increasing information has been provided to support specific and direct interaction with rRNA but size and complexity of this target [98] rendered structural elucidation extremely difficult. Two high resolution structures were reported only recently [99, 1001. They provide for the first time structural insight in molecular recognition between aminoglycosides and RNA. Further information was obtained by attempts to optimize either the low molecular weight aminoglycosides or the target RNA using chemical synthesis or affinity selection and amplification approaches, respectively. In addition to ribosomal RNA, aminoglycosides have been shown to interact with various, biologically relevant RNA structures. Among those are catalytic group I introns [ 101, 1021, hammerhead ribozymes [ 1031061, the rev-binding element of HIV [ 107-1091, and Hepatitis delta virus ribozyme [ 1101. 39.3.1. Aminoglycosides
Aminoglycosides are a class of aminosugar and aminocyclitol containing antibiotics that inhibit prokaryotic cell growth by interfering with ribosomal protein biosynthesis [ 111-1 141 (Scheme 4). They have been in clinical use for several decades and lack of oral bioavailability, toxicity and frequent occurrence of resistance limit their current use in humans. They are known to perturb translational events, causing a marked decrease in the fidelity of translation and/or inhibiting translocation. A variety of experimental findings led to an increased understanding of aminoglycoside function. Antibiotic resistance was not only found by enzymatic modifications of the aminoglycosides or mutations in ribosomal proteins but also by direct changes of the rRNA sequence [ 1151, indicating points of direct contact. The small 30s ribosomal subunit of procaryotes consists of 16s ribosomal RNA (1540 nucleotides in length) and 21 proteins. Sequence analysis of this RNA from aminoglycoside resistant strains indicated functionally important areas for binding. Streptomycin resistance in Escherichia coli was originally shown to be governed by ribosomal protein S12. More recently, however, mutants have been found with a C to U transition at position 912 of 16s rRNA that was directly correlated with the observed aminoglycoside resistance [ 116, 1171. This finding indicated that streptomycin (12) directly binds to RNA rather than proteins. Experimental support for this assumption was reported from RNA footprinting studies of intact 70s ribosomes and sequence analysis of the 16s rRNA: chemical modification by dimethylsulfate was strongly suppressed in the presence of streptomycin (12) at adenosines 913-915 and weak but significant protection was noted for both, uracil 91 1 and cytosine 912. This protection or shielding from chemical modification surrounds and includes the same nucleotide that was mutated in a resistant strain thereby indicating the importance of streptomycin binding at this RNA site for antibiotic function. The neomycin family of aminoglycosides displays a different pattern of RNA protection and mutations associated with resistance. For example paromomycin resistance has been shown to result from single mutations in both
1114
39 Carbohydrate-Nucleic Acid Interactions N
-r
h
8 e0 FII 2 A oc
II
I
0
Y
I"
z
I"
z \
39.3 Carbohydrates Binding to RNA
1115
yeast mitochondria1 [ 1 181 and Tetrahymena rRNA [ 119, 1201. In accordance to the numbering of E. coli 16s ribosomal RNA, both identified mutations aim at the disruption of the base pair between positions 1409 and 1491 (Scheme 5) that is part of the A-site involved in the decoding by binding aminoacyl-tRNAs. As described for streptomycin (12), an excellent correlation was found with chemical footprinting pointing at the A-site as binding site for paromomycin (16): adenosine 1408 and guanosine 1494 are strongly protected from alkylation at N-1 and N-7, respectively. Similar behavior was detected for neomycin and gentamycin, whereas the protection for the smaller kanamycin is less complete at both nucleotides. An additional clue is provided by antibiotic producing microorganisms in which the resistance is achieved by methylation of specific nucleotides on rRNA. The cloned methylases can be expressed in heterologous organisms like E. coli which they render resistant towards selected aminoglycosides by transforming either guanosine 1405 or adenosine 1408 in the A-site of 16s rRNA [121]. This site was mimicked by a self-structured oligoribonucleotide that bound both antibiotic and RNA ligands of the 30s subunit in a similar manner as compared to the normal subunit [ 1221. This oligoribonucleotide was further reduced in size to the 27-mer 21 (Scheme 5) that was shown to specifically bind paromomycin (16) with similar binding affinity and protection pattern as found for E. coli 16s rRNA in the 30s subunit [ 123, 1241. This complex turned out to be suitable for structure elucidation by NMR spectroscopy, providing insight in molecular interactions between aminoglycoside and target RNA [99, 125, 1261 (Figure 6a). Paromomycin (16) binds in the major groove of the A-site within a pocket that is formed by the asymmetric internal loop, thereby changing the local structure of the bound RNA [126]. Not bound to RNA, paromomycin (16) has in solution rather rigid ring conformations with certain flexibility at the glycosidic linkages connecting the four rings. In the bound form, however, paromomycin adopts a L-shaped conformation with rings 11, I11 and IV linearly arranged. Both, rings I and I1 are conformationally highly conserved; they adopt chair conformations placing all the substituents at the 6-membered ring in equatorial positions with a number of direct contacts to the RNA (see Figure 6b). Ring I stacks on the purine ring of guanosine 1491 and its substituents interact with the phosphodiesters of guanosine 1491 and adenosines 1492 and 1493. Both hydroxy groups, 3j-OH and 4/-OH, are not essential for aminoglycoside function [ 1271 but the replacement of the pro-R oxygens of the phosphates which point into the minor groove at positions 1492 and 1493 with sulfur interfere with paromomycin binding [ 1281. The 6/-OH group might possibly not only interact with the proximal phosphodiester of adenosine 1493 but also with its N-7 of the purine base. The only amino group at C-2’ strongly forms an intramolecular hydrogen bond with ring I11 and might additionally interact with the phosphodiester of guanosine 1491. Both amino groups of ring I1 (2-deoxystreptamine) directly interact with N-7 of guanosine 1494 and 0 - 4 of uracil 1495 and possibly with the phosphodiester between adenosines 1492 and 1493. The CG base pair between 1407 and 1494 is essential for aminoglycoside binding [ 1231, and carboxyethylation at N-7 of guanosine 1494 interferes with paromomycin binding. These crucial interactions between paromomycin (16) and the A-site model oligoribonucleotide 21 have been largely confirmed through an affinity chromatography
1116
1400
39 Carbohydrate-Nucleic Acid Interactions
c
G-C C :A’
..
C U C-G 1405
GVc 1495 U G C A A A C-G 1410 A- u 1490 C-G C-G
I t
3
5’
A
AG Ci5 U U lo U-A
u
G-C G-C C-G 1405 G-C U
1495
G
C
A
A
A
c -G 1410 A- u 1490 C-G C-G
U
G
u c 21
F C A G-C *O A-U
G-C 5C-G A-U C-G25 G-C G-C s
3’
22
20 Scheme 5. Secondary structure on Escherichia coli 16s RNA in the region of decoding A-site 20 and its shortened hairpin version (27-mer model oligoribonucleotide 21) used for the structure determination with bound paromomycin (16) [ 1251. Nucleotides which are protected from chemical modification by bound aminoglycosides, methylated in resistant strains or essential for aminoglycoside binding are shown in bold. For more detailed discussion refer to text. The sequence of the 27-mer RNA aptamer 22 with its secondary structure is shown. The consensus sequence identified for this family of aptamers is highlighted in bold [141].
based assay looking at binding interference between immobilized paromomycin (16) and various, base or backbone modified RNAs [ 1281. Rings I11 and IV of paromomycin (16) extend into the minor groove and in particular ring IV contributes through positive charges to the overall binding affinity without adding much to the specificity. This is supported by the fact that both, ribostamycin (14) and neamine (13), lacking ring IV and rings 111 and IV, respectively, bind to 16s rRNA and cause miscoding, albeit at higher concentration. Measured dissociation constants by surface plasmon resonance [I081 are 19 nM, 25 pM, and 7.8 pM for neomycin B (15), ribostamycin (14), and neamine (13), respectively (paromomycin (16): 0.20 pM) [129]. In addition, ring IV is partially disordered in the ensemble of 20 NMR structures providing the least confidence for conformational determination. Exchanging ring IV (2,6-dideoxy-2,6-diamino-P-~-
39.3 Carbohydrates Binding to R N A
11 17
a
,
Figure 6. Minimized average stereo representation of paromomycin (16) binding to the model 27mer oligoribonucleotide 21 (Scheme 5 ) [99, 1251 as retrieved from the Brookhaven database (Ipbr). The hydrogen atoms are omitted to simplify the representation of the complex. a) Complex with DNA in black and paromomycin (16) as a CPK model with carbon atoms shown in dark grey and oxygenlnitrogen atoms in light grey. b) Part of the same structure with the non-interacting base pairs and the loop removed. Carbon and phosphorous atoms are shown in black, oxygen in light grey and nitrogen in dark grey. Heteroatoms potentially involved in hydrogen bonding are drawn as small balls.
11 18
39 Carbohydrate-Nucleic Acid Interactions
idopyranose) by either simple amines or deaminated idoses revealed the importance of charges for high affinity binding even though flexible amines with identical charge were not fully able to substitute for the hydroxy group bearing and preorganized ring IV [130]. A library approach was chosen by Wong and co-workers to identify new A-site binding molecules based on the modification of ring I of neomycin B (15), containing a core 1,3-hydroxyamine motif [ 131, 1321. Despite various binding affinities (between 10 pM and >500 pM), no molecules were identified containing either a high binding affinity or selectivity for the target A-site model RNA, presumably due to lack of sufficient conformational preorganization. These aminoglycoside antibiotics of the neomycin family act preferentially on prokaryotic organisms. Comparison between ribosomes of either eukaryotic or prokaryotic origin reveals a 10-15-fold higher sensitivity of the latter. The main difference is the substitution of adenosine 1408 in prokaryotes for a guanosine at this position in eukaryotes. The adenosine pair 1408-1491 was shown to be essential for aminoglycoside binding, causing a specific binding pocket that accommodates ring I. As mentioned before, the base pair between positions 1409 and 1491 is essential for aminoglycoside binding as it forms the floor of the antibiotic binding pocket. Disruption of this base pair in prokaryotes leads to aminoglycoside resistance. Higher eukaryotic organisms, including humans, have both disruptions in their cytoplasmic rRNA sequences and as a consequence these aminoglycosides cannot bind the ribosomal target with high affinity. The perfect optimization of a RNA binding ligand is tedious and as experience demonstrates is not that straightforward. Independently, the groups of Gold [ 1331, Joyce [134],and Szostak 113.51 described a new technique that allowed optimization of the RNA (or DNA) binding partner rather than the small molecule ligand. This elegant and powerful approach, often referred to as ‘SELEX’, allows the optimization of certain properties from either a diverse RNA or DNA pool by repeating cycles of affinity selection and amplification of selected nucleic acid pools. Within weeks it became possible to identify optimized DNA or RNA sequences from 1014-1016of initial members with optimized properties not only to bind a variety of different molecules but also for catalyzing specific reactions (for reviews see [ 136-1381). Under conditions of high selection stringency, usually conserved RNA sequences are identified with low nanomolar binding affinities for their target molecule. This in vitro selection process has been applied to optimize RNA sequences for binding neomycin [ 1391, kanamycin [ 1401, and tobramycin (19) [ 1411. For the latter target aminoglycoside, on particular clone (named 56) was isolated coding for a 60-mer oligoribonucleotide insert with a dissociation constant for tobramycin (19) of 0.77 nM [142]. Key structural component of this RNA was shown to be a small stem loop that was incorporated in a 27-mer oligoribonucleotide 22 subjected to structure elucidation by NMR in the tobramycin (19) bound form (Figure 7a, [loo]). This structure is of particular interest as the affinity of tobramycin (19) to this RNA aptamer is roughly three orders of magnitude greater as compared to the ribosomal RNA target site and structural insight should provide the answer of how tighter binding to RNA can be achieved by aminoglycosides. Unfortunately, the resolution of the reported structure is not sufficient to locate specific intermolecular interactions at atomic level with possibly the exception of a
39.3 Carbohydrates Binding to RNA
1119
a
b Figure 7. Structure of tobramycin (19) bound to RNA apatmer 22 [loo]. From the co-ordinates of the 7 structures deposited in the Brookhaven database ( I tob), only the last was chosen to visualize interactions. a) Representation of the whole complex with the RNA displayed in surface view. b) Stick model of the actual binding sites with direct interactions by potential hydrogen bonds highlighted. Carbon and phosphorous atoms are shown in black, oxygen in light grey and nitrogen in dark grey.
1 120
39 Carbohydrate-Nucleic Acid Interactions
hydrogen bond between one of the amino groups at ring I and N-7 of guanosine 9 (Figure 7b). However, tobramycin (19) is well resolved with all three rings adopting a chair conformation connected by well-defined torsion angles. Some of the key features responsible for this specific and tight interaction can be summarized as follows: i) the increased width of the major groove at the binding site is governed by the mismatch between uracils 11 and 16 as part of a well defined and conserved loop that is critical for tobramycin binding; ii) cytosine 15 that is flapping over ring I11 of the bound tobramycin (19) and contributes to the large surface area (52%) that is covered by the RNA ligand in the complex; iii) the defined floor of the major groove that is formed by the edges of base pairs interacting with the ligand; and finally iv) the overall shape complementarity between tobramycin (19) and RNA aptamer 22. The work discussed above clearly indicates the importance of RNA as molecular target for small molecules to specifically (or non-specifically) interfere with crucial biological functions, an aspect that was recently reviewed by Michael and Tor [ 1431. A standard RNA duplex or stem with its wide, shallow minor groove and deep, narrow major groove is not ideally shaped to accommodate a low molecular weight compound. Distortions like bulges or internal loops often create the necessary binding pockets required for the binding event. The modified carbohydrates discussed here greatly helped to understand interactions between this class of molecules and structured RNA at the molecular level. Based on limitations like hydrophilicity and multiple positive charges, however, general designing rules for RNA binding molecules that would be of therapeutic use are difficult to make and the question if neutral molecules might specifically bind a specified target RNA with high affinity remains largely unanswered. First experimental evidence for such an approach are provided by Mei and Czarnik with the identification of neutral molecules disrupting either tat protein-TAR RNA interaction [ 144, 1451 or inhibiting a self-splicing group I intron ribozyme [ 1441. Interestingly, they demonstrated by footprinting techniques that binding is not necessarily competitive and can disrupt protein binding by induction of conformational changes on the RNA level. Additional evidence for the feasibility of neutral molecules to bind RNA with high affinity was provided by the identification of aptamers designed to bind a variety of different molecules. This approach, however, is extremely powerful as ‘perfect’ sequences can be selected from large pools containing roughly l O I 5 individual members and it might prove to be more difficult designing small molecules with drug-like properties for a given RNA target of biological relevance. References 1. R.C. Hawley, L.L. Kiessling, S.L. Schreiber. Proc. Nut1 Acud. Sci. U.S.A. 1989, 86, 11051109.
References 2. 3. 4. 5. 6. 7. 8. 9.
1121
D. Kahne. Chem. Biol. 1995,2, 7-12. J. Hunziker. Chimiu 1996, 50, 248-256. P. Sears, C.H. Wong. Proc. Nutl Acad. Sci. U.S.A. 1996, 93, 12086-12093. P. Sears, C.H. Wong. Chem. Commun. 1998, 1161-1170. M.D. Lee, G.A. Ellestad, D.B. Borders. Arc. Chem. Res. 1991, 24, 235-243. R.A. Kumar, N. Ikemoto, D.J. Patel. J. Mol. Biol. 1997,265, 187-201. R.A. Kumar, N. Ikemoto, D.J. Patel. J. Mol. Biol. 1998,265, 173-186. J. Golik, J. Clardy, G. Dubay, G. Groenewold, H. Kawaguchi, M. Konishi, B. Krishnan, H. 1987, 109, 3461-3462. Ohkuma, K. Saitoh, T.W. Doyle. J. Am. Chem. SOC. 10. J. Golik, G. Dubay, G. Groenewold, H. Kawaguchi, M. Konishi, B. Krishnan, H. Ohkuma, K. Saitoh, T.W. Doyle. J. Am. Chem. Soc. 1987,109, 3462-3464. 11. M.D. Lee, T.S. Dunne, M.M. Siegel, C.C. Chang, G.O. Morton, D.B. Borders. J. Am. Chem. SOC. 1987,109, 3464-3466. 12. M.D. Lee, T.S. Dunne, C.C. Chang, G.A. Ellestad, M.M. Siegel, G.O. Morton, W.J. McGahren, D.B. Borders. J. Am. Chem. Soc. 1987, 109, 3466-3468. 13. S.L. Schreiber, L.L. Kiessling. J. Am. Chern. SOC.1988, 110, 631--633. 14. S.J. Danishefsky, N.B. Mantlo, D.S. Yamashita, G. Schulte. J. Am. Chem. Soc. 1988, 110, 6890-6891. 15. W.L. Jorgensen. Chemtracts: Org. Chem. 1988, I, 485-487. 16. A.S. Kende, C.A. Smith. Tetrahedron Lett. 1988,29,4217-4220. 17. K.C. Nicolaou, Y. Ogawa, G. Zuccarello, E.J. Schweiger, T. Kumazawa. J. Am. Clzem. Soc. 1988, 110, 4866-4868. 18. P. Magnus, P.A. Carter. J. Am. Chem. Soc. 1988, 110, 1626-1628. 19. K. Tomioka, H. Fujita, K. Koga. Tetruhedron Lett. 1989, 30, 851-854. 20. S.M. Kerwin, C.H. Heathcock. Chemtracts: Org. Chem. 1989,2, 21-23. 21. B.H. Long, J. Golik, S. Forenza, B. Ward, R. Rehfuss, J.C. Dabrowiak, J.J. Catino, S.T. Musial, K.W. Brookshire, T.W. Doyle. Proc. Nutl Acud. Sci. U.S.A. 1989, 86, 2-6. 22. Y. Sugiura, Y. Uesawa, Y . Takahashi, J. Kuwahara, J. Golik, T.W. Doyle. Proc. Nut/ Acud. Sci, U.S.A . 1989, 86, 7612-7676. 23. M. Uesugi, Y . Sugiura. Biochemistry 1993, 32, 4622-4627. 24. L. Yu, J. Golik, R. Harrison, P. Dedon. J. Am. Chem. Soc. 1994, 116, 9733-9738. 25. N . Ikemoto, R.A. Kumar, P.C. Dedon, S.J. Danishefsky, D.J. Patel. J. Am. Chem. Soc. 1994, 116, 938779388, 26. J. Drak, N. Iwasawa, S. Danishefsky, D.M. Crothers. Proc. NatlAcad Sci. U S A . 1991, 88, 7464-7468. 27. J. Aiyar, S.J. Danishefsky, D.M. Crothers. J. Am. Chem. Soc. 1992, 114, 7552-1554. 1992, 114, 7555-7557. 28. K.C. Nicolaou, S.C. Tsay, T. Suzuki, G.F. Joyce. J. Am. Chem. SOC. 29. N. Zein, A.M. Sinha, W.J. McGahren, G.A. Ellestad. Science (Washington, D. C.) 1988,240, 1198-1201. 30. N . Zein, M. Poncin, R. Nilakantan, G.A. Ellestad. Science (Washington, D.C.) 1989, 244, 697-699. 31. S. Walker, J. Murnick, D. Kahne. J. Am. Chem. SOC. 1993, 115, 7954-7961. 32. S.L. Walker, A.H. Andreotti, D.E. Kahne. Tetrahedron 1994, 50, 1351-1360. 33. L.G. Paloma, J.A. Smith, W.J. Chazin, K.C. Nicolaou. J. Am. Chem. Soc. 1994, 116, 36973708. 34. G. Bifulco, A. Galeone, L. Gomez-Paloma, K.C. Nicolaou, W.J. Chazin. J. Am. Chem. Soc. 1996, 118, 8817-8824. 35. S. Walker, K.G. Valentine, D. Kahne. J. Am. Chem. SOC. 1990, 112, 6428-6429. 36. S. Walker, D. Yang, D. Kahnc, D. Gange. J. Am. Chenz. Soc. 1991,113,4716-4717. 37. S. Walker, V. Gupta, D. Kahne, D. Gange. J. Am. Chem. Soc. 1994, 116, 3197-3206. 38. T. Li, Z. Zeng, V.A. Estevez, K.U. Baldenius, K.C. Nicolaou, G.F. Joyce. J. Am. Chem. Soc. 1994, 116, 3709-3715. 39. S.N. Ho, S.H. Boyer, S.L. Schreiber, S.J. Danishefsky, G.R. Crabtree. Proc. Nut1 Acad. Sci. U.S.A. 1994, 91, 9203-9207. 40. K.C. Nicolaou, K. Ajito, H. Komatsu, B.M. Smith, T. Li, M.G. Egan, L. Gomez-Paloma. Angew. Chem., Int. Ed. Engl. 1995, 34, 576-518.
1122
39 Carbohydrate-Nucleic Acid Interactions
41. K.C. Nicolaou, K. Ajito, H. Komatsu, B.M. Smith, P. Bertinato, L. Gomez-Paloma. Chem. Commun. 1996, 1495-1496. 42. K.C. Nicolaou, B.M. Smith, J. Pastor, Y. Watanabe, D.S. Weinstein. Synletters 1997, 401410. 43. K.C. Nicolaou, B.M. Smith, K. Ajito, H. Komatsu, L. Gomez-Paloma, Y. Tor. J. Am. Chem. Soc. 1996, 118,2303-2304. 44. G. Bifulco, A. Galeone, K.C. Nicolaou, W.J. Chazin, L. Gomezpaloma. J. Am. Chem. SOC. 1998,120, 7183-7191. 45. C. Liu, B.M. Smith, K. Ajito, H. Komatsu, L. Gomez-Paloma, T. Li, E.A. Theodorakis, K.C. Nicolaou, P.K. Vogt. Proc. Nut1 Acad. Sci. U.S.A. 1996, 93, 940-944. 46. S.B. Shuker, P.J. Hajduk, R.P. Meadows, S.W. Fesik. Science (Washington, D.C.) 1996,274, 1531-1 534. 47. P.J. Hajduk, et al. J. Am. Chem. Soc. 1997,119, 5818-5827. 48. P.J. Hajduk, R.P. Meadows, S.W. Fesik. Science (Washington, D.C.) 1997, 278, 497--499. 49. J.W. Lown. Chem. Soc. Rev. 1993,22, 165-176. 50. W.R. Strohl, M.L. Dickens, V.B. Rajgarhia, A.J. Woo, N.D. Priestley. Drugs Pharm. Sci. 1997, 82, 577-657. 51. F.M. Arcamone. Biochimie 1998, 80, 201-206. 52. A.H. Wang. Adv. DNA Sequence Specific Agents 1996,2, 59-100. 53. F. Arcamone, G. Franceschi, P. Orezzi, S. Penco, R. Mondelli. Tetrahedron Lett. 1968, 33493352. 54. R.H. Iwamoto, P. Lim, N.S. Bhacca. Tetrahedron Lett. 1968, 3891-3894. 55. F. Arcamone, G. Franceschi, S. Penco, A. Selva. Tetrahedron Lett. 1969, 1007-1010. 56. P.B. Jensen, B.S. Soerensen, M. Sephested, E.J. Demant, E. Kjeldsen, E. Friche, H.H. Hansen. Biochem. Pharmacol. 1993, 45, 2025-2035. 57. G.J. Quigley, A.H. Wang, G. Ughetto, G. van der Marel, J.H. van Boom, A. Rich. Proc. Natl Acad. Sci. U.S.A. 1980, 77, 7204-7208. 58. A.H. Wang, G. Ughetto, G.J. Quigley, A. Rich. Biochemistry 1987, 26, 1152-1163. 59. M.H. Moore, W.N. Hunter, D. Langlois, 0. Kennard. J. Mol. Biol. 1989,206, 693-705. 60. G.A. Leonard, T.W. Hambley, K. McAuley-Hecht, T. Brown, W.N. Hunter. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1993, 049, 458-467. 61. Y. Gao, H. Robinson, E.R. Wijsman, G. van der Marel, J.H. van Boom, A.H. Wang. J. Am. Chem. SOC.1997,119, 1496-1497. 62. S. Neidle, G. Taylor. Biochim. Biophys. Acta 1977, 479, 450-459. 63. J.B. Chaires, J.E. Herrera, M.J. Waring. Biochemistry 1990, 29, 6145-6153. 64. C.J. Shelton, M.M. Harding, A S . Prakash. Biochemistry 1996, 35, 7974-7982. 65. K.R. Fox. Anti-Cancer Drug Des. 1988, 3, 157-168. 66. S. Kunimoto, Y. Takahashi, T. Uchidsa, T. Takeuchi, H. Umezawa. J. Antibiot. 1988, 41, 655-659. 67. J.B. Chaires, F. Leng, T. Przewloka, I. Fokt, Y.H. Ling, R. Perez-Soler, W. Priebe. J. Med. Chem. 1997, 40, 261-266. 68. G.G. Hu, X. Shui, F. Leng, W. Priebe, J.B. Chaires, L.D. Williams. Biochemistry 1997, 36, 5940-5946. 69. H. Robinson, W. Priebe, J.B. Chaires, A.H. Wang. Biochemistry 1997, 36, 8663-8670. 70. K.M. Depew, S.M. Zeman, S.H. Boyer, D.J. Denhart, N. Ikemoto, S.J. Danishefsky, D.M. Crothers. Angew. Chem., Int. Ed. Engl. 1997: 35, 2797--2800. 71. R.P. Hertzberg, P.B. Dervan. Biochemistry 1984, 23, 3934-3945. 72. P.F. Wiley, F.A. MacKellar, E.L. Caron, R.B. Kelly. Tetrahedron Lett. 1968, 663-668. 73. M.S. Searle, J.G. Hall, W.A. Denny, L.P. Wakelin. Biochemistry 1988, 27, 4340-4349. 74. Y.C. Liaw, Y.G. Gao, H. Robinson, G. van der Marel, J.H. van Boom, A.H. Wang. Biochemistry 1989, 28, 9913-9918. 75. X. Zhang, D.J. Patel. Biochemistry 1990, 29, 9451-9466. 76. L.D. Williams, M. Egli, Q. Gao, P. Bash, G. van der Marel, J.H. van Boom, A. Rich, C.A. Frederick. Proc. Nut1 Acad. Sci. U.S.A. 1990, 87, 2225-2229. 77. Y.G. Gao, Y.C. Liaw, H. Robinson, A.H. Wang. Biochemistry 1990,29, 10307-10316. 78. M. Egli, L.D. Williams, C.A. Frederick, A. Rich. Biochemistry 1991, 30, 1364-1372.
References
1123
H. Robinson, D. Yang, A.H. Wang. Gene 1994, 149, 179-188. C.K. Smith, G.J. Davies, E.J. Dodson, M.H. Moore. Biochemistry 1995, 34, 415-425. C.K. Smith, J.A. Brannigan, M.H. Moore. J. Mol. Biol. 1996,263, 237-258. S.K. Arora. J. Am. Chem. Soc. 1983, 105, 1328-1332. M.R. Hansen, L.H. Hurley. Acc. Chem. Res. 1996,29,249-258. W. Kersten, H. Kersten, F.E. Steiner, B. Emmerich. Z. Physiol. Chem. 1967,348, 1415-1423. W.E. Mueller. Pharmacol. Ther., Part A 1977, I , 451-474. Ward, D. C., Reich, E., and Goldberg, Irving H. Science (Washington, D.C.) 1965, 12591263. 87. M.W. Van Dyke, P.B. Dervan. Biochemistry 1983,22, 2373-2377. 88. K.R. Fox, N.R. Howarth. Nucleic Acid3 Res. 1985, 13, 8695-8714. 89. B.M. Cons, K.R. Fox. Nucleic Acids Res. 1989, 17, 5441-5459. 90. A. Stankus, J. Goodisman, J.C. Dabrowiak. Biochemistry 1992, 31, 9310-9318. 91. X. Gao, P. Mirau, D.J. Patel. J. Mol. Bid. 1992, 223, 259-279. 92. D.L. Banville, M.A. Keniry, M. Kam, R.H. Shafer. Biochemistry 1990, 29, 6521-6534. 93. J.G. Pelton, D.E. Wemmer. Proc. Nut1 Acad. Sci. U.S.A. 1989, 86, 5723-5727. 94. C.L. Kielkopf, S. White, J.W. Sewczyk, J.M. Turner, E.E. Baird, P.B. Dervan, D.C. Rees. Science ( Washington, D.C.) 1998, 282, 111-1 15. 95. K.D. Rice, R.D. Tanaka, B.A. Katz, R.P. Numerof, W.R. Moore. Curr. Pharm. Des. 1998,4, 381-396. 96. D. Moazed, H.F. Noller. Nature (London) 1987, 327, 389-394. 97. J. Woodcock, D. Moazed, M. Cannon, J. Davies, H.F. Noller. EMBO J. 1991, 10, 30993103. 98. E.V. Puglisi, J.D. Puglisi. Mod. Cell Bid. 1997, 17, 1-21. 99. D. Fourmy, M.I. Recht, S.C. Blanchard, J.D. Puglisi. Science (Washington, D.C. ) 1996,274, 1367-1371. 100. L. Jiang, A.K. Suri, R. Fiala, D.J. Patel. Chem. Biol. 1997, 4, 35-50. 101. U. von Ahsen, J. Davies, R. Schroeder. J. Mol. Bid. 1992, 226, 935-941. 102. U. von Ahsen, H.F. Noller. Science (Washington, 0 . C . ) 1993,260, 1500-1503. 103. T.K. Stage, K.J. Hertel, O.C. Uhlenbeck. R N A 1995, I , 95-101. 104. H. Wang, Y. Tor. Angew. Chem., Znt. Ed. 1998,37, 109-111. 105. H. Wang, Y. Tor. J. Am. Chem. Soc. 1997, 119, 8734-8735. 106. T. Hermann, E. Westhof. J. Mol. Bid. 1998, 276, 903-912. 107. M.L. Zapp, S. Stern, M.R. Green. Cell (Cambridge, Muss.) 1993, 74, 969-978. 108. M. Hendrix, E.S. Priestley. G.F. Joyce, C.H. Wong. J. Am. Chem. Soc. 1997,119, 3641-3648. 109. S.H. Wang, P.W. Huber, M. Cui, A.W. Czarnik, H.Y. Mei. Biochemistry 1998, 37, 55495557. 110. J.S. Chia, H.L. Wu, H.W. Wang, D.S. Chen, P.J. Chen. J. Biomed. Sci. (Basel) 1997, 4, 208216. 11 1. Puglisi, Joseph D. ‘Structural basis foy aminoglycoside antibiotic action’ in Many Faces RNA, (8th SmithKline Beecham Pharm. Res. Symp.), ed. by D.S. Eggleston, Ed. Academic Press, San Diego, CA 1998, p. 97. 112. Schroeder, U. von Ahsen. Nucleic Acid,? Mol. Biol. 1996, 10, 53-74. 1 13. Wank, Herbert and Schroeder, Renee. ‘Antibiotics that interfere with R N A J R N A interactions’ in Ribosomal RNA Group I Introns, ed. by R. Green and R. Schroeder, Eds. Landes, Austin, TX 1996, p. 129. 114. C. Bochaton, S. Rochegude, R. Roubille. Lyon Pharm. 1997, 48, 226-239. 115. P. Chakrabarti. Proc. Natl Acad. Sci., India, Sect. B 1997, 67, 169-179. 116. P.E. Montandon, R. Wagner, E. Stutz. EMBO J. 1986,5, 3705-3708. 117. P.E. Montandon, P. Nicolas, P. Schuermann, E. Stutz. Nucleic Acid.y Res. 1985, 13, 42994310. 118. M. Li, A. Tzagoloff, K. Underbrink-Lyon, N.C. Martin. J. Biol. Chem. 1982,257, 5921-5928. 119. E.A. Spangler, E.H. Blackburn. J. Bid. Chem. 1985,260, 6334-6340. 120. M. Li, A. Tzagoloff, K. Underbrink-Lyon, N.C. Martin. J. Biol. Chem. 1982,257, 5921-5928. 121. A.A. Beauclerk, E. Cundliffe. J. Mol. Bid. 1987, 193, 661-671. 122. P. Purohit, S. Stern. Nature (London) 1994,370, 659-662. 79. 80. 81. 82. 83. 84. 85. 86.
1 124
39 Carbohydrate-Nucleic Acid Interactions
123. M.I. Recht, D. Fourmy, S.C. Blanchard, K.D. Dahlquist, J.D. Puglisi. J. Mol. Biol. 1996, 262, 421-436. 124. H. Miyaguchi, H. Narita, K. Sakamoto, S. Yokoyama. Nucleic Acids Res. 1996, 24, 37003706. 125. D. Fourmy, M.I. Recht, J.D. Puglisi. J. Mol. Biol. 1998,277, 347-362. 126. D. Fourmy, S. Yoshizawa, J.D. Puglisi. J. Mol. Bid. 1998, 277, 333-345. 127. R. Benveniste, J. Davies. Antimicrob. Agents Chemother. 1973, 4, 402-409. 128. S.C. Blanchard, D. Fourmy, R.G. Eason, J.D. Puglisi. Biochemistry 1998, 37, 7716-7724. 129. C.H. Wong, M. Hendrix, E.S. Priestley, W.A. Greenberg. Chem. Biol. 1998, 5, 397-406. 130. P.B. Alper, M. Hendrix, P. Sears, C.H. Wong. J. Am. Chem. Soc. 1998, 120, 1965-1978. 131. M. Hendrix, P.B. Alper, E.S. Priestley, C.H. Wong. Angew. Chem., Int. Ed. Engl. 1997, 36, 95-98. 132. C.H. Wong, M. Hendrix, D.D. Manning, C. Rosenbohm, W.A. Greenberg. J. Am. Chem. SOC. 1998, 120, 8319-8327. 133. C. Tuerk, L. Gold. Science (Washington, D.C.) 1990,249: 505-510. 134. D.L. Robertson, G.F. Joyce. Nature (London) 1990,344,467-468. 135. A.D. Ellington, J.W. Szostak. Nature (London) 1990,346, 818-822. 136. G.F. Joyce. Curr. Opin. Struct. Bid. 1994, 4, 331-336. 137. L. Gold, B. Polisky, 0. Uhlenbeck, M. Yarus. Annu. Rev. Biochem. 1995,64, 763-797. 138. J.R. Lorsch, J.W. Szostak. Ace. Chem. Res. 1996,29, 103-110. 139. M.G. Wallis, U. von Ahsen, R. Schroeder, M. Famulok. Chem. Biol. 1995,2, 543-552. 140. S.M. Lato, A.R. Boles, A.D. Ellington. Chem. Bid. 1995,2, 291-303. 141. Y. Wang, R.R. Rando. Chem. Biol. 1995,2, 281-290. 142. Y. Wang, J. Killian, K. Hamasaki, R.R. Rando. Biochemistry 1996, 35, 12338-12346. 143. K. Michael, Y. Tor. Chem. Eur. J. 1998, 4, 2091-2098. 144. H.Y. Mei, M. Cui, S.M. Lemrow, A.W. Czarnik. Bioorg. Med. Chem. 1997, 5, 1185-1195. 145. H.Y. Mei, M. Cui, A. Heldsinger, S.M. Lemrow, J.A. Loo, K.A. Sannes-Lowery, L. Sharmeen, A.W. Czarnik. Biochemistry 1998, 37, 14204-14212.
Part I1 Volume 3
i
Biosynthesis of Glycoconjugates
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
1 Metabolism of Sugars and Sugar Nucleotides Hudson H. Freeze
1.1 Introduction Glycoconjugate biosynthesis requires activation of monosaccharides to sugar nucleotides. Specific glycosyltransferases then donate the activated sugars to various acceptors. To achieve this, monosaccharides must be imported into the cell, salvaged from degraded glycoconjugates or derived from other monosaccharides within the cell. Most glycosylations occur in the lumen of the ER or Golgi, but monosaccharide metabolism occurs in the cytoplasm. Sugar nucleotide specific transporters carry the activated donors into the Golgi. This chapter explores various pathways leading to the synthesis and delivery of precursors for glycosylation. Most of the biosynthetic reactions are covered in standard biochemistry texts, but this chapter will point out some recent observations suggesting that salvage pathways make substantial contributions to glycoconjugate synthesis. This bedrock of biochemistry may have undiscovered layers.
1.2 Basic Principles In 1951 Nobel Laureate L.F. Leloir showed that a nucleotide triphosphate such as UTP reacts with a glycosyl-1-P forming a high energy donor for glycoconjugate synthesis [l]. There are a few variations on the theme, but all sugars must either be activated by a kinase (reaction 1 below) or generated from a previously synthesized activated sugar nucleotide (reactions 2 and 3 below):
-
NTP PPi Sugar + ATP +Sugar-P
L?
Sugar-NDP
4
1 Metabolism of Sugars and Sugar Nucleotides
Sugar(A)-NDP Sugar(A)-NDP
+
Sugar(B)-NDP Sugar(B)-l-P Sugar(B)NDP
+ Sugar(A)-l-P
(2) (3)
All the donors are dinucleotides, except for sialic acid which is activated as a mononucleotide, CMP-Sia. Iduronic acid does not have a sugar nucleotide parent since it is formed by epimerization of glucuronic acid after this sugar is incorporated into the glycosaminoglycan chains. Some reactions use uncommon nucleotides as donors which are formed from the usual ones by a nucleotide exchange reaction (reaction 3 above).
1.3 Transporters Deliver Monosaccharides to Cells Carbohydrate degrading enzymes (such as a-amylase, disaccharidases, etc.) are secreted into the digestive tract or localized to the brush border of intestinal epithelial cells. These enzymes generate monosaccharides for sugar transporters located at the plasma membrane. Glucose and fructose account for most of the monosaccharide traffic in mammals. Glucose is transported from the digestive tract using energydependent sodium-glucose transporters, (SGLT) found on the intestinal epithelial cell surface. This transporter is also found on kidney tubules where it reabsorbs glucose from the kidney filtrates. Their K,s for glucose are < 1 mM [2]. Hexoses enter other cells via a set of facilitated diffusion transporters that mostly transport glucose (hence they are called GLUT transporters) 13-51. K,s for glucose are in the 2-20 mM range. Fructose is transported by one of the GLUT family members, GLUT 5. Even though the GLUT transporters can recognize other hexoses such as mannose and galactose, only glucose or fructose are in sufficiently high concentrations to be physiologically relevant. Other sugar-specific transporters occur in higher organisms and they may have physiological importance. Two types of mannose transporters are known [6-111. One is an energy-dependent transporter analogous to the SGLT variety used for glucose and located on the apical surface of enterocytes and kidney tubule epithelial cells [7, 9, 111. It presumably transports mannose liberated from digested macromolecules and retrieves mannose from the kidney filtrates, analogous to SGLT used for glucose. The second type of transporter is a highly mannose-specific, energyindependent transporter found on the surface of many types of mammalian cells [6, 8, 101. It is practically insensitive to inhibition by glucose and its is about 50 pM, which is near the average concentration of mannose in the blood of mammals (average 75 pM, range 30-120 pM). Following intravenous injection in rats, mannose is primarily taken up by the liver and intestine [ 101. Hepatoma cells and fibroblasts preferentially use mannose for up to 80% of their glycoprotein synthesis when given physiological concentrations of both mannose (50 pM) and glucose (5 mM) [lo, 121. Direct conversion from glucose (see below) is a relatively minor pathway in these cells. This is quite striking considering the 100-fold molar excess of
1.4 Intracellulur Sources of Sugars
5
glucose in the blood. The relative contributions of mannose and glucose in different tissues and organs in intact animals is unknown. A fucose-specific transporter [ 131 has also been reported in mammalian cells. The Kuptakeis -250 pM, which is much higher than the fucose concentration in human blood (4pM). We do not know if this transporter is physiologically important, but much of what is taken up can be converted into GDP-Fuc and incorporated into glycoproteins. In cell culture, most GDP-Fuc is thought to be derived from GDP-Man [ 141 (see below). No cell surface transporters specific for galactose, amino sugars, or sialic acids are known in higher organisms, but about 25%) of radiolabeled glucosamine injected into the peritoneum of rats or rabbits is incorporated into serum glycoproteins as GlcNAc and sialic acid [15].
1.4 Intracellular Sources of Sugars 1.4.1 Salvage
Monosaccharides can also be salvaged from glycoconjugates degraded within the same cell. Salvage pathways have not been systematically explored, however, their contribution to glycosylation may be quite substantial (see Table 1). For instance, -80'%1 of radiolabeled GlcNAc derived from a glycoprotein degraded in liver lysosomes is converted into UDP-GlcNAc [ 161. At least a third of that amount is used to synthesize secreted glycoproteins. Also, glycoconjugates endocytosed by fibroblasts are degraded in the lysosomes and about 50% of the amino sugars are used for new glycoprotein synthesis [ 171. Thyroid glands blocked in de novo synthesis of UDP-GlcNAc from fructose-6-P degrade thyroglobulin and use the liberated GlcNAc to maintain protein glycosylation [ 181. Salvage is not limited to GlcNAc. Glycosylation-impaired mutant CHO cells (fdl-D)that require supplements of Gal and GalNAc for normal 0-glycosylation can endocytose and degrade glycoproteins from the 5-10% fetal bovine serum in the culture medium, and use the liberated Table 1. Evidence for the importance of monosaccharide salvage pathways. Amino Sugars * More than 50'1/0of label reused for new glycoconjugates (GalNAc, GlcNAc) * Approximately 70% of 3H-GlcNAc incorporated into sugar nucleotides in rat liver * 15-909'0 Sialic acids recycled in some cells * GlcNAc/ManNAc kinase present in many tissues * GalNAc-I-kinase distinct from Gal-1-kinase Hexose and Fucose * Mannose transporter uses physiological concentrations of Man * Cells lacking UDP-Gal/GalNAc-4-epimerase can use serum glycoproteins for Gal and GalNAc * Fucose transporter identified * Correction of GDP-Man-GDP-Fuc deficiency with exogenous Fuc
6
I Metabolism of’ Sugars and Sugar Nucleotides
monosaccharides for glycosylation [ 19, 201. It is likely that 30-900/0 of sialic acids released in the lysosome can be reutilized for new glycoconjugate synthesis [21, 221. Since glycoconjugate degradation occurs in the lysosome, and monosaccharide activation in the cytosol, reutilization of liberated monosaccharides requires their exit from the lysosome. To do this, the lysosome has separate carriers for neutral hexoses, N-acetylated amino sugars, and acidic hexoses [23]. The neutral hexose carrier has a K, of 50-75 mM and also transports fucose and xylose [23]. Although these sugars could diffuse through the lysosomal membrane, their efflux rate may not be high enough to keep up with synthetic demands. The N-acetylhexosamine carrier with a 4.4 mM K, cannot use non-acetylated amino sugars. Sialic and glucuronic acids will not diffuse out of the lysosome. Their carrier has a relatively low K, (-300-550 pM) to prevent accumulation of these sugars in the lysosome [23]. Once the sugars reach the cytoplasm, they can be activated to sugar nucleotide donors as described below. Is sugar transport from the lysosome associated with sugar activation? This has not been demonstrated, but a small amount of GlcNAc kinase is found in lysosomal preparations from rat liver [24]. The quantitative contributions of exogenous sugar, salvage pathways, and interconversions probably vary with the cell type and amount of glycoproteins they synthesize. This is clearly the situation seen in the case of CMP-sialic acid synthesis described later in this chapter. Most of the work on monosaccharide and sugar nucleotide metabolism centers on the direct activation or interconversions of various sugars, regardless of their source. Nearly all cell culture studies supply growth media that contains only glucose and 10-20% serum. It is usually assumed that glucose generates all of the other monosaccharides. Certainly, it can, but does it normally do so? Metabolic radiolabeling with glucose is inefficient, in part because most glucose enters the glycolytic pathway, but few experiments have directly assessed the amount of glucose converted to other monosaccharides and then into various glycans [12, 25, 261. The relative contributions of diet, salvage, and various interconversions is really not known [25-271. Recent studies suggest some key biosynthetic enzymes are quite restricted in their distribution in different organs [28]. Other studies show that the clinical conditions of patients with a human glycosylation disorder (Carbohydrate Deficient Glycoprotein Syndrome, CDGS) can be improved substantially by providing the missing monosaccharides as dietary supplements [29, 301. Clearly, these pathways can play an important role in maintaining health.
1.4.2 Activation and Interconversion of Monosaccharides The major pathways for sugar activation and monosaccharide interconversions are shown in Figure 1. Glycogen Glycogen is the major storage polysaccharide in animal cells. This mega-dalton-size molecule contains up to 100,000 glucose units, arranged in Glcal,4Glc repeating
7
1.4 Intracellukar Sources of Sugars
Tm- t v
Gal-1-P
GlycTn
$,
UTPI UDP-Glc
inir
G-;P il J,
Glycolysis
-
I
I
I
I 2,Fry-6-P I
-
-J
$131ACC~A
ATP
Man-6-P J,
ATP F :-1-P
Man-I-P
I GTP
GTP
GIcNAc-6-P
IUTP
bMaANAc
/
ManNAc-6-P
1
NADP
PEP
*-
Figure I . Biosynthesis and interconversion of monosaccharides. The relative contributions of each and (-donors; =-monosaccharides; under physiological conditions are unknown. control points (Used with the permission of the Consortium of Glycobiology Editors).
a
disaccharides with periodic ul,6Glc branches. Glycogen synthesis and degradation (glycogenolysis) are highly regulated for energy utilization. Some of the glucose used to synthesize other sugars may be derived from glycogen as well as from glucose transported into the cell. Glycogen is synthesized by addition of single glucose units from UDP-Glc, and it is degraded by glycogen phosphorylase. The non-ATP dependent reaction forms Glc-1-P which can be used directly to form UDP-Glc or epimerized to Glc-6-P for glycolysis. Glycogen is actually a glycoprotein. The core protein, called glycogenin, comprises < 1% of the mass of the molecule and has the unusual property of being selfglucosylating. Up to seven glucose residues are polymerized on a single Tyr (Tyr 194) of the protein. Glycogen synthase extends the primed glycogenin into huge glycogen molecules [31-33].
Glucose Glucose is convertible into all other monosaccharides (Figure 1). Hexokinase begins the glycolytic pathway generating Glc-6-P which can then either form Fru-6-P by phosphoglucose isomerase or Glc- 1-P by phosphoglucomutase. Reaction of Glc-1-P
8
I Metabolism of Sugars and Sugar Nucleotides
with UTP generates UDP-Glc. The large UDP-Glc pool is used for glycogen synthesis or glucose-containing molecules such as glucosylceramide and dolichol-P-Glc in N-linked oligosaccharide biosynthesis. Some proteins with EGF-like modules contain P-Glucose in 0-linkage to Ser/Thr [34]. Glucuronic acid Oxidation of UDP-Glc to UDP-glucuronic acid requires NAD. This acidic sugar is used primarily for glycosaminoglycan biosynthesis. Its importance is emphasized in developmental mutants of Drosophila (sugarless, sgl) that are defective in this enzyme [35-38]. UDP-GlcA is used to add GlcA to N - and 0-linked oligosaccharides and glycolipids, for the glucuronidation of bile acids [39-411 and detoxification of xenobiotic compounds in the ER [42]. Some evidence sugests that UDP-GlcA is derived from glycogenolysis rather than gluconeogenesis [43]. Iduronic acid This uronic acid (IdoA), the 5-epimer of GlcA, is found only in glycosaminoglycans. IdoA is not directly synthesized from a sugar nucleotide donor. Instead, it arises by epimerization of GlcA after its incorporation into the GAG chain. Xylose Decarboxylation of UDP-GlcA produces UDP-Xyl which is used primarily to initiate glycosaminoglycan synthesis, but it is also found as an extension to the 0-PGlucose residues found on some proteins with EGF-like modules [34]. The decarboxylation reaction that generates UDP-Xyl appears to occur in the lumen of the ER or early in the Golgi, as well as in the cytoplasm [44, 451. Mannose Mannose is a key sugar used for N-linked oligosaccharide and GPI-anchor synthesis. GDP-Man can be formed in two ways. First, directly from mannose beginning with hexokinase, or in some invertebrates, by a mannokinase [46].The second, and by far the best known way, is by conversion of Fru-6-P to Man-6-P through the enzyme phosphomannose isomerase (Figure 1). Genetic loss of a majority of this enzyme produces a potentially fatal human disease called CDGS (Type lb) [29, 30, 47, 481. However, the defect can be rescued by providing the patient with oral mannose supplements. In yeast, loss of PMI is lethal, but it can also be rescued with exogenous mannose [49]. Mannose is essential for glycosylation, but at least for bees, too much mannose is lethal [ 501. This curious phenomenon, called the “honeybee syndrome”, occurs when bees feed on mannose instead of sucrose or glucose. When mannose is the sole energy source for the bees, it serves as an ATP sink. Hexokinase converts mannose to Man-6-P, but further metabolism via glycolysis requires conversion to Fru-6-P. Honeybees have relatively low PMI activity compared to hexokinases which creates a bottleneck, and excess Man-6-P accumulates. It is quickly degraded by phospha-
1.4 Intracellular Sources of' Sugars
9
tase to free mannose, and again phosphorylated with the ever-diminishing supply of ATP. Very high concentrations of mannose are teratogenic in rats for a similar reason [51, 521. Early in development, the embryo relies more on glycolysis than on oxidative phosphorylation. At high concentrations, mannose enters cells via the typical glucose transporters. but once inside, the low PMI activity again creates a bottleneck in energy production, causing abnormal or arrested development. After Man-6-P is formed, it is converted to Man-1-P by phosphomannomutase. Two genes, P M M l and PMM2, produce two different isozymes in humans. Loss of PMM2 produces another form of CDGS (Type la) that results from underglycosylation of proteins [53, 541. Depletion of Man-6-P or Man-1-P limits the formation of GDP-Man and Dol-P-Man needed for lipid-linked oligosaccharide synthesis. Dol-P-Man is also the donor for synthesis of glycophospholipid anchors and the recently identified C-mannosylation reaction [55]. Another type of human Congenital disorder of glycosylation (CDG) results from defects in Dol-P-Man synthesis [56].
Fucose GDP-Fucose can be derived from GDP-Man by oxidation of the 6-OH of Mannose to CH3. This reaction is catalyzed by a three-step reaction that uses two enzymes. In the first step, the C-4 Man of GDP-Man is oxidized to a ketone, GDP-4-dehydro-6deoxy-mannose by the enzyme GDP-Man 4,6-dehydratase along with the reduction of NADP to NADPH. A single polypeptide called Fx catalyzes the next two reactions. GDP-4-keto-6-deoxy-mannose is first epimerized at C-3 and C-5 to form GDP-4-keto-6-deoxyglucose and then reduced with NADPH at C-4 to form GDPFuc [57-591. F x is well-conserved from bacteria and mammals. The first dehydration step is feedback inhibited by GDP-Fuc [60]. Free fucose can also be converted to GDP-Fuc after it is first converted to Fuc-I-P. Some mutant cell lines that cannot convert GDP-Man to GDP-Fuc form underfucosylated proteins [ 141, but the block can be bypassed with exogenous fucose. The human glycosylation disease called Leukocyte Adhesion Deficiency Type-I1 (LADII) may result from reduced GDPMan+GDP-Fuc conversion [61] or reduced utilization of GDP-Fuc. Regardless of the actual mechanism, underfucosylated glycoproteins result. These include sialyl Lewis' glycans that are required for selectin-mediated leukocyte rolling and extravasation into inflamed tissues [62]. Patients with LADII have chronic infections since they cannot effectively extravasate leukocytes to the sites of inflammation. They require constant antibiotic treatment. In addition, these patients show mental and psychomotor retardation and a severe failure to thrive. Fucose can be transported by mammalian cells, as mentioned above, and then converted to Fucose-I-P by a fucose kinase [63]. GDP-Fuc synthase generates the activated donor. The quantitative contribution of this fucose salvage pathway under normal conditions is not known, but one LADII patient was treated with oral fucose supplements and showed dramatic improvements in his clinical condition after only a few months of therapy [64]. Fucose was absorbed well. neutrophil levels returned to normal, infections ceased, antibiotics were discontinuted, and having a medically
10
I Metabolism of Sugars and Sugar Nucleotides
stable base, the patient showed significant psychomotor improvement. Clearly, there is much yet to learn about the importance of direct utilization of fucose by higher mammals. In pigs, fucose can also be fully oxidized by the liver, but this catabolic pathway is incomplete in rats [65]. Galactose
Galactose is activated to UDP-Gal in several ways. First, by direct phosphorylation to give Gal-1-P that reacts with UTP to form UDP-Gal. Alternatively, Gal-1-P is converted to UDP-Gal via a uridyl transferase exchange reaction with UDP-Glc displacing Glc-1-P. A deficiency in the uridyl transferase activity results in a severe human disease called galactosemia, leading to mental retardation, liver damage and eventual death [66]. Finally, UDP-Gal can be formed from UDP-Glc by the NADdependent reaction catalyzed by UDP-galactose 4-epimerase [67]. The 4-OH group is first converted to a 4-keto derivative with the formation of NADH from NAD. In the next step, NAD reforms and the hydroxyl group is regenerated producing one of the two 4-epimers. The same enzyme converts UDP-GalNAc to UDP-GlcNAc. N-Acetylglucosamine
Synthesis of UDP-GlcNAc begins with the formation of glucosamine-6-P from fru6-P by transamidation using glutamine as the -NH2 donor. Glucosamine-6-P is N acetylated via acetyl-CoA to form GlcNAc-6-P and then isomerized to GlcNAc-1-P via a 1,6 bis-phosphate intermediate. Similar to the other activation reactions, GlcNAc-1-P + UTP then yields UDP-GlcNAc and pyrophosphate. Alternatively, GlcNAc can be directly phosphorylated to form GlcNAc-6-P using a GlcNAc/ ManNAc kinase [24]. A mutase then converts GlcNAc-6-P to GlcNAc-1-P. This route may account for the efficient salvage of GlcNAc from lysosomal degradation. GlcN can be very efficiently used for liver glycoprotein synthesis in intact animals, showing that direct phosphorylation can be important [15]. Not all GlcN is necessarily used for glycoprotein synthesis, since it depends on the cell type. For instance, in high energy requiring tissues such as neurons and transporting epithelial cells in the kidney and intestine, glucosamine-6-P deaminase (GlcN-6-P- - - - - ->Fru-6P + NH4) (GNPDA) generates Fru-6-P for glycolysis [68]. This is not simply the reverse of the biosynthetic transamidation reaction which uses glutamine as a donor. The GNPDA reaction proceeds in the forward direction, since the concentration of NH4 required for GlcNH2 formation would be extremely high. This enzyme is essentially absent from liver where other sources of energy are available. Thus, UDP-GlcNAc can be formed from Fru-6-P or from GlcNAc via kinase. Separate pools of UDP-GlcNAc may arise for GalN and GlcN [69]. N-Acetylgalactosamine
UDP-GalNAc can arise from two routes. One is the direct reaction of GalNAc-1-P with UTP. GalNAc-1-P is formed by a specific kinase that probably uses ATP, but can also use ITP [70]. This enzyme is distinct from galactose-1-kinase. UDPGalNAc can also be formed by epimerization of UDP-GlcNAc using the same NAD-dependent epimerase that converts UDP-Glc to UDP-Gal [ 191.
1.5 Suyur Nucleotide Trunsportrrs
11
Sialic acids Sialic acid is the generic name given to a group of more than 30 different variations of the parent compound. The modifications include oxidation, single and multiple acetylations, sulfation, and methylation. Except for the formation of the glycolyl derivative as an activated sugar nucleotide, all the other modifications of sialic acid probably occur in the Golgi after transfer of the sialic acid to the acceptor glycan [71, 721. CMP-N-acetyl (or glycolyl) neuraminic acid is the immediate donor. It has a complicated de novo biosynthetic pathway that begins with UDP-GlcNAc which is converted to N-acetylmannosamine by a single enzyme with two catalytic activities. First, is the 2-epimerization and cleavage of the UDP to yield N-acetylmannosamine. Next, this same polypeptide uses ATP as a donor forming N-acetylmannosamine-6P. In the next step, this compound condenses with phosphoenolpyruvate forming Nacetylneuraminic acid 9-P. Phosphate is removed and reaction with CTP yields CMP-N-acetylneuraminic acid PPi. All the steps, except the last one, occur in the cytosol. CMP-Sialic acid is formed in the nucleus with subsequent export to the cytoplasm [71]. This is the only monosaccharide donor made in the nucleus. The reason for this unusual localization is not known. There is an alternate biosynthetic pathway leading to sialic acid. The epimerase/ kinase enzyme activity can only be detected in liver, salivary gland and intestinal mucosa [73]. The mRNA can only be detected in the liver [28]. It is clear that many other tissues contain sialylated glycoproteins and glycolipds, making it likely that other pathways exist. Salvage of sialic acids from other proteins is extremely efficient in some cells and may explain the absence of epimerase/kinase. Another possibility is that a widely distributed 2-epimerase converts GlcNAc to ManNAc. No ManNAc specific kinase has been described, but the GlcNAc kinase can also use ManNAc to form ManNAc-6-P although the K, for ManNAc (0.9 mM) is about 15-fold higher than GlcNAc (0.06 mM) [74].
+
1.5 Sugar Nucleotide Transporters Sugar nucleotides synthesized in the cytosol (or nucleus for CMP-sialic acid) are carried into the ER and Golgi using sugar nucleotide transporters, actually antiporters, that deliver sugar nucleotides into the lumen of these organelles. This occurs with simultaneous exit of nucleotide monophosphates which must first be derived from the nucleotide diphosphates. K , of the transporters range from 1-10 pM. Using in vitro systems, the transporters have been shown to increase the concentration of the sugar nucleotides within the Golgi lumen by 10-50 fold. This is usually sufficient to reach or exceed the calculated K, of sugar nucleotides for most glycosyltransferases [75, 761. Most of the antiporters are found in the Golgi, but some also occur in the ER [75, 761. They are organelle specific and their location usually corresponds to the location of the known glycosyltransferases. Their import into the Golgi is not energy
12
I Metabolism of Sugars and Sugar Nucleotides
dependent or affected by ionophores, however they are competitively inhibited by the corresponding nucleoside mono- and di-phosphates, but not by the monosaccharides. In addition to sugar nucleotide-specific transporters, separate transporters exist for ATP and PAPS, the donor for protein and carbohydrate sulfation. Glucuronidation of bile and xenobiotic compounds in the ER [41, 421 is consistent with the presence of the UDP-GlcA transporter in the ER, and the identification of reglycosylation of misfolded glycoproteins in the ER explains the need for an ER UDP-Glc transporter 1771. The ER UDP-Xyl transporter may be needed for initiating GAG chain synthesis there 144, 451. The existence of UDP-GlcNAc, and UDP-GalNAc transporters in the ER is more difficult to explain. For most glycosylation reactions, the sugar nucleotide donates the sugar residue resulting in the formation of nucleoside diphosphate, which must be converted into a monophosphate by the nucleoside diphosphatase that occurs in the Golgi lumen. In yeast a GDPase converts GDP to GMP. Disrupting the gene that codes for this Type I1 membrane protein reduces glycosylation of all the mannosylated glycoconjugates 1781. The antiporter system has the advantage of coupling the rate of sugar nucleotide utilization with its import. However, the Golgi can have a pool of transported, but unutilized, sugar nucleotides that can participate in glycosylation. This can be seen in in vitro Golgi preparations that are capable of glycosylating partially completed endogenous glycoproteins as well as freely diffusable glycoside acceptors 179, 801. The artificial acceptors enter the Golgi where they encounter glycosyl transferases and, in some cases, glycosylation proceeds even though no exogenous donors have been added [81]. Another advantage of using the antiporter system is that the precursor monophosphate is returned to the cytosol where it is available for another round of activation. This creates a highly efficient salvage system for the nucleotide precursors. Several transporters, including CMP-Sia, UDP-Gal and UDP-GlcA, have been reconstituted into proteoliposomes and used for functional assays and purification of the proteins. Several transporters have been cloned from mammals (CMP-Sia, UDP-Gal), yeast (UDP-GlcNAc) 175, 76, 821 and protozoa (GDP-Man) [83]. All are very hydrophobic and appear capable of spanning the membrane many times. They appear to function as homodimers. Several mutant mammalian cell lines lack specific sugar nucleotide transporters, e.g. UDP-Gal and CMP-Sia, and as a result they make incomplete sugar chains. However, they are “leaky” mutants. For instance, loss of the UDP-Gal in the Golgi of mutant MDCK cells, decreases the synthesis of galactosylated glycoproteins, glycolipids and keratan sulfate, but heparan and chondroitin sulfate synthesis is unaffected [84]. The galactosyl transferases that synthesize the core region tetrasaccharide common to GAG chains probably have low K,,, values for their sugar nucleotide donors. Mammalian cells do not have GDP-Man transporters but the trypanosomal parasite Leishmania donovani does [ 831 and it is required for the synthesis of lipophosphoglycan (LPG). Yeast also has a GDP-Man transporter which is essential for mannan synthesis 1821. In both microbes, specific inhibition of GDP-Man transporters might have potential therapeutic value. Theoretically, glycosylation could be controlled by regulating the presence or activity of sugar nucleotide transporters within the Golgi. At this time, the sub-
1.7 Possible Future Directions
13
compartmental location (cis, medial, trans) of the transporters in the Golgi is unknown as is their physical relationships to the various glycosyl transferases they service. Golgi localization has focused on glycosyltransferases, but there have been few revealing studies about how the actual glycosylation reactions occur within the Golgi. There is physical evidence for complexes containing several mannosyl transferase in yeast [ 851 and circumstantial evidence for association of selected glycosyltransferases in complexes in mammalian cells, but no studies have yet been able to determine whether the transporter is part of a complex.
1.6 Control of Sugar Nucleotide Levels This topic may be physiologically very important, but it is not well understood. A human genetic disorder called sialuria shows the importance of at least one prediction from in vitro studies. In this condition, large amounts of sialic acid (5-7 g/day) are secreted into the urine along with various intermediates in the UDPGlcNAc--->CMP-Sia pathway [86, 871. This was confirmed to be due to a defective feed-back inhibition of the 2-epimerase/ManNAc kinase, the first step in the pathway [88]. Most of the precursor pools turnover within a few minutes [12], and the size of various sugar nucleotide pools have been determined by methods with different reliability. But even with the best methods, it is hard to interpret the measured numbers and translate them into a clear picture because the relative distribution of the precursors in the cytosol and Golgi is not known. The average cellular concentration of sugar nucleotide precursors may not be very meaningful, since “cytosol” is an operational definition (100,000 g supernatant) that may not detect compartments of cytoplasmic organization or substrate channeling. Providing moderate amounts (< 1 mM) of glucosamine to muscle cells increases the size of their UDP-hexosamine pools and creates the biochemical phenotype of non-insulin dependent diabetes [89]. Glucosamine-incubated cells do not increase glucose uptake or form glycogen in response to insulin [90, 911. Glucosamine can induce a similar response when given to animals [91, 921. The pathway and mechanism for this effect is not known, but finding O-GlcNAc on many regulatory proteins may suggest a link [93, 941. Alternatively, since the UDP-GlcNAc concentration is second only to ATP (1-2 mM), some of the effects could simply be due to consumption of ATP in producing UDP-GlcNAc [95].
1.7 Possible Future Directions The production and utilization of glycoconjugate precursors is more complex than the metabolic chart indicates. Future studies may want to address various sources of monosaccharides, the organization of metabolic enzymes within the cells, and the
14
I Metabolism of Sugars and Sugar Nucleotides
influence of diet and environment on the metabolic control of complex sugar chain synthesis [26]. These studies will be especially important for patients with glycosylation disorders who are being treated with monosaccharide therapy [29, 30, 641.
References 1. Leloir, L.F. In: Phophorus Metabolism, 1st ed.; McElroy, W.D., Glass, B. (Eds.); Johns Hopkins Press: Baltimore, 1951; pp 67. 2. Thorens, B. Glucose transporters in the regulation of intestinal, renal, and liver glucose fluxes. Am. J. Physiol. 1996,270, G541LG553. 3. Could, G.W.; Bell, (3.1. Facilitative glucose transporters: an expanding family. Trends. Biochem. Sci. 1990, 15, 18-23. 4. Could, G.W.; Holman, G.D. The glucose transporter family: structure, function and tissuespecific expression. Biochem. J. 1993,295, 329-341. 5 . Walmsley, A.R.; Barrett, M.P.; Bringaud, F.; Could, G.W. Sugar Transporters from Bacteria, Parasites and Mammals: Structure-activity Relationships. TIBS 1998, 23, 476-48 1. 6. Panneerselvam, K.; Freeze, H.H. Mannose enters mammalian cells using a specific transporter that is insensitive to glucose. J. B i d Chem. 1996, 19t271, 9417-9421. 7. Ogier-Denis, E.; Blais, A,; Houri, J.J.; Voisin, T.; Trugnan, G.; Codogno, P. The emergence of a basolateral 1-deoxymannojirimycin-sensitivemannose carrier is a function of intestinal epithelial cell differentiation. Evidence for a new inhibitory effect of 1-deoxymannojirimycin on facilitative mannose transport. J. Bid. Chem. 1994, 269, 4285-4290. 8. Ogier-Denis, E.; Codogno, P.; Chantret, 1.; Trugnan, G. The processing of asparagine-linked oligosaccharides in HT-29 cells is a function of their state of enterocytic differentiation. An accumulation of Man9,8-GlcNAc2-Asn species is indicative of an impaired N-glycan trimming in undifferentiated cells. J. Biol. Chrm. 1988, 263, 6031-6037. 9. Ogier-Denis, E.; Trugnan, G.; Sapin, C.; Aubery, M.; Codogno, P. Dual effect of 1deoxymannojirimycin on the mannose uptake and on the N-glycan processing of the human colon cancer cell line HT-29. J. Bid. Chem. 1990,265, 5366-5369. 10. Alton, G.; Hasilik, M.; Niehues, R.; Panneerselvam, K.; Etchison, J.R.; Fana, F.; Freeze, H.H. Direct utilzation of mannose for mammalian glycoprotein biosynthesis. Glycohiologj~1998, 8, 285-295. 11 Silverman, M.; Aganon, M.A.; Chinard, F.P. Specificity of monosaccharide transport in dog kidney. Am. J. Physiol. 1970, 218, 743-750. 12 Panneerselvam, K.; Etchison, J.R.; Freeze, H.H. Human fibroblasts prefer mannose over glucose as a source of mannose for N-glycosylation. Evidence for the functional importance of transported mannose. J. Biol. Chem. 1997,272, 23123-23129. 13 Wiese, T.J.; Dunlap, J.A.; Yorek, M.A. L-fucose is accumulated via a specific transport system in eukaryotic cells. J. Biol. Chem. 1994, 269, 22705-2271 1. 14 Ripka, J.; Adamany, A.; Stanley, P. Two Chinese hamster ovary glycosylation mutants affected in the conversion of GDP-mannose to GDP-fucose. Arch. Biochem. Biophys. 1986, 249, 533545. 15 Shetlar, M.R.; Capps, J.C.; Hern, D.L. Incorporation of radioactive glucosamine into the serum proteins of intact rats and rabbits. Biochim. Biophys. Acta. 1964, 83, 93-101. 16 Aronson. N.N., Jr.; Docherty, P.A. Degradation of [6-3H]- and [ l-'4C]glucosaminelabeled asialo-alpha 1-acid glycoprotein by the perfused rat liver. J. Biol. Chem. 1983, 258, 4266-4271. 17 Rome, L.H.; Hill, D.F. Lysosomal degradation of glycoproteins and glycosaminoglycans. Efflux and recycling of sulphate and N-acetylhexosamines. Biochemical J. 1986, 235, 707713. 18. Trujillo, J.L.; Gan, J.C. Glycoprotein biosynthesis. VI. Regulation of uridine diphosphate N-acetyl-D-glucosamine metabolism in bovine thyroid gland slices. Biochim Biophys. Acta. 1973,304, 32-41.
References
15
19. Krieger, M.; Reddy, P.; Kozarsky, K.; Kingsley, D.; Hobbie, L.; Penman. M. Analysis of the synthesis, intracellular sorting, and function of glycoproteins using a mammalian cell mutant with reversible glycosylation defects. Met1zod.y Cell B i d . 1989, 32, 57-84. 20. Kingsley, D.M.; Kozarsky, K.F.; Hobbie, L.; Krieger, M. Reversible defects in 0-linked glycosylation and LDL receptor expression in a UDP-Gal/UDP-GalNAc 4-epimerase deficient mutant. Cell 1986, 44, 749 -759. 21, Schauer, R. Sialic acids as antigenic determinants of complex carbohydrates. Adc. Expeu. Med. B i d . 1988, 228, 47-72. 22. Chigorno, V.; Tettamanti, G.; Sonnino, S. Metabolic processing of gangliosides by normal and Salla human fibroblasts in culture. A study performed by administering radioactive GM3 ganglioside. J. Biol. Chem. 1996. 271, 21738-21744. 23. Lloyd, J.B. Metabolite efflux and influx across the lysosome membrane. Subcell. Biochem. 1996,277, 361-386. 24. Van Rinsum, J.; Van Dijk, W.; Hooghwinkel, G.J.; Ferwerda, W. Subcellular localization and tissue distribution of sialic acid precursor-forming enzymes. Biochemical Jouunul1983,211),2128. 25. Martin, A,; Rambal, C.; Berger, V.; Perier, S.; Louisot, P. Availability of specific sugars for glyconjugate biosynthesis --a need for further investigations in man. Biochimie 1998,8O, 75-86. 26. Berger, B.; Perier, S.; Pachiaudi, C.; Normand, S.; Louisot, P.; Martin, A. Dietary specific sugars for serum protein enzymatic glycosylation in man. Metabolism 1998, 47, 1499-1503. 27. Freeze, H.H. Human Glycosylation Disorders and Sugar Supplement Therapy. Bioclienz. Biophys. Res. Commun. 1999,255, 189- 193. 28. Hinderlich, S.; Stasche. R.; Zeitler, R.; Reutter, W. A bifunctional enzyme catalyzes the first two steps in N-acetylneuraminic acid biosynthesis of rat liver. Purification and characterization of UDP-N-acetylglucosamine 2-epimerase/N-acety~mannosdmine kinase. J. Bid. Cliem. 1997, 272, 2431 3-24318. 29. Niehues, R.; Hasilik, M.; Alton, G.; Korner, C.; Schiebe-Sukumar, M.; Koch, H.G.; Zimmer, K.P.; Wn, R.; Harms, E.; Reiter, K.; Von Figura, K.; Freeze, H.H.; Harms, H.K.; Marquardt, T. Carbohydrate-deficient glycoprotein syndrome type Ib. Phosphomannose isomerase deficiency and mannose therapy. J. Clin. Inwst. 1998, 101, 1414-1420. 30. Babovic-Vuksanovic, D.; Patterson, M.C.; Schwenk, W.F.; O’Brien, J.F.; Vockley, J., Freeze, H.H.; Mehta, D.P.; Michels, V.V. Severe hypoglycemia as a presenting symptom of carbohydrate-deficient glycoprotein syndrome. J Pediatr. 1999 Dec; 135(6): 775-81. 31. Baque, S.; Guinovart, J.J.; Ferrer, J.C. Glycogenin, the primer of glycogen synthesis, binds to actin. FEBS Lett. 1997, 41 7, 355-359. 32. Lomako, J.; Lomako, W.M.; Whelan. W.J. Glycogen metabolism in quail embryo muscle. The role of the glycogenin primer and the intermediate proglycogen. Euu. J. Biockem. 1995, 234, 343-349. 33. Lomako, J.; Lomako, W.M.; Whelan, W.J. A self-glucosylating protein is the primer for rabbit muscle glycogen biosynthesis. FASEB J. 1988, 2, 3097-3103. 34. Harris, R.J.; Spellman, M. W. 0-linked fucose and other post-translational modifications unique to EGF modules. Glycobiolmqj 1993, 3, 219- 224. 35. Lin, X.; Buff, E.M.; Perrimon, N.; Michelson, A.M. Heparan sulfate proteoglycans are essential for F G F receptor signaling during Drosophila embryonic development. Development. 1999 Sep; 126(17): 3715-23. 36. Benevolenskaya, E.V., Frolov, M.V.; Birchler, J.A. The sugarless mutation affects the expression of the white eye color gene in Drosophila melanogaster. Mol Gen Genet. 1998 Nov; 260(2-3): 131-43. 37. Hacker, U.; Lin, X.; Perrimon, N. The Drosophila sugarless gene modulates Wingless signaling and encodes an enzyme involved in polysaccharide biosynthesis. Development 1997, 124, 35653573. 38. Cumberledge, S.; Reichsman, F. Glycosaminoglycans and WNTs: just a spoonful of sugar helps the signal go down. Trends Genet. 1997, 13, 421-423. 39. Yuen, C.T.; Chai, W.; Loveless, R.W.; Lawson, A.M.; Margolis, R.U.; Feizi, T. Brain contains HNK-I immunoreactive 0-glycans of the sulfoglucuronyl lactosamine series that terminate in 2-linked or 2,6-linked hexose (mannose). J. Bid. Chem. 1997, 272, 8924-8931.
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I Metabolism of Sugars and Sugar Nucleotides
40. Tereyama, K.; Seiki, T.; Nakamura, A,; Matsumori, K.; Ohta, S.; Oka, S.; Sugita, M.; Kawasaki, T. Purification and characterization of a glucuronyltransferase involved in the biosynthesis of the HNK-1 epitope on glycoproteins from rat brain. J. Biol. Chem. 1998,273, 30295-30300. 41. Kim, B.H.; Takahashi, M.; Tada, K.; Bosma, P.J.; Roy-Chowdbury, J.; Roy-Chowdbury, N. Cell and gene therapy for inherited deficiency of bilirubin glucuronidation. J. Perinatol. 1996, 16, S67-S72. 42. Burchell, B.; Brierley, C.H.; Rance, D. Specificity of human UDP-glucuronosyltransferases and xenobiotic glucuronidation. Life Sci. 1995, 57, 1819-1831. 43. Banhegyi, G.; Garzo, T.; Antoni, F.; Mandl, J. Glycogenolysis-and not gluconeogenesis-is the source of UDP-glucuronic acid for glucuronidation. Biochim. Biophys. Acta 1988, 967, 429-435. 44. Vertel, B.M.; Walters, L.M.; Flay, N.; Kearns, A.E.; Schwartz, N.B. Xylosylation is an endoplasmic reticulum to Golgi event. J. Biol. Chem. 1993,268, 11 105--11112. 45. Kearns, A.E.; Vertel, B.M.; Schwartz, N.B. Topography of glycosylation and UDP-xylose production. J. Biol. Chem. 1993,268, 11097-1 1104. 46. Stetten, M.R.; Goldsmith, P.K. Two hexokinases of Homarus americanus (lobster), one having great affinity for mannose and fructose and low affinity for glucose. Biochirn. Biophys. Acta 1981, 657,468-481. 47. Freeze, H.H. Disorders in protein glycosylation and potential therapy: Tip of an iceberg? J. Pediatr. 1998, 133, 593-600. 48. Jaeken, J.; Matthijs, G.; Saudubray, J.M.; Dionisivici, C.; Bertini, E.; Delonlay, P.; Henri, H.; Carchon, H.; Schollen, E.; Vanschaftingen, E. Phosphomannose isomerase deficiency-A carbohydrate-deficient glycoprotein syndrome with hepatic-intestinal presentation. Am. J. Hum. Genet. 1998, 62, 1535-1539. 49. Smith, D.J.; Proudfoot, A,; Friedli, L.; Klig, L.S.; Paravicini, G.; Payton, M.A. PMI40, an intron-containing gene required for early steps in yeast mannosylation. Mol. Cell. Biol. 1992, 12, 2924-2930. 50. Freinkel, N.; Lewis, N.J.; Akazawa, S.; Roth, S.1.; Gorman, L. The honeybee syndrome-implications of the teratogenicity of mannose in rat-embryo culture. N. Enr). J. Med. 1984, 310, 223-230. 51. Buchanan, T.; Freinkel, N.; Lewis, N.J.; Metzger, B.E.; Akazawa, S. Fuel-mediated teratogenesis. Use of D-mannose to modify organogenesis in the rat embryo in vivo. J. Clin. Invest. 1985, 75, 1927-1934. 52. Moore, D.C.; Stanisstreet, M.; Beck, F.; Clarke, C.A. The effects of mannose on rat embryos grown in vitro. Life Sci. 1987, 41, 1885-1893. 53. Krasnewich, D.; Gahl, W.A. Carbohydrate-deficient glycoprotein syndrome. Adu. Pediutr. 1997, 44, 109-140. 54. Matthijs, G.; Schollen, E.; Van Schaftingen, E.; Cassiman, J.J.; Jaeken, J. Lack of homozygotes for the most frequent disease allele in carbohydrate-deficient glycoprotein syndrome type 1A. Am. J. Hum. Genet. 1998, 62, 542-~550. 55. Doucey, M.A.; Hess, D.; Cacan, R.; Hofsteenge, J. Protein C-mannosylation is enzymecatalysed and uses dolichyl-phosphate-mannose as a precursor. Mol. Bid. Cell 1998, 9, 291300. 56. Kim, S.; Westphal, V.; Srikrishna, G.; Mehta, D.P.; Peterson, S.; Filiano, J.; Karnes, P.S.; Patterson, M.C.; Freeze, H.H. Dolichol phosphate mannose synthase (DPM1) mutations define congenital disorder of glycosylation Ie (CDG-Ie). J Clin Invest. 2000 Jan; 105(2): 191-8. 57. Sullivan, F.X.; Kumar, R.; Kriz, R.; Stahl. M.; Xu, G.Y.; Rouse, J.; Chang. X.J.; Boodhoo, A,; Potvin, B.; Cumming, D.A. Molecular cloning of human GDP-mannose 4,6-dehydratase and reconstitution of GDP-fucose biosynthesis in vitro. J. Biol. Chem. 1998,273, 8193-8202. 58. Chang, S.; Duerr, B.; Serif, G. An Epimerase-Reductase in L-Fucose Synthesis. J. Biol. Chem. 1988,263, 1693-1697. 59. Tonetti, M.; Sturla, L.; Bisso, A,; Benatti, U.; De Flora, A. Synthesis of GDP-L-fucose by the Human FX Protein. J. Biol. Chem. 1996, 271, 27274427279, 60. Kornfeld, R.H.; Givsburg, V. Control of Synthesis of Guanosine 5’-Diphosphate D-Mannose and Guanosine 5‘-Diphosphate L-Fucose in Bacteria. Biochim. Biophys. Actu 1966, 117, 7987.
References
17
61. Sturla, L.; Etzioni, A,; Bisso, A,; Zanardi, D.; Deflora, G.; Silengo, L.; Deflora, A.; Tonetti, M. Defective intracellular activity of GDP-D-Mannose-4,6-Dehydratase in leukocyte adhesion deficiency type I1 syndrome. FEBS Lett. 1998, 429, 274-278. 62. Etzioni, A,; Phillips, L.M.; Paulson, J.C.; Harlan, J.M. Leukocyte adhesion deficiency (LAD) TI. Ciba Found. Symp. 1995,189, 51-58. 63. Park, S.H.; Pastuszak, I.; Drake, R.; Elbein, A.D. Purification to apparent homogeneity and properties of pig kidney L-fucose kinase. J. Bid. Chem. 1998, 273, 5685-5691. 64. Marquardt, T.; Luhn, K.; Srikrishna, G.; Freeze, H.H.; Harms, E.; Vestweber. D. Fucose Therapy for Leukocyte Adhesion Deficiency Type I1 (LAD TI). Blood 1999, 94. 3976-3985. 65. Chan, J.Y.; Nwokoro, N.A.; Schachter. H. L-Fucose metabolism in mammals. The conversion of L-fucose to two moles of L-lactate, of L-galactose to L-lactate and glycerate, and of Darabinose to L-lactate and glycollate. J. Biol. Chenz. 1979, 2.54, 7060-7068. 66. Holton, J.B. Galactosaemia: pathogenesis and treatment. J. Inherit. Metab. Di,x 1996, 19, 3-7. 67. McDowell, G.; Gahl, W.A. Inherited disorders of glycoprotein synthesis: cell biological insights. Proc. Soc. Exp. Biol Med. 1997. 215, 145-157. 68. Wolosker, H.; Kline, D.; Bian, Y.; Blackshaw. S.; Cameron, A.M.; Fralich, T.J.; Schnaar, R.L.; Snyder, S.H. Molecularly coned mammalian glucosamine-6-phosphate deaminase localizes to transporting epithelium and lacks oscillin activity. FASEB J. 1998, 12, 91-99. 69. Pels Rijcken, W.R.; Ferwerda, W.; Van den Eijnden, D.H.; Overdijk, B. Influence of Dgalactosamine on the synthesis of sugar nucleotides and glycoconjugates in rat hepatocytes. Gll'cobiology 1995, 5, 495- 502. 70. Pastuszak, I.; Drake, R.; Elbein, A.D. Kidney N-acetylgalactosamine (GalNAc)-1-phosphate kinase, a new pathway of GalNAc activation. J. Bid. Chem. 1996, 271, 20776-20782. 71. Kean, E.L. Sialic acid activation. Glycabiologj>1991, 1, 441-447. 72. Varki, A. Diversity in the sialic acids [published erratum appears in Glycobiology 1992 Apr; 2(2): following 1681. GlycobiologjJ 1992, 2. 25-40. 73. Reuter, G.; Gabius, H.J. Sialic acids structure-analysis-metabolism-occurrence-recognition. Biol. Chern. Hoppe-Seyler 1996, 377, 325-342. 74. Allen, M.B.; Walker, D.G. Kinetic characterization of N-acetyl-D-glucosamine kinase from rat liver and kidney. Bioclwm. J. 1980, 185, 577 582. 75. Hirschberg, C.B.; Robbins, P.W.; Abeijon, C. Transporters of nucleotide sugars, ATP, and nucleotide sulfate in the endoplasmic reticulum and Golgi apparatus. Annu. Reu. Biochem. 1998,67,49-69. 76. Abeijon. C.; Mandon, E.C.; Hirschberg, C.B. Transporters of nucleotide sugars, nucleotide sulfate and ATP in the Golgi apparatus. 7 i m t l . r Biodzem. Sci. 1997, 22, 203-207. 77. Trombetta, E.S.; Helenius, A. Lectins as chaperones in glycoprotein folding. Curr. Opin. Stvuct. Bid. 1998, 8, 587-592. 78. Abeijon, C.; Yanagisawa, K.; Mandon, E.C.; Hausler, A,; Moremen, K.: Hirschberg, C.B.; Robbins, P.W. Guanosine siphosphatase is required for protein and shpingolipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae. J. eel/ Biol. 1993, 122, 307-323. 79. Varki. A. Factors controlling the glycosylation potential of the Golgi apparatus. Trends Cell Biol. 1998, 8, 34-40. 80. Hayes; B.K.; Freeze, H.H.; Varki, A. Biosynthesis of oligosaccharides in intact Golgi preparations from rat liver. Analysis of N-linked glycans labeled by UDP-[6-3H]N-acetylglucosamine. J. Biol. Chenz. 1993, 268, 16 1 39- 16 154. 81. Etchison, J.R.; Freeze. H.H. A new approach to mapping co-localization of multiple glycosyl transferases in functional Golgi preparations. Glj~cohiology1996, 6 , 177-1 89. 82. Dean, N.; Zhang, Y.B.; Poster, J.B. The VRG4 Gene is Required for GDP-mannose Transport cereuisiue. J. Bid. Client 1997, 272, into the Lumen of the Golgi in the Yeast, Sacc~l~tivornyces 319O8--31914. 83. Ma, D.; Russell, D.G.; Beverley, S.M.; Turco, S.J. Golgi GDP-mannose uptake requires Leishmania LPG2. A member of a eukaryotic family of putative nucleotide-sugar transporters. J. Bid. Cliem. 1997, 272, 3799-3805. 84. Toma, L.; Pinhal, M.A.; Dietrich, C.P.; Nader, H.B.; Hirschberg. C.B. Transport of UDPgalactose into the Golgi lumen regulates the biosynthesis of proteoglycans. J. Bid. Che~z.1996, 271. 3897-3901.
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1 Metabolism of Sugars and Sugar Nucleotides
85. Jungmann, J.; Munro, S. Multi-protein complexes in the cis golgi of Saccharomyces cerevisiae with alpha-l,6-mannosyltransferaseactivity. EMBO J. 1998, 17, 423-434. 86. Thomas, G.H.; Reynolds, L.W.; Miller, C.S. Overproduction of N-acetylneuraminic acid (sialic acid) by sialuria fibroblasts. Ped. Res. 1985, 19, 451-455. 87. Strecker, G. Genetic disorders of N-acetylneuraminic acid metabolism: sialurias and sialidoses. Comp. Rend. Seances Soc. Bid. Fil. 1999, 179, 567-576. 88. Weiss, P.; Tietze, F.; Gahl, W.A.; Seppala, R.; Ashwell, G. Identification of the metabolic defect in sialuria. J. Biol. Chem. 1989, 264, 17635-17636. 89. Hawkins, M.; Angelov, I.; Liu, R.; Barzilai, N.; Rossetti, L. The tissue concentration of UDPN-acetylglucosamine modulates the stimulatory effect of insulin on skeletal muscle glucose uptake. J. Biol. Chem. 1997, 272. 4889--4895. 90. Spiro, R.G. The effect of N-acetylglucosamine and glucosamine on carbohydrate metabolism in rat liver slices. J. Biol. Chem. 1958, 233, 546-550. 91. Hawkins, M.; Barzilai, N.; Liu, R.; Hu, M.; Chen, W.; Rossetti, L. Role of the glucosamine pathway in fat-induced insulin resistance. J. Clin. Invest. 1997, 99, 2173-2182. 92. Rossetti, L.; Hawkins, M.; Chen, W.; Gindi, J.; Barzilai, N. In uivo glucosamine infusion induces insulin resistance in normoglycemic but not in hyperglycemic conscious rats. J. Clin. Invest. 1995, 96, 132-140. 93. Snow, D.M.; Hart, G.W. Nuclear and cytoplasmic glycosylation. Intl Rev. Cyfol. 1998, 181, 43-74. 94. Yki-Jarvinen, H.; Vogt, C.; Lozzo, P.; Pipek, R.; Daniels, M.C.; Virkamaki, A,; Makimattila, S.; Mandarino, L.; DeFronzo, R.A.; McClain, D.; Gottschalk, W.K. UDP-N-acetylglucosamine transferase and glutamine: Fructose 6-phosphate amidotransferase activities in insulin-sensitive tissues. Diabetologia 1997, 40, 76-8 1. 95. Hresko, R.C.; Heimberg, H.; Chi, M.M.Y.; Mueckler, M. Glucosamine-induced insulin resistance in 3T3-Ll adipocytes is caused by depletion of intracellular ATP. J. Biol. Chem. 1998, 273, 20658-20668.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
2 Nucleotide Sugar Transporters Rita Gerardy-Schahn and Matthias Eckhardt
2.1 Introduction The cellular loci for the production of secreted and membrane bound glycoproteins and glycolipids are the endoplasmic reticulum (ER) and the Golgi apparatus. Both compartments are sealed vesicular structures that require active in- and outward transport of metabolites that are substrates or products of catalytic reactions taking place at the luminal side. Studies on the secretory pathway carried out in the 1960s, together with the delineation of the steps involved in N-glycosylation in the 1970s, made the need for specific transport systems apparent. Transport was demonstrated in vitro in isolated Golgi vesicles, and genetic proof for the existence of specific nucleotide sugar transporters was obtained when Chinese hamster ovary (CHO) cell mutants with asialo- or asialo-agalacto-surfaces were shown to be inactive in translocating CMP-sialic acid and UDP-galactose, respectively, from the cytoplasm into the Golgi lumen. Further biochemical analysis of the transport process demonstrated that nucleotide sugars are specifically recognized by individual translocators that work in a temperature-dependent, and saturatable manner. Nucleotide sugars can be concentrated in the organellar lumen up to 50-fold. The energy required for this active transport results from exchanging luminal nucleoside monophosphates with cytosolic nucleotide sugars. The nucleoside monophosphates thereby follow their concentration gradients. Attempts to purify nucleotide sugar transporters led to highly enriched and functionally active protein fractions, however, homogeneous purification is a very recent accomplishment and is discussed in this Chapter. Cloning of the first nucleotide sugar transporter genes in 1996 identified a family of highly hydrophobic multimembrane spanning proteins, with molecular masses of 34-42 kDa. Ten transmembrane domains have been demonstrated in the CMP-sialic acid transporter and due to similarities in sequence and hydrophobicity, it seems plausible to assume that the membrane topology is the same for most, if not all, nucleotide sugar transporters. Progress in studying nucleotide sugar transporters over the last few years stimu-
20
2 Nucleotide Suyur Transporters
lated a series of excellent reviews that are recommended to the interested reader 11-51. In this communication, the focus will be on advance made in identifying structure-function relationships in this important protein family. Furthermore, we discuss recent observations that suggest nucleotide sugar transporters may be involved in the regulation of glycosylation, and in the manifestation of a new form of carbohydrate deficiency glycoprotein syndrome (CDGS). Finally, we address the future perspectives of nucleotide sugar transporters in generating improved cell systems for use in the field of biotechnology.
2.2 General considerations The glycocalyx is a well known characteristic of the animal cell and has been recognised for more than 100 years. Its “outstanding” position per se implies that the sugar rim plays an important role in cellular functions associated with communication and contact. Nevertheless, deciphering the genetic code and the development of numerous powerful molecular biological techniques, concentrated research efforts on genes and on their linear translation products, the proteins. The subsequent observation that in many cases, recombinant proteins were also active in the absence of their physiological carbohydrate additives, substantiated the widely accepted view that carbohydrates provide a kind of luxury decoration to functionally active proteins. This view was reversed when sugars were found to be directly involved in a large variety of biological reactions 16, 71 including the stability and immunological properties of recombinant proteins [ 81.
2.3 The Requirement for Nucleotide Sugar Transporters and Their Mechanism of Function: A Comprehensive Overview of the Last 20 Years The addition of carbohydrates to secreted and membrane-bound proteins and to lipids starts in the ER and continues along the secretory pathway up to the transGolgi-network [9, 101. Maturation of N-glycans involves the interplay of two classes of enzymes: the trimming glycosidases and the elongating glycosyltransferases (for review, see 1111). In contrast, 0-glycosylation of proteins and the transfer of glycans to lipids, is the result of sequentially active glycosyltransferases, exclusively [ 121. The spatial organisation of the processing enzymes within the membranes of ER and Golgi corresponds to their position in the reaction sequence of oligosaccharide maturation [ 131. While the majority of ER glycosyltransferases use monosaccharides that are activated via dolichol phosphate, some ER glycosyltransferases and the glycosyltransferases of the Golgi apparatus use nucleotide sugars as donor substrates. Nu-
2.3 The Requirenicntjor Nuclrotidp Sugar Transporters
21
NDPase
UDP-Gal
I I"1" "Wlr ~
Galactose-I-phosphate uridylyltransferase Gal-1-P
JL
Glc-I-P
UDP-Glc
/
/
synthetase
Cytosol
Figure 1. Synthesis and transport of UDP-galactose and CMP-sialic acid. This scheme demonstrates three terminal steps in the synthesis of sialylated glycoproteins. The nucleotide sugar donors UDP-galactose (UDP-Gal) and CMP-sialic acid (CMP-Sia) are synthesized in the cytosol and in the nucleus, respectively. From the cytosol, the nucleotide sugars are transported into the lumen of the Golgi apparatus, the cellular locus where secreted and membrane bound glycoproteins and glycolipids are produced. The transport of nucleotide sugars across the Golgi membrane requires specific transporter proteins (black boxes). Nucleotide sugar transporters act as antiporters. The counter-transported substrates are the nucleoside monophosphates UMP and CMP. CMP is a product of the lumenal sialyltransferase (Sia-T), while UMP production requires an additional enzymatic step, the hydrolysis of UDP by the nucleoside diphosphatase (NDPase). The second product in the NDPase reaction is phosphate (Pi). The existence of a specific phosphate transporter has not been demonstrated yet.
cleotide sugars are generated in the cytosol, with the exception of CMP-sialic acid, which is synthesised in the nucleus [14, 151 (Figure 1). Translocation systems that bring the activated monosaccharides into the reaction compartments of the glycosyltransferases have therefore been postulated, and were demonstrated to exist, using in vitro studies [16, 171. Transport assays were carried out on purified ER and Golgi vesicles and on proteoliposomes (nucleotide sugar transporter enriched protein fractions reconstituted into artificial phospholipid vesicles) to investigate the kinetic and mechanistic properties of these proteins. The results of these studies demonstrate: 1) each nucleotide sugar is recognised by a specific protein; 2) nucleotide sugar transporters act in a temperature-dependent and saturatable manner with apparent K , values of 1-10 pM; 3 ) substrates can be actively transported against steep concentration gradients; 4) the driving force for the intra-organelle accumulation of nucleotide sugars is provided by the simultaneous outward transport of nucleoside monophosphates down their concentration gradients [ 161. Hence, nucleotide sugar transporters are believed to act as antiporters [2, 18, 191. Clear evidence for the antiport mechanism has been obtained in the case of GDP-fucose
22
2 Nucleotide Sugur Trunsporter.r
transport, where an equimolar exchange of [ ''C]GDP-fucose and [ 3H]GMP was measured in rat liver Golgi vesicles after preincubation with tritiated GDP-fucose, followed by exposure to the [ ''C]-labelled nucleotide sugar [ 191. In a later study, the exchange of UDP-GlcNAc against UMP was demonstrated to reach an optimum when preloading of proteoliposomes was carried out at pH 7.5, indicating that the dianionic form of UMP is counter-transported with UDP-GlcNAc [20]. Because uptake of nucleotide sugars into proteoliposomes was stimulated when vesicles were preloaded with the respective nucleoside monophosphates, and transport was inhibited after disruption of nucleoside monophosphate gradients, the antiport mechanism can be regarded as the general mechanism used by nucleotide sugar transporters [2, 191. Only in the case of CMP-sialic acid is the nucleoside monophosphate a direct product of the glycosyltransferase reaction. All other sugars are activated as nucleoside diphosphates, and these diphospho-nucleosides are the products released by glycosyltransferases. The Golgi-resident nucleoside diphosphatases have therefore been hypothesized to complete the cycle of nucleotide sugar import. Guanosine diphosphatase was purified from Succhuromyces cerevisiue [21] and the gene (gdul) was cloned [22]. Genetic disruption of the gdul locus interfered with N- and 0glycosylation, and with sphingolipid synthesis. Golgi vesicles isolated from gene targeted cells were found to have a five-fold reduced GDP-mannose uptake capacity [23]. A second nucleoside diphosphatase (YND1) with a broader substrate specificity has been cloned very recently from S. cerevisiur [24, 251. Over-expression of the YNDl protein in GDAl deficient strains was able to partially complement the defect, while the double mutantion Agdul, Ayndl generated a lethal phenotype [24, 251. Although precise data on the function and subcellular localisation of YNDl are presently not known the current data suggest the existence of two guanosine diphosphatases in S. cereuisiue which, under physiological conditions, may co-operate in producing GMP and so may be able to influence the velocity of GDP-fucose uptake. The speculation that nucleotide sugar transport into the Golgi lumen is metabolically controlled is in good correlation with earlier experiments demonstrating that the extra-vesicular concentration of nucleoside mono-, di-, and triphophates influences transport activity [ 1g]. Moreover, cross inhibition has been observed between nucleotide sugars. For example, UDP-GlcNAc translocation can be modulated by variations in the concentrations of UDP-glucose and UDPGalNAc [26], CMP-sialic acid transport is sensitive against elevated UDP-GlcNAc concentrations [ 18, 271 and GDP-fucose transport against GDP-mannose [ 181. It is, however, worth mentioning that inhibition is most efficient between sugar derivatives that are activated via the same nucleotide.
2.4 Molecular Cloning of Nucleotide Sugar Transporters Somatic mutants exhibiting defects in cell surface glycosylation have been isolated from mammalian cell lines as clones that withstand the cytotoxic activity of plant
2.4 Molecular Cloning of Nucleotide Sugur Trunsporters
23
lectins (for review see [28]) or acquired resistance towards Newcastle disease virus infection [29, 301. Cell fusion experiments allowed the subdivision of individual clones into genetic complementation groups [31]. Two of more than 40 complementation groups identified so far comprise mutants that are defective in Golgi nucleotide sugar transport systems. Lec2 cells are unable to translocate CMP-sialic acid [32] and cells of the complementation group Lec8/Had-1 are impaired in the Golgi-associated transport of UDP-galactose [33, 341. Additional mutants, lacking specific nucleotide sugar transport activities, have been identified in yeast [ 17, 35371, protozoa, [38] and in Caenorhahditis e1egun.r [39-411. Complementation cloning in these mutants identified the first nucleotide sugar transporter genes [36, 37, 42-48]. The cDNAs for both hamster and mouse CMPsialic acid transporter were isolated using Lec2 cells as recipients for wild type cDNA libraries [43, 441. Complementation to wild type cells was monitored via the cell surface marker polysialic acid, a post-translational modification of the neural cell adhesion molecule that is expressed in wild type CHO cells [49]. Detection of polysialic acid with the monoclonal antibody 735 [50] provides a useful strategy, because the antibody recognises polysialic acid chains with unusual high sensitivity. A helical segment of nine a2,8-linked sialic acid residues forms the epitope [51, 521. Since polymerization degrees of n > 50 have been determined, a single polysialic acid chain contains a multitude of 735 epitopes [52] and explains the sensitivity of the antibody reaction. Monitoring polysialic acid re-expression is a sufficient strategy to isolate any gene whose inactivation causes a severe reduction in sialic acid addition to cell surface glycans, such that polysialic acid is not synthesized [15, 43, 44, 491. Primary sequence analysis of nucleotide sugar transporters revealed that all are hydrophobic type I11 membrane proteins with molecular masses of 34-42 kDa (see [53] and literature cited therein). Database searches identified a large number of structurally related proteins of as yet unidentified specificity in human, Drosophilu, C. elegans and yeast. The dendrogram shown in Figure 2 summarizes all the sequences that were found to exhibit significant similarity to experimentally identified nucleotide sugar transporters. In good correlation with the nine different nucleotide sugars that need to be translocated, 7-9 clusters are discernible on the basis of primary sequence similarities. In the C. eleyuns genome, which has been completely sequenced, 14 genes encoding putative nucleotide sugar transporters have been identified. Of course, the functional activity of these genes awaits verification, but strong evidence has already been obtained that the gene U50135 (identical to sqv-7 [40]) encodes a nucleotide sugar transporter [41]. Another interesting observation that arises from primary sequence comparisons in the group of nucleotide sugar transporters, is that UDP-GlcNAc transporters isolated from Kluyveromyces luctis and MDCK cells are only 22% identical, while the mammalian transporters for CM P-sialic acid, UDP-galactose, and UDPGlcNAc are very similar, suggesting that these genes have developed from a common ancestor. Despite the low conservation between transporters of identical specificity in distant species, all the information required for the formation of a substrate specific transporter is contained within the primary sequence. Functional expression of the
Hsapiens
C.elegans (AFO16438)
S.pombe (AL021816)
hapiens (D87989)
R.norvegicus (D87991)
M.musculus (D87990)
rc
C.elegans (AF003383)
C.elegans (AF045639)
C.elegans (AFO245W)
C.elegans (AFO16438)
I
UDPGlcNAc
GDP-Man GDP-Man
C.elegans (270750)
C.elegans (U50135)
H.sapiens (D87449)
H.sapiens (AJOO5866)
L.donovani
S.cerevisiae
Scerevisiae (P40027)
C.elegans (268215)
C.elegans (AFO36696)
S.pombe (AL031856)
K.ladis
S.cerevisiae (P40004)
C.elegans (UOCC6-I)
Figure 2. Dendrogram identifying putative nucleotide sugar transporters. The dendrogram shows relationships among identified and putative nucleotide sugar transporters. Putative nucleotide sugar transporters were identified by BLAST searches against the sequences of known transporters. The evolutionary tree was generated using Megalign (DNAStar). The substrates of identified transporters are indicated. In all other cases, the data base accession numbers are included. The accession numbers of the identified transporters are (from top to bottom): AF057365, AL022598, P78381, 461420, 008520, P78382, U48413, P40107, U26175.
I
CMPSia
C.griseus C.elegans ((102334)
CMPSia CMPSia
M.musculus
C.elegans (281102)
L n
I ’
UDPGal
C.elegans (282288)
H.sapiens
Dmelanogaster (AL023874)
UDPGlcNAc UDPGal
Spornbe
C.elegans (AF016674) C.familiaris
ru
P
t3
2.5 The Structure of Nucleotide Sugar Tr~insporters
25
murine CMP-sialic acid [54] and the human UDP-galactose transporter [55] on the zero background of S. cerevisiae, and in transporter deficient CHO cells [64]demonstrated that the cloned cDNAs in fact, encode the transporters and not accessory proteins. In line with this, expression cloning in heterologeous systems identified additional family members, although the genes that have been isolated show very low conservation to their orthologues. The canine UDP-GlcNAc transporter was isolated in the UDP-GlcNAc transporter negative clone of K. Zactis [56] and a plant cDNA that encodes a putative UDP-galactose transporter was identified by phenotypic correction of CHO Lec8 cells [57]. These studies point the way to rapidly isolating a large panel of functionally related molecules that will improve the basis for studies aimed at enlightening structure-function relationships in the family of nucleotide sugar transporters.
2.5 The Structure of Nucleotide Sugar Transporters Hydrophobicity analyses and secondary structure predictions suggested that 6- 10 transmembrane domains exist in the different nucleotide sugar transporters [4]. As shown in Figure 3, ten transmembrane domains have been identified in the CMPsialic acid transporter by using an epitope insertion approach [58]. Complementary DNAs carrying epitope tags inserted into putative hydrophilic loop structures were transiently expressed in CHO cells, and the orientation in relation to the Golgi membrane was determined by indirect immunofluorescence in semi-permeabilized cells [58]. It will be interesting to see whether all nucleotide sugar transporters exhibit a common membrane topology. The high similarity observed in hydrophobicity plots and the conservation of structural elements between the different transporters argue for this possibility. At the theoretical level, ten hydrophobic domains can be clearly distinguished in the human UDP-galactose [46], canine UDP-GlcNAc [56], and GDP-mannose transporters of Leishmania donovani [47]. In addition the ten transmembrane domain model is compatible with the majority of the sequences shown in Figure 2. Only the genes designated as Q02334 and P40027 that have been identified in C. elegrxns and 5'. ceuevisiae, respectively, lack regions corresponding to the first two transmembrane domains. Because functional data on these putative transporters are not available so far, it remains unclear if these genes encode functional proteins, A mutant of the hamster CMP-sialic acid transporter that lacks the first two transmembrane domains was found to be functionally inactive [59]. Future experiments are needed to reveal whether ten transmembrane domains are the rule in nucleotide sugar transporters. The oligomeric state of functional nucleotide sugar transporters has been proposed to be the homodimer. This hypothesis was established on the basis of structural analogy existing between nucleotide sugar transporters and the Golgi transporter for activated sulfate, which is a dimer in its active state [60]. The identification of potential leucine zipper motifs in some of the cloned nucleotide sugar transporters supported this hypothesis [3]. However, the importance of the leucine zipper motifs
Figure 3. Membrane topology of the CMP-sialic acid transporter. Using an epitope insertion approach, ten transmembrane domains have been identified in the CMP-sialic acid transporter. Positions in the primary sequence where the HA-epitope was inserted are indicated by triangles. All constructs summarised in this figure were correctly transported to the Golgi apparatus. The orientation of the epitope in relation to the Golgi membrane was analyzed by indirect immunofluorescence. Filled triangles (HA14, HA7. HA6) indicate insertion mutants that were unable to translocate CMP-sialic acid. Transport active constructs are marked with open triangles. Polar and charged residues are indicated by filled circles and t,-, respectively.
H z6
2
‘5
2.6 The Subcellular Distrihution qf Nucleotide Sugur Transporters
27
is still questionable. Experimental inactivation of the leucine zipper in the CMPsialic acid transporter did not change the transport activity of the protein, demonstrating that the motif is not required for the assembly of an active CMP-sialic acid transporter [59]. Homogeneous purification has now been achieved for the rat UDP-N-acetylglactosamine transporter [ 531. The purified protein retained functional activity. In SDS-PAGE analysis, a protein band with an apparent molecular mass of 43 kDa was found, but in native glycerol gradients, the sedimentation behavior is identical to a protein of 80-90 kDa [ 531. These data demonstrate that the transporter is a dimer in this soluble state. The final decision on the aggregation state of the membrane bound nucleotide sugar transporters and on whether the transport active unit is a mono-, di-, or oligomer requires direct proof such as that provided recently for the yeast phosphate carrier [61].
2.6 The Subcellular Distribution of Nucleotide Sugar Transporters The distribution of nucleotide sugar transport activities over the membranes of ER and Golgi have been determined in vitro by measuring transport activities on purified ER and Golgi vesicles (for review, see [4]).The data summarized in Table 1 demonstrate that all transport activities can be found in the Golgi apparatus and only some are translocated into the ER. The transporter patterns show species specific variations. Measuring transport activity on vesicle fractions isolated from animal cells, the UDP-galactose transporter has been restricted to Golgi membranes (Table 1). A very recent study on the subcellular localization of UDP-galactose:
Table 1. Summary of nucleotide sugar transport activities identified in Golgi- and ER fractions and genes that have been isolated so far. Nucleotide sugar
Localization
Cloned genes
CMP-sialic acid
Golgi
UDP-galactose
Golgi
UDP-N-Acetylglucosamine
Golgi
UDP-N-Acetylgalactosamine UDP-glucose UDP-x ylose UDP-glucuronic acid GDP-fucose GDP-mannose
Golgi + ER (Golgi) + ER Golgi + ER Golgi + ER Golgi Golgi
mouse [43] human [45] hamster [44] human [46] Scl~i~o.succhoron~yee.~ ponqbe [ 371 Kluyuerorriyces Iuctis [42] dog [561
+ ER
Leishrnunia donovani [38] Succhuromyces cerevisiue [48]
28
2 Nucleotide Sugar Transporters
ceramide galactoyltransferase (CGalT) carried out by [62], however, suggests that UDP-galactose is also transported into the ER lumen. CGalT synthesises galactosylceramide, a major component of myelin, from ceramide and UDP-galactose. With the aid of immunological techniques, CGalT has been shown to be an ER resident [62]. The catalytic domain of the class I integral membrane protein is orientated towards the ER lumen [62]. In the UDP-galactose transporter negative Lec8 cell [33], CGalT activity is blocked [62]. These data strongly suggest the existence of the UDP-galactose transporter in ER membranes. Sensitive immunological techniques are required to conclusively identify the subcellular expression pattern also for other nucleotide sugar transporters. Furthermore, additional studies are required to find out whether nucleotide sugar transporters expressed in the ER and Golgi membranes represent identical proteins, alternative splice variants, or are encoded by different genes. Two splice variants that differ at the carboxyl terminus have been isolated in the case of the human UDP-galactose transporter. Both isoforms are active but only one contains the putative ER-retrieval sequence K(X)KXX [45]. Five of the sequences shown in the dendrogram (Figure 2) exhibit a K(X)KXX motif at the carboxyl terminus. The question on the subcellular distribution of nucleotide sugar transporters should soon be solved, since cDNAs are available that enable the production of specific immunoreagents [63].
2.7 Molecular Defects that Cause Inactive UDP-Galactose and CMP-Sialic Acid Transporters As mentioned above cloning of nucleotide sugar transporters was achieved by complementation of cells that previously had been shown to exhibit defects in the respective nucleotide sugar transport system. UDP-galactose transporter negative Had-1 clones have been isolated from the mouse mammary tumor cell line FM3A [29, 301. Clones belonging into the CMP-sialic acid transporter negative Lec2 group were isolated by lectin resistance [28] or immunoselection on the mAb 735 [49]. Individual clones of both genetic complementation groups have been analyzed at the molecular level. The majority of the clones contain mutations that lead to internal deleted or truncated translation products [59, 651. In two mutants, however, the inactivation of transport was found to be caused by single amino acid exchanges. Inactivation of transport in the FM3A subclone Had-lm can be explained by exchange of glycine-178 to aspartic acid [65]. The Lec2 subclone 9D3 carries a mutation that leads to replacement of glycine-189 with glutamic acid [59]. in both cases, the inactive proteins are correctly targeted to the Golgi-apparatus, demonstrating that the mutations do not interfere with protein folding and subcellular transport [59, 651, but concern recognition or transport of the nucleotide sugar. If mutated positions were replaced by alanine, transport activity was reconstituted [ 59, 651, while the introduction of large or charged amino acid residues in position 189 of the CMP-sialic acid transporter phenocopied the mutant phenotype [59]. These data together with the fact that glycine-178 and -189 are highly conserved residues
2.9 Inuolrmicnt o j Nuclwtide Sugar Trunxp0rter.r
29
allow us to speculate that these positions belong to structural elements that are required for the transport process.
2.8 Association Between Defects in Nucleotide Sugar Transporters and Diseases An observation made by three collaborating German groups [66] provides the first evidence for the association between a nucleotide sugar transport defect and a carbohydrate deficiency glycoprotein syndrome. Leukocytes and fibroblasts isolated from a patient suffering from a new type of leukocyte adhesion deficiency syndrome (LAD 11) express drastically reduced levels of fucosylated carbohydrates [67,68].The patient suffers from multiple infections and hence shows very high leukocyte counts [ 681. Immunological analyses demonstrated deficiencies of several fucosylated carbohydrates that are generated by different fucosyltransferases in activated leukocytes and endothelial cells [68]. The defect in fucose metabolism could be partially corrected by oral supplementation of fucose. After five months of fucose therapy, leukocyte counts turned to normal levels and psychomotor capabilities of the patient improved [69]. Biochemical studies carried out on leukocytes and primary fibroblasts from the LAD I1 patient demonstrated that the enzymes involved in GDP-fucose synthesis are active at normal levels [70]. In contrast, the transport of GDP-fucose into Golgi vesicles was found to be reduced by 80% and the cytoplasmic concentration of GDP-fucose was increased. Control studies demonstrated normal Golgi-update for UDP-Gal and normal GDPase activity [71]. The data obtained so far allow the conclusion that the defect in this patient concerns the Golgi GDP-fucose transporter or an element indirectly involved in GDP-fucose translocation. Attempts to isolate the cDNA that rescues the transport deficient cells are underway and will define the molecular defect that causes this new form of LAD 11 syndrome.
2.9 Involvement of Nucleotide Sugar Transporters in the Regulation of Glycosylation The pattern of glycan structures produced by a given cell depends on its glycosyltransferases. In accord with this, changes in carbohydrate structures that occur during onto- and onco-genetic development [74, 751 and in relation with different stages of cellular activity [6, 76, 771, are accompanied by variations in the expression of glycosyltransferases. Despite this situation, attempts to control N-glycan processing by overexpressing specific glycosyltransferases have met with variable success [78, 791 and suggest that prevalence of glycosyltransferases is not the sole determinator of glycosylation. Metabolic control also seems related to the availability and transport of nucleotide sugars into the respective compartment [22, 27,
30
2 Nucleotide Sugar Transporters
SO]. In a proteoglycan-producing MDCK cell mutant 1801, where availability of UDP-galactose in the Golgi lumen is limited, the synthesis of keratan sulfate drops down to a low level, while chondroitin sulfate and heparan sulfate are produced at normal concentrations [SO]. Possible explanations for this observation are: 1) Biosynthesis of the different glycoconjugates occurs in isolated compartments that are supplied with nucleotide sugar transporters of different kinetic properties. 2) Glycosyltransferases of different substrate specificity exhibit different K, values. 3) Glycosyltransferases form functional complexes with nucleotide sugar transporters and this quaternary organisation may cause a hierarchical order in substrate availability. Very interestingly in this context, is the observation that fucose therapy in the LAD I1 patient described above 1681 partially restores the functional form of Pselectin ligand PSGL-1 on neutrophils but not E-selectin binding to neutrophils 1691. Fucosylation of ESL-1 is mediated by FucT IV, while fucose transfer to PSGL-1 depends on FucT VII 1731. Cloning the GDP-fucose transporter and analyzing its subcellular and functional organisation will therefore be of major interest also in this aspect.
2.10 Future Perspectives Since cDNAs became available in 1996, research with nucleotide sugar transporters has progressed quickly and some of the questions that have been described as future aims in recent reviews [4,641, have already been answered [53, 58, 59, 641. Insights into structure-function relationships have been obtained by defining the membrane topology of the CMP-sialic acid transporter 1581 and by analyzing the sedimentation behaviour of homogeneously purified UDP-GlcNAc transporter from rat liver Golgi membranes 1531. The results of these studies suggest nucleotide sugar transporters to contain ten transmembrane spanning domains [58] and form dimers in the active state [53]. Analyzing loss-of-function mutants in the complementation groups Lec2 [59] and Had-1 1651 suggest two conserved glycine residues (position 189 of the CMP-sialic acid transporter, position 179 in the UDP-Gal transporter) take part in the transport cycle. Nevertheless, structure-function studies are only beginning, and much more information is required to define the molecular architecture of nucleotide sugar transporters. Protein domains involved in specific substrate recognition, in mediating the translocation process, and in regulating their activity need to be determined. Related studies have been carried out with a large variety of transporters that localize to plasma membranes and inner mitochondria1 membranes of mammalian, yeast, and bacterial cells [81-83]. Published experimental protocols can be used to design future experiments aimed at determining the secondary, tertiary and quaternary organization of nucleotide sugar transporters. As shown in Table 1, some transporters are found in Golgi membranes exclusively, others localise to both ER and Golgi. Poly- and monovalent immunoreagents against these proteins shall soon be available and can be used to reinvestigate and potentially complete the topography of nucleotide sugar transporters. It will be
2.10 Future Perspectives
31
interesting to see whether transporters that are targeted to different membranes are also different at protein level and whether ER and Golgi resident forms exhibit different kinetic properties, providing an additional level for the regulation of glycosylation patterns. Nucleotide sugar transporters are the first multiple membrane spanning residents of the Golgi apparatus identified so far and provide new model proteins to study Golgi-targeting, trafficking, and retention. Heterologous expression of mammalian transporters for CMP-sialic acid [54] and UDP-galactose [55] on the zero background of S. cereuisiae showed a substantial proportion of the recombinant proteins correctly targeted to the Golgi membranes, indicating that the information for subcellular destination is contained in the primary sequence. Moreover, these data provide initial evidence that the targeting machinery is, at least in part, conserved amongst different species. Identifying the physiological functions and the subcellular localisation of the many genes that have been identified as transporter-related in the sequence data bases (see Figure 2) should largely contribute to understanding structure-function relationships. There is increasing evidence that glycoconjugate synthesis is controlled at the metabolic level [3, 27, SO]. Recent support for this hypothesis has been obtained by analyzing the fucosylation patterns in the LAD I1 patient described above [69]. Oral supplementation therapy partially restored expression of the functional form of PSGL-I and core fucosylation of secreted proteins, while reconstitution of Eselectin binding was not obtained [69]. These observations suggest that increased expression of nucleotide sugar transporters in eukaryotic cells can be used to modulate and improve glycosylation pathways e.g. in biotechnological processes [27]. Last but not least, functional expression of mammalian nucleotide sugar transporters in yeast opens new perspectives to modulate glycosylation pathways in these organisms. The first example for successful neo-galactosylation has been described by [84]. Two heterologeous genes, ymuZ2+ encoding u 1,2-galactosyltransferase from Schizosuccharomyces pornbe and UGT2 encoding the human UDP-galactose transporter, were co-expressed in galactosylation negative Saccharomyces cerenisiue cells. An efficient transfer of galactose to N- and O-glycans was observed, if cells were supplied with the donor sugar and acceptor substrates. Again, the level of UDP-galactose transporter expression and not the level ul,2-galactosyltransferase was found to limit formation of galactosylated structures. Obviously, we are approaching the time when cellular systems expressing desired glycoforms can be designed for use in biotechnological processes.
Acknowledgments We are most grateful to our colleagues for contributing to this article by sharing unpublished data: C. Korner, K. von Figura, D. Vestweber, T. Marquardt and their research teams are kindly acknowledged for providing data on the newly defined LAD I1 syndrome patient, M. Kawakita and N. Ishida for informations on Had-l mutants, C. Hirschberg and P. Berninsone for information on the C. eleyans nucleotide sugar transporter, and H. Bakker for information on the plant UDP-
32
2 Nucleotide Sugur Trunsporters
galactose transporter. We thank C. Hirschberg, A. Miinster, A. Cook, and K. Baker for helpful discussions and critical remarks on the manuscript. Work in the authors’ laboratory was supported by grants from the Deutsche Forschungsgemeinschaft and the European Community.
References 1. Abeijon C, Mandon EC, Hirschberg CB. Transporters of nucleotide sugars, nucleotide sulfate and ATP in the Golgi apparatus. Trends Biochern. Sci. 1997 22:203-207. 2. Hirschberg CB. Transporters of nucleotides and nucleotide derivatives in the endoplasmic reticulum and Golgi apparatus. Soc. Gen. Physiol. Ser. 1996 51:105-120. 3. Hirschberg CB. Transporters of nucleotide sugars, nucleotide sulfate and ATP in the Golgi apparatus membrane: where next? G/ycobio/oqy 1997 7: 169- 171. 4. Hirschberg CB, Robbins PW, Abeijon C. Transporters of nucleotide sugars, ATP, and nucleotide sulfate in the endoplasmic reticulum and Golgi apparatus. Annu. Rev. Biochem. 1998 67:49-69. 5. Kawakita M, Ishida N , Miura N, Sun-Wada GH, Yoshioka S. Nucleotide sugar transporters: elucidation of their molecular identity and its implication for future studies. J. Biochern. ( T o k y o ) 1998 123:717-785. 6. Varki A. Biological roles of oligosaccharides: all of the theories are correct. Glycohioloy/y. 1993 397-130. 7. Zhang JX, Braakman I, Matlack KE, Helenius A. Quality control in the secretory pathway: the role of calreticulin, calnexin and BiP in the retention of glycoproteins with C-terminal truncations. Mol. Biol. Cell 1997 8:1943- 19.54. 8. Jenkins N, Curling EM. Glycosylation of recombinant proteins: problems and prospects. Enzyme Microb. Technol. 1994 16:354-364. 9. Palade G. Intracellular aspects of the process of protein synthesis. Science 1975 189:347-358. 10. Neutra M, Leblond CP. Synthesis of the carbohydrate of mucus in the golgi complex as shown by electron microscope radioautography of goblet cells from rats injected with glucose-H3. J. Cell Bid. 1966 30:119-136. 1 1 . Cacan R, Duvet S, Kmiecik D, Labiau 0, Mir AM, et al. ‘Glyco-deglyco’processes during the synthesis of N-glycoproteins. Biochirnie 1998 80:59-68. 12. Van den Steen P, Rudd PM, Dwek RA, Opdenakker G. Concepts and principles of 0-linked glycosylation. Crit.Rev. Biochern. Mol. Biol. 1998 33:151-208. 13. Munro S. Localization of proteins to the Golgi apparatus. Trends Cell Bid. 1998 8:11-15. 14. Kean EL. Sialic acid activation. Glycohiologv 1991 1:441-447. 1.5. Miinster AK, Eckhardt M, Potvin B, Miihlenhoff M, Stanley P, et al. Mammalian cytidine 5’monophosphate N-acetylneuraminic acid synthetase: a nuclear protein with evolutionarily conserved structural motifs. Proc. Nut1 Acad. Sci. U.S.A. 1998 95:9140-9145. 16. Hirschberg CB, Snider MD. Topography of glycosylation in the rough endoplasmic reticulum and Golgi apparatus. Annu. Rev. Biockeni. 1987 56:63-87. 17. Abeijon C, Orlean P, Robbins PW, Hirschberg CB. Topography of glycosylation in yeast: characterization of GDPmannose transport and lumenal guanosine diphosphatase activities in Golgi-like vesicles. Proc. Nut1 Acad. Sci. U.S.A. 1989 86:6935-6939. 18. Capasso JM, Hirschberg CB. Effect of nucleotides on translocation of sugar nucleotides and adenosine 3’-phosphate 5’-phosphosulfate into Golgi apparatus vesicles. Biochirn. Biophys. Actu 1984 777:133-139. 19. Capasso JM, Hirschberg CB. Mechanisms of glycosylation and sulfation in the Golgi apparatus: evidence for nucleotide sugar/nucleoside monophosphate and nucleotide sulfate/nucleoside monophosphate antiports in the Golgi apparatus membrane. Proc. Nut1 Acad. Sci. U.S.A. 1984 81:7051 -5. 20. Waldman BC, Rudnick G. UDP-GlcNAc transport across the Golgi membrane: electroneutral exchange for dianionic UMP. Biochemistry 1990 29:44-52.
21. Yanagisawa K, Resnick D, Abeijon C, Robbins PW, Hirschberg CB. A guanosine diphosphatase enriched in Golgi vesicles of Saccharomyces cerevisiae. Purification and characterization, J. Riol. Chern. 1990 265:19351-19355. 22. Abeijon C, Yanagisawa K, Mandon EC, Hausler A, Moremen K, et al. Guanosine diphosphatase is required for protein and sphingolipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae. J. Cell Biol. 1993 122:307-323. 23. Berninsone P, Miret JJ, Hirschberg CB. The Golgi guanosine diphosphatase is required for transport of GDP-mannose into the lumen of Saccharomyces cerevisiae Golgi vesicles. J. Biol. Chem. 1994 269:207-21 I . 24. Gao XD, Jigami Y. Yeast YNDZ gene encodes a new nucleoside diphosphatase required for maturation of N - and 0-glycosylation. G1j~cohiolo;qy1997 7: 1044. 25. Gao XD, Kaigorodov V, Jigami Y. Y N D 1 , a homologue of CDAZ, encodes membrane bound apyrase required for Golgi N- and 0-glycosylation in Succhnron?jws wrerisiue. J. Biol. C!7ern. 1999 274:21450-21456. 26. Traynor AJ, Hall ET, Walker G, Miller WH, Melancon P, et al. Inhibition of UDP-Nacetylglucosamine import into Golgi membranes by nucleoside monophosphates. J. Med. Client 1996 39:2894-2899. 27. Pels Rijcken WR, Overdijk B, Van den Eijndcn DH, Ferwerda W. The effect of increasing nucleotide-sugar concentrations on the incorporation of sugars into glycoconjugates in rat hepatocytes. Biochem. J. 1995 305:865-870. 28. Stanley P. Membrane mutants of animal cells: rapid identification of those with a primary defect in glycosylation. Mol. Cell Bid. 1985 5923 929. 29. Hara T, Endo T, Furukawa K, Kawakita M. Kobata A. Elucidation of the phenotypic change on the surface of Had-1 cell, a mutant cell line of mouse FM3A carcinoma cells selected by resistance to Newcastle disease virus infection. J . Biochem. (Tokyo) 1989 106:236-247. 30. Hara T, Hattori S, Kawakita M. Isolation and characterization of mouse FM3A cell mutants which are devoid of Newcastle disease virus receptors. J. Virol. 1989 63:182-188. 3 I . Stanley P, Ioffe E. Glycosyltransferase mutants: key to new insights in glycobiology. FASEB J. 1995 91436- 1444. 32. Deutscher SL. Nuwayhid N, Stanley P, Briles EI, Hirschberg CB. Translocation across Golgi vesicle membranes: a CHO glycosylation mutant deficient in CMP-sialic acid transport. Cell 1984 39:295- 299. 33. Deutscher SL, Hirschberg CB. Mechanism of galactosylation in the Golgi apparatus. A Chinese hamster ovary cell mutant deficient in translocation of UDP-galactose across Golgi vesicle membranes. .I. Bid. Chcm 1986 261:96 100. 34. Taki T: Ogura K, Rokukawa C, Hara T, Kawakita M, et al. Had-I, a uridine 5’diphosphogalactose transport-defective mutant of mouse mammary tumor cell FM3A: composition of glycolipids, cell growth inhibition by lactosylceramide, and loss of tumorigenicity. Cancer Rex 1991 51:1701-1707. 35. Abeijon C, Mandon EC, Robbins PW. Hirschberg CB. A mutant yeast deficient in Golgi transport of uridine diphosphate N-acetylglucosamine. J. Biol. Clzern. 1996 271:8851 8854. 36. Dean N, Zhang YB, Poster JB. The VRG4 gene is required for GDP-mannose transport into the lumen of the Golgi in the yeast, Saccharomyces cerevisiae. J. Bid. Cheni. 1997 27,7:3190831914. 37. Tabuchi M, Tanaka N, Iwahara S, Takegawa K. The Schizosaccharomyces pombe gmsl+ gene encodes an UDP-galactose transporter homologue required for protein galactosylation. Biochem. Biophys. Res. Cominun. 1997 232: 121 125. 38. Descoteaux A, Luo Y, Turco SJ, Beverley SM. A specialized pathway affecting virulence glycoconjugates of Leishmania. Science 1995 269: 1869-1 872. 39. Herman T, Hartwieg E, Horvitz HR. sqv mutants of caenorhabditis elegans are defective in vulval epithelial invagination. Proc. Not1 Acud Sci. U.S.A. 1999 Y6:968- 913. 40. Herman T, Horvitz HR. Three proteins involved in Caenorhabditis elegans vulval invagination are similar to components of a glycosylation pathway. Puoc. i V d Acud. Sci. U.S.A. 1999 96:974-979. 41. Berninsone P, Hirschberg CB. Pcrsonal communication. -
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2 Nucleotide Sugar Transporters
42. Abeijon C, Robbins PW, Hirschberg CB. Molecular cloning of the Golgi apparatus uridine diphosphate-N- acetylglucosamine transporter from Kluyveromyces lactis. Proc. Nutl Acud. Sci. U.S.A . 1996 Y35963-5968. 43. Eckhardt M, Muhlenhoff M, Bethe A, Gerardy-Schahn R. Expression cloning of the Golgi CMP-sialic acid transporter. Proc. Natl Acad. Sci. U.S.A . 1996 93:7572-7576. 44. Eckhardt M, Gerardy-Schahn R. Molecular cloning of the hamster CMP-sialic acid transporter. Eur. J. Biochem. 1997 248:187-192. 45. Ishida N, Miura N, Yoshioka S, Kawakita M. Molecular cloning and characterization of a novel isoform of the human UDP-galactose transporter, and of related complementary DNAs belonging to the nucleotide-sugar transporter gene family. J. Biochem. (Tokyoj 1996 120:1074-1078. 46. Miura N, Ishida N, Hoshino M, Yamauchi M, Hara T, et al. Human UDP-galactose translocator: molecular cloning of a complementary DNA that complements the genetic defect of a mutant cell line deficient in UDP-galactose translocator. J. Biochem. (Tokyo) 1996 120:236241. 47. Ma D, Russell DG, Beverley SM, Turco SJ. Golgi GDP-mannose uptake requires Leishmania LPG2. A member of a eukaryotic family of putative nucleotide-sugar transporters. J. Biol. Chem. 1997 272:3799-3805. 48. Poster JB, Dean N. The yeast VRG4 gene is required for normal Golgi functions and defines a new family of related genes. J. Bid. Chem. 1996 271:3837-3845. 49. Eckhardt M, Muhlenhoff M, Bethe A, Koopman J, Frosch M, et al. Molecular characterization of eukaryotic polysialyltransferase-1. Nature 1995 373:715-718. 50. Frosch M, Gorgen I, Boulnois GJ, Timmis KN, Bitter-Suermann D. NZB mouse system for production of monoclonal antibodies to weak bacterial antigens: isolation of an IgG antibody to the polysaccharide capsules of Escherichia coli K1 and group B meningococci. Proc. Nutl Acud. Sci. U.S.A. 1985 82:1194-1198. 5 1. Baumann H, Brisson JR, Michon F, Pon R, Jennings HJ. Comparison of the conformation of the epitope of alpha(27-8) polysialic acid with its reduced and N-acyl derivatives. Biochemistry 1993 32:4007-4013. 52. Jennings, H. J. N-Propionylated Group B Meningococcal Polysaccharide Glycoconjugate Vaccine against Group B Meningococcal Meningitis. Znt. J. Infect. Dis.1997 1:158-164. 53. Puglielli L; Mandon EC, Rancour DM, Menon AK, Hirschberg CB. Identification and Purification of the Rat Liver Golgi Membrane UDP-N-acetylgalactosamine Transporter. J. Biol. Chem. 1999 274:4474-4479. 54. Berninsone P, Eckhardt M, Gerardy-Schahn R, Hirschberg CB. Functional expression of the murine Golgi CMP-sialic acid transporter in saccharomyces cerevisiae. J. Biol. Chem. 1997 272:12616-12619. 55. Sun-Wada GH, Yoshioka S, Ishida N, Kawakita M. Functional expression of the human UDP-galactose transporters in the yeast Saccharomyces cerevisiae. J. Biochem. (Tokyoj 1998 123:912-917. 56. Guillen E, Abeijon C, Hirschberg CB. Mammalian Golgi apparatus UDP-N-acetylglucosamine transporter: molecular cloning by phenotypic correction of a yeast mutant. Proc. Nutl Acud. Sci. U.S.A. 1998 95:7888-7892. 57. Bakker H. Personal communication. 58. Eckhardt M, Gotza B. Gerardy-Schahn R. Membrane topology of the mammalian CMP-sialic acid transporter. J. Biol. Chem. 1999 27453779-8787. 59. Eckhardt M, Gotza B, Gerardy-Schahn R. Mutants of the CMP-sialic acid transporter causing the Lec2 phenotype. J. Biol. Chern. 1998 273:20189-20195. 60. Mandon EC, Milla ME, Kempner E, Hirschberg CB. Purification of the Golgi adenosine 3’phosphate 5’-phosphosulfate transporter, a homodimer within the membrane. Proc. Nut1 Acad. Sci. U.S.A. 1994 YI:10707-10711. 61. Schroers A, Burkovski A, Wohlrab H, Kramer R. The phosphate carrier from yeast mitochondria. Dimerization is a prerequisite for function. J. Bid. Chem. 1998 273: 14269- 14276. 62. Sprong H, Kruithof B, Leijendekker R, Slot JW, van Meer G. et al. UDP-ga1actose:ceramide galactosyltransferase is a class I integral membrane protein of the endoplasmic reticulum. J. Biol. Chem. 1998 273:25880-25888.
ReJiwnces
35
63. Yoshioka S, Sun-Wada GH, Ishida N, Kawakita M. Expression of the human UDP-galactose transporter in the Golgi membranes of murine Had-I cells that lack the endogenous transporter. J. Biochem. (Tokyo) 1997 12269 1-695. 64. Ishida, N., Ito, M., Yoshioka, S., Sun-Wada, G.H., and Kawakita, M. Functional expression of human golgi CMP-sialic acid transporter in the Golgi complex of a transporter-deficient Chinese hamster ovary cell mutant. J. Biochmz. (Tokyo) 1998 124:171-178. 65. Ishida N, Yoshioka S, Iida M, Sudo K, Miura N , Aoki K, Kawakita M. Indispensability of transmembrane domains of Golgi UDP-Galactose transporter as revealed by analysis of genetic defects in UDP-Galactose transporter-deficient murine Hud-I mutant cell lines and construction of deletion mutants. J. Biochem. (Tokyo) 1999 126:1107-1117. 66. Kurt von Figura, Institut fur Biochmie Universitlt Gottingen, Germany. Dietmar Vestweber, Institut fur Zellbiologie, Universitat Munster, Germany. Thorsten Marquardt, Klinik und Polyklinik fur Kinderheilkunde, Universitiit Munster, Germany. 67. Etzioni, A., Frydman, M., Pollack, S., Avidor, l., Phillips, M.L., Paulson, J.C., and GershoniBaruch, R. Brief report: recurrent severe infections caused by a novel leukocyte adhesion deficiency. N. Engl. J. M a ! 1992 327: 1789- 1792. 68. Marquardt T, Brune T, Luhn K, Zimmer P, Korner C, Fabritz L, vander Werft N, Vormoor J, Freeze HH, Louwen F, Biermann B, Harms E,von Figura K, Vestweber D, Koch HG. A new patient with LeukocyteAdhesion Deficiency (LAD) IT Syndrome. J. Pediutr. 1999 f34:681-688. 69. Marquardt T, Luhn K, Srikrishna G, Freeze HH, Harms E, Vestweber D. Correction of leukocyte adhesion deficiency type I1 with oral fucose Blood 1999 943976-3985, 70. Korner C, Linnebank M, Koch HG, Harms E, von Figura K, Marquardt T. Decreased availability of GDP-L-fucose in a patient with LAD I1 with normal GDP-D-mannose dehydratase and FX protein activities J. Leukoc. Biol. 1999 66:95-98. 7 1. Liibke T, Marquardt T, von Figura K, Kdrner C. A new type of carbohydrate-deficient glycoprotein syndrome due to a decreased import of GDP-fucose into the Golgi. J. Bid. Chem. 1999 27425986-25989. 72. Marquardt T, Luhn K, Srikrishna G, Freeze HH, Harms E, Vestweber D. Manuscript submitted. 73. Huang MC, Zollner 0, Moll T, Maly P, Thall AD, Lowe JB, Vestweber D. Manuscript in preparation. 74. Fukuda M. Possible roles of tumor-associated carbohydrate antigens. Cancer Res. 1996 56:2237-2244. 75. Hakomori, S. Tumor malignancy defined by aberrant glycosylation and sphingo(g1yco)lipid metabolism. Cancer Res. 1996 56:5309-5318. 76. Noda K, Miyoshi E, Uozumi N, Yanagidani S, Ikeda Y, et al. Gene expression of alphal-6 fucosyltransferase in human hepatoma tissues: A possible implication for increased fucosylation of alpha-fetoprotein. Heputoloyy 1998 28:944-952. 77. Borges E, Pendl G, Eytner R, Steegmaier M, Zollner 0, et al. The binding of T cell-expressed P-selectin glycoprotein ligand-1 to E- and P-selectin is differentially regulated. J. Bid. Chem. 1997 272:28786-28792. 78. Minch SL, Kallio PT, Bailey JE. Tissue plasminogen activator coexpressed in Chinese hamster ovary cells with alpha(2,6)-sialyltransferase contains NeuAc alpha(2,6)Gal beta(l,4)Glc-NAcR linkages. Biotechnol. Proy. 1995 I I :348-35 1. 79. Grabenhorst E, Hoffmann A, Nimtz M. Zettlmeissl G, Conradt HS. Construction of stable BHK-21 cells coexpressing human secretory glycoproteins and human Gal(beta 1-4)GlcNAc-R alpha 2,6-sialyltransferase alpha 2,6-linked NeuAc is preferentially attached to the Gal(beta l-4)GlcNAc(beta I-2)Man(alpha 1-3)-branch of diantennary ohgosaccharides from secreted recombinant beta-trace protein. Eur. J. Biochem. 1995 232:7 18-725. 80. Toma L, Pinhal MA, Dietrich CP, Nader HB, Hirschberg CB. Transport of UDP-galactose into the Golgi lumen regulates the biosynthesis of proteoglycans. J. Bid. Chem. 1996 271 :38973901, 81. Greenberger, L.M.. Collins, K.I., Annable, T., Boni, J.P., May, M.K., Lai, F.M., Kramer, R., Citeralla, R.V., Hallett, W.A., and Powell, D. alpha-(3,4-dimethyoxyphenyl)-3,4-dihydro6,7-dimethoxy-alpha-[(4-methylphenyl)thio1-2( 1H)-isoquinolineheptanenitrile(CL 329,753): a
36
82. 83.
84.
85.
2 Nucleotide Sugar Transporters novel chemosensitizing agent for P-glycoprotein-mediated resistance with improved biological properties compared with verapamil and cyclosporine A. Oncol. Res. 1996 K:207-218. Kramer R. Analysis and modeling of substrate uptake and product release by prokaryotic and eukaryotic cells. A h . Biochem. Eny. Biotechnol. 1996 54:31-74. Klingenberg M. Dialectics in carrier research: the ADP/ATP carrier and the uncoupling protein. J. Bioenery. Biomembr. 1993 25:441-451. Kainuma M, Ishhida N, Yoko-o T, Yoshioka S, Takeuchi M, Kawakita M, Jigami Y. Coexpession of al,2 galactosyltransferase and UDP-galactose transporter efficiently galactosylates N- and 0-glycans in Saccharomyces cerevisiae. Glycohioloyy 1999 9:133-142 Zollner 0, Vestweber D. The E-selectin ligand-1 is selectively activated in Chinese hamster ovary cells by the alpha(l,3)-fucosyltransferasesIV and VII. J. Biol. Clzeni. 1996 271:3300233008.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
3 Biosynthesis of Oligosaccharyl Dolichol Slzuron S. Kray
3.1 General Overview Oligosaccharyl dolichol is a biosynthetic intermediate in the process of attaching an oligosaccharide to particular asparagine residues in proteins via an N-glycosidic bond. These glycoproteins, often referred to as N-linked glycoproteins or asparagine-linked glycoproteins, generally have 1-20'%) carbohydrate by weight which represents 1- 10 oligosaccharides attached to the protein backbone. N-linked glycoproteins are membrane-associated or soluble products of a biosynthetic system found in the secretory pathway of eukaryotic cells. These glycoproteins are diverse in structure and function and often have other co-/post-translational modifications such as lipids or sugars linked via 0-glycosidic bonds. For example, the insulin receptor, low-density lipoprotein receptor, ovalbumin, thyroglobulin, transferrin, immunoglobulins, lysosomal hydrolases, HMG-CoA reductase, and many Golgi glycosy1 transferases are N-linked glycoproteins. The function of the oligosaccharide moiety on these N-linked glycoproteins varies from one N-linked glycoprotein to another. These functions include having a role in the folding of the protein, being important for the biological half-life, being an important component of ligand-receptor interactions, affecting three-dimensional structure. and affecting solubility (see [ 11 and later Chapters of this volume). The cotranslational addition of oligosaccharide from oligosaccharyl dolichol to protein is necessary for life, as will be discussed below in this Chapter. Oligosaccharyl transferase, a multi-subunit, ER enzyme, catalyzes the transfer of oligosaccharide from oligosaccharyl dolichol to asparagine residues in an Asn-X-Ser(Thr) consensus sequence of protein (see next chapter and [2, 31). Thus, the core of each oligosaccharide of N-linked glycoproteins is not synthesized by a step-wise addition of monosaccharides onto a protein substrate, as is the case for oligosaccharides on many types of glycoproteins. Instead, the oligosaccharide core of N-linked glycoproteins is first assembled step-wise on a lipid carrier, transferred en bloc to protein, and then modified by numerous glycosidases, glycosyltransferases, and sulfo-
38
3 Biosynthesis of' Oligosaccharyl Dolichol
Figure 1. Structure of oligosaccharyl dolichol. The structure of this amphipathic phosphopolyisoprenyl glycolipid is given in this Figure. As mentioned in the text, the major isomer of dolichol in hamsters is N = 17 (4) while the major isomer of dolichol in S. pombe is N = 14,15 ( 5 ) . M = mannose; GlcNAc = N-acetylglucosamine; G = glucose.
transferases (most of which are subjects of other chapters in this volume) in order to reach its final. mature structure.
3.2 Oligosaccharyl Dolichol Oligosaccharyl dolichol is a large, amphipathic phosphopolyisoprenyl glycolipid; its structure is shown in Figure 1. This glycolipid has solubility properties similar to large, complex glycosphingolipids, in that it is not soluble in the standard lipid solvent, chlorofomxmethanol 2 : 1 but is soluble in a more polar solvent, chloroform: methanokwater 10 : 10 : 3 . The lipid portion of oligosaccharyl dolichol in any particular species is a mixture of different lengths of isoprene units. For example, in hamster cells, the dolichol moiety in oligosaccharyl dolichol ranges from 17 to 22 isoprene units with the predominant chain length being 19 isoprenyl units [4],while in Schizosacchavomyces pombe the dolichol moiety ranges from 14 to 18 isoprene units with the predominant chain length being 16 or 17 isoprene units [ 5 ] . Oligosaccharyl dolichol contains two phosphate molecules, in a pyrophosphoryl linkage. Oligosaccharyl dolichol also contains two N-acetylglucosamine (GlcNAc) residues, nine mannose (Man) residues, and three glucose (Glc) residues (Figure 1). The linkages and anomeric configurations of these sugars are identical to those found in N-linked glycoproteins that have undergone little or no processing [6]. Synthesis of the lipid moiety dolichol occurs in the cytoplasm and endoplasmic reticulum, beginning with steps identical to those of cholesterol biosynthesis, up to and including the formation of farnesyl pyrophosphate [7]. At this point, the synthesis pathways of cholesterol and dolichol branch, with dolichol synthesis proceeding with the addition of isoprenyl units to farnesyl pyrophosphate by cisprenyltransferase, yielding polyprenylpyrophosphate as the product. An important step in the synthesis of dolichol is the reduction of polyprenol, with a double bond in every isoprene unit, to dolichol, in which the ct isoprene, the one nearest the hydroxyl unit, is saturated (Figure 1 and [7]). The importance of the reduction of polyprenol to dolichol, catalyzed by polyprenol reductase, is clear from the study of Chinese hamster ovary (CHO) cells that lack polyprenol reductase activity [ 81. These cells only synthesize polyprenol and utilize polyprenol rather than dolichol for oligosaccharyl lipid and for the various
3.3 Kejs Enzytnatir. Steps in the Assembly Process
39
monoglycosylated lipids involved in the assembly of oligosaccharyl lipid (see below). The molecular basis of the lack of polyprenol reductase activity in these CHO mutants is not known yet, but the assumption is that the functional allele for polyprenol reductase is defective in these mutants; CHO cells are functionally haploid at many loci [9]. The result of a lack of polyprenol reductase activity is that glycosylation of N linked proteins is altered. Both non-glycosylation of certain sites and altered mature structures on certain sites of N-linked glycoproteins are observed in these mutants [lo]. The level of oligosaccharyl polyprenol is reduced in these mutants, due to the inability of the first enzyme in the assembly pathway (see below) to use polyprenyl phosphate [ 1 I]. Finally, the structure of the oligosaccharyl moiety of the most abundant oligosaccharyl polyprenol is truncated in these mutant cells due to differences in the abilities of various biosynthetic enzymes in the assembly pathway to use polyprenol derivatives [ 81. Recently, observations in two other systems have been reported that corroborate the importance of the structure of the lipid carrier in N-linked glycoprotein biosynthesis. A glycosylation mutant of Tryprrnosoma brucei has been shown to have reduced polyprenol reductase activity [ 121. Also, cells from patients with Carbohydrate Deficient Glycoprotein Syndrome (CDGS) were shown to have reduced polyprenol reductase activity [13]. Interestingly, in both these systems, the amount of dolichol and polyprenol was about equal, suggesting that only one of the two alleles of polyprenol reductase was mutated, and that polyprenol reductase activity is a rate-limiting step in dolichol synthesis [ 141. As expected from substrate specificity studies with membranes of CHO cells, the lipid utilized in these systems as a carrier of the oligosaccharide moiety was dolichol, since the first enzyme in the assembly pathway strongly prefers dolichyl phosphate to polyprenyl phosphate as a substrate [ 1 11. Nonetheless, these Trypuno.somi and CDGS cells displayed altered glycosylation of N-linked glycoproteins, perhaps because they had lower amounts of oligosaccharyl dolichol. The synthesis of the oligosaccharide of oligosaccharyl dolichol, detailed below, involves membrane-associated enzymes using either sugar nucleotides (UDPGlcNAc or GDP-Man) or monoglycosylated dolichols (dolichyl phosphate mannose, DPM, or dolichyl phosphate glucose, DPG) as sugar-donating substrates. The use of two types of sugar donors for the assembly of oligosaccharyl dolichol is reminicent of the biosynthesis of complex glycans in bacteria, such as peptidylglycan and lipopolysaccharide (see Chapter 26 in this volume). The metabolism of sugars and sugar nucleotides has been covered in Chapter 1 in this volume. Different enzymatic activities involved in the biosynthesis of oligosaccharyl dolichol occur on different faces of the endoplasmic reticulum of cells, and the challenges of this topology of the biosynthetic pathway will be discussed below.
3.3 Key Enzymatic Steps in the Assembly Process There are at least 14 enzymatic steps directly involved in the assembly of the oligosaccharide onto dolichyl phosphate to make oligosaccharyl dolichol. The levels
40
3 Biosynthesis of Oligosuccliuryl Dolichol
of the glycosyl transferase enzymes are thought to be very low, and the transferases are all membrane-associated. Thus, none of the transferases involved in these reactions have been studied as purified proteins from the ER membrane. The information we have about these transferases and the reactions they catalyze comes from genetics, the use of specific inhibitors, and in vitro reactions utilizing membrane preparations. The assembly begins with the addition of GlcNAc-P from UDP-GlcNAc to dolichyl phosphate, generating dolichol-P-P-GlcNAc. This reaction is catalyzed by the enzyme UDP-N-acetylglucosaminy1:dolichyl phosphate N-acetylglucosaminyl phosphoryl transferase (L-G 1PT). Studies on L-G 1PT have been performed primarily using membrane preparations and techniques such as in vitro mutagenesis, epitope tagging, peptide antibodies, and plasmid shuffling since quantities of purified, stable enzyme have thus far been impossible to obtain 11.51. Sequences of the cDNA of L-GlPT are known from hamster and mouse, and the sequences and/or structure of the gene are known from hamster, mouse, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Leishmania amazonensis ([ 161 and references therein). L-G1PT is a hydrophobic protein with its active site on the cytoplasmic face of the ER membrane 1171. L-G1PT has at least five transmembrane regions 118, 191, three residues known to be essential for enzymatic activity (arginine 303, aspartic acid 252, and asparagine 185) [18, 191, and five regions, conserved in L-GlPT and two bacterial enzymes with analogous functions, which appear important for activity and/or stability of the protein 1201. L-GlPT oligomerizes within the membrane, an unusual property for a protein with multiple transmembrane domains As mentioned above, L-G1PT has a strict substrate preference for dolichyl phosphate [ 1 I]. Its activity is stimulated specifically in vitro by mannosylphosphoryldolichol (MPD) and by phospholipids [22], although the function of this stimulation is not yet clear. Although L-G1PT appears to be constitutively expressed in all cells, its level is higher in actively growing cells. The amount of L-G1PT and its activity and the amount of L-GIPT mRNA increases when B-cells are activated by LPS [23] and in mammary gland tissue during lactation 1241; the amount of mRNA increases when growth-arrested Saccharomyces cerevisiae begin to proliferate [25]. LTGIPT is inhibited by a fungal antibiotic tunicamycin (TM), which has a structure that suggests it is a bisubstrate analog [26]. Therefore, TM has been used to inhibit N-linked glycosylation in a large number of systems since it is specific for this initial enzyme in the assembly pathway of oligosaccharyl dolichol [27]. TM is cytotoxic to cells, suggesting that N-linked glycosylation is crucial for cell viability 1281. Haploid S. cerevisiae cells or Schizosaccharomyces pombe cells with a disruption in the gene for L-G1PT are not viable [16, 291. Transgenic mice with a disrupted L-G1PT have been generated (see Chapter 55 in this volume), and embryos die between days 4-5 post-fertilization, again indicating the essential role of LGlPT in cell viability [30]. Mammalian cells treated with TM for as short a time as 4 h die by apoptosis, suggesting that altered glycosylation may be an endogenous signal for induction of apoptosis [ 3 11. The next six steps in the pathway, resulting in the elongation of dolicho1-P-P-
3.3 Key Enzymatic Steps in the Assembly Process
41
GlcNAc to dolichol-P-P-GlcNAc2M an5, are catalyzed by membrane-associated transferases that utilize sugar nucleotides as the sugar donors. Intermediates in this pathway normally do not accumulate, as one rarely detects any labeled products smaller than dolichol-P-P-GlcNAcZMan5 during metabolic labeling experiments with [2-3H]Man in parental cells. What is known about these transferases comes from studies of the yeast mutants akq1 (accumulates dolichol-P-P-GlcNAc2) and alg2 (accumulates dolichol-P-P-GlcNAc:Man*) [32]. Deletion of these genes is lethal. The addition of the final four Man residues to dolichol-P-P-GlcNAcZMans to form dolichol-P-P-GlcNAc2Mang is catalyzed by membrane-associated mannosyl transferases, products of ALQ, ALG9, and ALGl2 [32]. These genes are not essential for cell viability. The mannosyl transferases encoded by these genes utilize DPM as the sugar donor. DPM is synthesized in cells by DPM synthase, an endoplasmic reticular membrane enzyme, that catalyzes the addition of Man to dolichyl phosphate from GDP-Man. The structure of DPM synthase appears to be either a single polypeptide of about 26 KDa (encoded by D P M l ) in Saccharomyces cevevisiae, T. brucei and Ustilage maydis [33] or two polypeptides, dpmlp and dpm2p in Schizosaccharomyces pombe, humans, hamster, mouse, and nematode [33, 341. Dpmlp presumably contains the catalytic site. Sequence analysis of human D P M l revealed a conserved serine moiety and neighboring conserved residues that met criteria for a consensus site for phosphorylation by CAMP-dependent kinase [33], consistent with earlier biochemical studies [ 351 that suggested that the activity of DPM synthase was stimulated by phosphorylation. These biochemical studies. done with membrane fractions, showed a two-fold increase in DPM synthase activity in membranes either isolated from isoproterenol-treated cells or incubated in vitro with CAMP-dependent protein kinase. Dpm2p is a hydrophobic protein of only 84 amino acids and may function to correctly localize and stabilize dpmlp [36]. It is important to note that cells in culture can grow without DPM synthase activity. For example, Thy1 thyoma cells, with a mutation in DPMl [34], and Lecl5.1 (B4-2-1 [58])or Lec15.2 fibroblasts [36], with mutations in DPM2, survive, although the expression of cell-surface molecules (Thy antigen or mannose 6phosphate receptor) in these cells is altered. B4-2-1 cells do die at elevated temperatures [31]. However, while DPM synthase activity is not necessary for cell viability, lack of DPM synthase activity has serious consequences at the organism level. Cells of CGDS patients lacking DPM synthase activity have been found [37]. The final three steps in the assembly of oligosaccharyl dolichol are catalyzed by three different membrane-associated glucosyltransferases that utilize DPG as the sugar donor. A lack of these glucosyltransferase activities either in yeast (alg6, aly8, alglO, references [32, 381) or in mammalian cells [39, 591 does not result in cell death. Again, however, there appears to be serious consequences to the organism of a lack of glucosyltranferase activity, as cells of CGDS patients lacking glucosyltransferase activity have been found [40, 411. A dogma in the field had been that the presence of glucose in oligosaccharyl dolichol was essential for the transfer of oligosaccharide from oligosaccharyl dolichol to protein [42]. While in vivo studies using mutants synthesizing a truncated oligosaccharyl dolichol showed a preference for the transfer of glucosylated oligosac-
42
3 Biosynthesis of Oligosaccharyl Dolichol
charides [43, 441, it is clear from the work on alg6 and MI8-5 mutants that transfer of non-glucosylated GlcNAcZMang is only slightly less (no more than two-fold) than glucosylated oligosaccharide [38, 391. The function of the presence of glucose in oligosaccharyl dolichol is not clear at the present time, but glucose residues may be involved in determining the level of oligosaccharyl dolichol.
3.4 Topology of the Assembly Process One of the intriguing, yet complicating aspects of the assembly of oligosaccharyl dolichol is that it occurs on both faces of the ER [45]. That is, the first seven steps of the assembly process are thought to occur on the cytoplasmic face [46].For example, as mentioned above, the catalytic site of L-G1PT has been localized to the cytoplasmic side of the ER [ 171. The intermediate dolichol-P-P-GlcNAczMan5 is thought to be translocated through the bilayer to face the lumenal side of the endoplasmic reticulum, and subsequently elongated to dolichol-P-P-GlcNAc2MangGlc3. The oligosaccharyl transferase active site is thought to be on the lumenal side of the ER [2]. In addition, other molecules involved in the assembly process must be translocated. For example, DPM and DPG are synthesized on the cytoplasmic face and then translocated to the lumenal face. Dolichyl pyrophosphate or dolichyl phosphate, generated on the lumenal face by transfer of the oligosaccharide from oligosaccharyl dolichol to protein, must be translocated back to the cytosolic face in order to be used in the L-G1PT reaction to begin the assembly process again. While it is generally thought that these translocation events are protein-mediated, little experimental data exists to document these activities. The best work in this area is being done with water-soluble analogs of dolichol by Waechter and coworkers [47, 481. Their data are consistent with the presence of a membrane-associated “flippase” that mediates transbilayer movement of the saccharyl dolichols.
3.5 Utilization of Oligosaccharyl Dolichol As has been mentioned earlier in this chapter, oligosaccharyl dolichol is a substrate for oligosaccharyl transferase, generating glycoprotein and dolichyl pyrophosphate [2]. The latter is hyrolyzed by a phosphatase, regenerating dolichyl phosphate for use in subsequent assembly cycles [49]. The level of dolichyl phosphate is clearly important in the regulation of the amount of assembly of oligosaccharyl dolichol that occurs [7, 501. Oligosaccharyl dolichol is the major dolichol form in growing cells, with DPM, GPM, dolichol, and dolichyl phosphate being present in lower amounts [51]. Besides being a substrate for oligosaccharyl transferase, oligosaccharyl dolichol is
References
43
also catabolized to free oligosaccharide in the lumen of the ER [52, 531. Shorter, non-glucosylated oligosaccharyl dolichols, such as dolichol-P-P-GlcNAc2Mans, are substrates for a pyrophosphatase present on the cytoplasmic face of the ER, yielding free oligosaccharidyl phosphates [ 531. Thus it is clear that oligosaccharyl dolichol is catabolized by at least two pathways. The function of this catabolism is perhaps to regulate the level of oligosaccharyl dolichol in cells [54]. There is a transport system for non-glucosylated free oligosaccharides out of the ER [55]. Cytoplasmic catabolism of oligosaccharides is rapid, yielding GlcNAcMan5 [ 561, which is specifically transported into the lysosome [57].
Acknowledgment
The author gratefully acknowledges support from NIH grant R01 CA20421. References 1. 2. 3. 4. 5.
A. Varki, Glycobioloyy, 1993, 3, 97-130. S. Silberstein, and R. Gilmore, FASEB J . , 1996, 10. 849-858. R. Knauer and L. Lehle, Biochim. Biophys. Actu, 1999, 1426, 259-273. J . Stoll, A.G. Rosenwald, and S.S. Krag, J. B i d Chem., 1988, 263, 10774-10782. G.J. Quellhorst, Jr., J.S. Piotrowski, S.E. Steffen, and S.S. Krag, Biochenz. Bioplzys. Research Conzm., 1998, 244, 546-550. 6. E. Li, 1. Tabas, and S. Kornfeld, J. Biol. Clzem. 1978, 253, 7762-7770. I. A. Kaiden and S.S. Krag, Trends G1yco.sc.i. Glycoteclinol., 1991, 3, 275-287. 8. S.S. Krag, Biochem. Biophys. Rex Comm., 1998, 243, 1-5. 9. G.M. Adair and M.J. Siciliano, Somutic Cell Mol. Gen., 1986, 12, 11 1-1 19. 10. A.G. Rosenwald, P. Stanley, and S.S. Krag, Mol. C d . Biol., 1989, 9, 914-924. 11. K.R. McLachlan and S.S. Krag, Glycohioloyj~,1992, 2, 313-319. 12. A. Acosta-Serrano, K.-Y. Hwa, G.J. Jr. Quellhorst, H. Lei, S.S. Krag, and P.T. Englund, Mol. Parrcsit. Mtg VIII, 1997, Woods Hole, MA. 13. T. Ohkura, K. Fukushima, A. Kurisaki, H. Sagami, K. Ogura, K. Ohno, S. Hara-Kuge, and K. Yamashita, J. Bid. Chtw., 1997, 272, 6868-6875. 14. A.G. Rosenwald, P. Stanley, K.R. McLachlan, and S.S. Krag, Glycohiology, 1993,3, 481-488. 15. K. Shailubhai. B. Dong-Yu, E.S. Saxena, and I.K. Vijay, .I. Biol. Chem., 1988, 263, 1596415972. 16. J. Zou, J.R. Scocca, and S.S. Krag, Arch. Biochem. Biophys., 1995, 317, 487-496. 17. E.L. Kean, J. Biol. Chem., 1991,266, 942-946. 18. N. Dan, R.B. Middleton, and M.A. Lehrman, J. Biol. Chem.. 1996,271, 30717-30724. 19. J.R. Scocca and S.S. Krag, Glycohiokocjy, 1997, 7, 1181-1191. 20. A.R.D. Nogare, N. Dan, and M.A. Lehrman, Gljmhiology, 1998, 8, 625-632. 21. N . Dan and M.A. Lehrman, J. Bid. Clzem., 1997, 272, 14214-14219. 22. E.L. Kean, J. Bid. Chern., 1985,260, 12561-12571. 23. D.C. Crick, J.R. Scocca, J.S. Rush, D.W. Frank, S.S. Krag, andC.J. Waechter, J. Bid. Chenz., 1994,269, 10559-10565. 24. J. Ma, H. Saito, T. Oka, and I.K. Vijay, J. Biol. Chem., 1996, 271, 11197-11203. 25. M.A. Kukuruzinska and K. Lennon-Hopkins, Bioclzim. Biophys. Acta, 1999, 1426, 359-372. 26. A. Takatsuki, K. Kawamura, M. Okina, Y. Kodama, T. Ito, and G. Tamura, Agric. Biol. Chem., 1977,41,2307-2309. 27. Y.T. Pan and A.D. Elbein, 1995, in Glycoproteins, Montreuil, J., Schachter, H., and Vliegenthard, J.F.G., eds. Elsevier Science, Amsterdam, pp. 415-454.
44 28. 29. 30. 31. 32. 33.
3 Biosynthesis of Oligosaccharyl Dolichol
B.A. Criscuolo and S.S. Krag. J. Cell Biol., 1982, 94, 586-591. M.A. Kukuruzinska and P.W. Robbins, Proc. Nut1 Acad. Sci. USA, 1987,84, 214552149, K.W. Marek, I.K. Vijay, and Marth, J.D., Glycohiology, 1999, 9, 1263-1271. B.K. Walker, H. Lei, and S.S. Krag. Biochem. Biophys. Rex Comrnun., 1998. 250, 264-270. P. Burda and M. Aebi, Biochim. Biophys. Acta, 1999, 1426, 239-257. P.A. Colussi, C.H. Taron, J.C.Mack, and P. Orlean, Proc. Nut1 Acad. Sci. USA, 1997, 94, 7873-7878. 34. S. Tomita, N. Inoue, Y. Maeda, K. Ohishi, J. Takeda, and T. Kinoshita, J. Biol. Chem., 1998, 273, 9249-9254. 35. D.K. Banerjee, E.E. Kousvelari, and B.J. Baum, Proc. Vat1 Acad. Sci. USA, 1987, 84, 63896393. 36. Y. Maeda, S. Tomita, R. Watanabe, K. Ohishi, and T. Kinoshita, EMBO J., 1998, 17, 49204929. 37. S. Kim, D. Mehta, G. Srikrishna, S. Murch, and H. Freeze, Glycobioloyy, 1998,8, abstract 153. 38. G. Reiss, S. te Heesen, J. Zimmerman, P.W. Robbins, and M. Aebi, Glycohioloyy, 1996, 6, 493-498. 39. G.J. Quellhorst, Jr., J.L. O’Rear, R. Cacan, A. Verbert, and S.S. Krag, GIycohiology, 1999, 9: 65-72. 40. P. Burda, L. Borsig, J. de Rijk-van Andel, R. Wevers, J. Jaeken, H. Carchon, E.G. Berger, and M. Aebi, J. Clin. Invest., 1998, 102, 647 652. 41. T. Imbach, P. Burda, P. Kuhnert, R.A. Wevers, M. Aebi, E.G. Berger, and T. Hennet. Proc. Natl Acad. Sci. USA, 1999, 96, 6982-6987. 42. L.A. Murphy and R.G. Spiro, J. Biol. Chem., 1981,256, 7487-7494. 43. S. Kornfeld, W. Gregory, and A. Chapman, .I. Biol. Chew?.,1979,254, 11649-11654. 44. J. Stoll, R. Cacan, A. Verbert, and S.S. Krag, Arch. Biochem. Biophys., 1992, 299, 225-231. 45. C.B. Hirschberg and M.D. Snider, Annu. Rev. Biockenz., 1987, 56, 63-87. 46. C. Abeijon and C.B. Hirschberg, J. Bid. Chern., 1990,265, 14691-14695. 47. J.S. Rush and C.J. Waechter, J. Cell Biol., 1995, 130, 529-536. 48. J.S. Rush, K. van Leyen, 0. Ouerfelli, B. Wolucka, and C.J. Waechter, GljXdJiohgy,1998, 8, 1195-1205. 49. D. W. Frank and C.J. Waechter, J. Bid. Chern., 1998, 273, 11791-11798. 50. A.G. Rosenwald, J. Stoll, and S.S. b a g , J. Biol. Chem., 1990, 265, 14544-14553. 51. A. Kaiden and S.S. Krag, Biochem. Cell Biol., 1992, 70, 385S389. 52. M.J. Spiro and R.G. Spiro, J. Bid. Chem., 1991, 266, 5311-5317. 53. R. Cacan, C. Villers, M. Belard, A. Kaiden, Krag, S.S. and A. Verbert, Glycobioloyy, 1992. 2, 127- 136. 54. R. Cacan and A. Verbert, Trends Gl.ycosci. Glycotechn., 1997, 9, 365-377. 55. S.E.H. Moore, C. Bauvy, and P. Codogno. EMBO J.. 1995,14, 6034-6042. 56. D. Kmiecik, V. Herman, C.J.M. Stroop, J.-C. Michalski, A.-M. Mir, 0. Labiau, A. Verbert, and R. Cacan, Glycobiology, 1995. 5, 483-494. 57. A. Saint-Pol, C. Bauvy, P. Codogno, and S.E.H. Moore, J. Cell Biol., 1997, 136, 45-59. 58. L. Pu, J.R. Scocca, and S.S. Krag, unpublished. 59. G.J. Quellhorst, Jr., J.L. O’Rear, R. Cacan, A. Verbert, and S.S. Krag, unpublished.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
4 Biochemistry and Molecular Biology of the N-Oligosaccharyltransferase Complex Roland Knauer and Ludwig Lehle
4.1 Introduction Protein glycosylation is one of the most common types of eukaryotic protein modification [I-51. There is clear evidence that it also occurs in many archaea [6, 71, whereas in eubacteria it is found only exceptionally [8]. Both N-linked (9-1 11 and O-linked [ 121 glycans are essential for cell viability and can have a profound role in the biological function and in physicochemical properties of many secreted and integral membrane proteins [ 131. The first commited step of the N-glycosylation pathway is the en bloc transfer of the preassembled high mannose core oligosaccharide Glc3MangGlcNAc2 from dolichyl pyrophosphate onto selected Asn-XSer/Thr acceptor sites of nascent polypeptide chains during or shortly after their translocation into the lumen of the endoplasmic reticulum. It is catalyzed by the hetero-oligomeric membrane-bound enzyme complex N-oligosaccharyltransferase (OST) (Figure 1). It is these processes, the assembly of the lipid-linked oligosaccharide and the so called “core glycosylation” reaction of the protein at the ER, that are highly conserved during evolution, whereas subsequent diversification of the primary glycoprotein conjugate occurs along the secretory pathway, mainly in the Golgi, in a species and cell type specific manner. Previous attempts to purify and characterize OST have been hindered by the lability of the enzyme upon solubilization [ 14-16] and probably also because of its hetero-oligomeric nature. Meanwhile, however, OST complexes have been purified from different sources, such as dog [17, 181, yeast [19-231, hen [24], human [25, 261 and pig [27]. The subunit composition of the various isolated complexes and the protein sequences so far obtained reveal indeed a high degree of conservation. In this review we will try to summarize the current knowledge of biochemistry and molecular biology of the OST from lower and higher eukaryotes. Sequence search in databases indicate also homologous proteins in archaea that may be involved in the N-glycosylation of these organisms. The reader is also referred to previous reviews dealing with this topic 128-3 11.
46
4 Biochemistry and Molecular Biology Ribosome
OST
m
Cytosol
ER lumen
Man
Man
Man I G,Ic G,Ic Glc
Figure 1. The glycosylation reaction catalyzed by oligosaccharyltransferase. The unique core oligosaccharide Glc3MangGlcNAcz is transferred from dolichyl pyrophosphate to selected asparagine residues in an Asn-X-Ser/Thr consensus sequon of nascent polypeptides. The dolichol moiety contains an saturated a-isoprenoid unit and may vary in chain length. The OST complex from yeast is shown. Subunits in bold face are essential for yeast growth; SEC61 represents the yeast protein translocation complex.
4.2 Biochemistry of OST OST catalyzes an unusual reaction, rare in mechanistic enzymology, in which the nucleophilicity of the amide nitrogen of the asparagine residue must be enhanced to make a covalent bond with the oligosaccharide (see below). Before purified OST became available, basic biochemical characteristics of the enzyme were determined using microsomal membranes or detergent solubilized extracts. OST exhibits a requirement for divalent cations with a preference for Mn2+ over Mg2+ [15, 16, 27, 32-34]. The pH optimum of the enzyme is between 6.5 and 7.5 [15, 27, 341. OST from different sources has been shown to be strongly stabilized by addition of phos-
4.2 Biochemistry of OST
47
pholipids upon detergent solubilization [17, 21, 23, 27, 35, 361, sucrose or glycerol [15, 271. OST activity is determined in vitro using an appropriate lipid-linked saccharide as glycosyl donor [ 151 and a peptide acceptor containing the Asn-X-Thr/Ser recognition motif, whereby X can be every amino acid except proline [37].
4.2.1 Lipid-Saccharide Donor Full length core oligosaccharide DolPP-GlcNAczMang Glc3 is the optimal glycosyl donor both in vitro [15, 27, 33, 38-40] and in vivo [41-441. The minimal glycosyl donor in vitro is DolPP-GlcNAcz; no transfer is observed from DolPP-GlcNAcl [ 151. The glucose residues enhance glycosyl transfer presumably by adopting a favourable conformation contributing to oligosaccharide recognition by the OST [45, 461 and thus affecting the apparent binding affinity for the acceptor substrate [15, 271. For the yeast enzyme a ten times lower K, value for the acceptor hexapeptide YNLTSV was determined, when DolPP-GlcNAc2MangGlc~is the donor compared to DolPP-GlcNAc2 [15]. Thus OST may display two substrate recognition sites, the chitobiose site and a glucose binding domain [ 15, 461. Transfer of incomplete core oligosaccharides was originally observed in vitro [15, 47-49], but later shown also to occur in vivo [50]. In alg (asparaginelinked glycosylation) yeast mutants [9, 50, and reviewed in 511 which have a defect in lipid-linked oligosaccharide assembly, shorter glycan chains are transferred to proteins: e.g. Man*-zGlcNAc2 in a1q2 [52, 531; MansGlcNAc2 in 4 3 [42, 43, 541; MangGlcNAcz in alg.5 and alg6 [55-571; Man7GlcNAcz in alg12 [58]; Glcl MangGlcNAcz in a478 [59]; Man6GlcNAc2 in aly9 [60] or GlczMangGlcNAc2 in alglO [61]. Depending on the protein, not only truncated saccharides, but also fewer chains may be transferred, and to selected glycosylation sites [42, 43, 50, 62651. The basis for this underglycosylation appears to be the reduced affinity of the OST towards truncated oligosaccharides. Glucosylation is not essential for viability of yeast cells under laboratory growth conditions, but double mutants with a simultaneous defect in glucose addition and OST activity [59, 661 may display a temperature-sensitive growth phenotype. Nonglucosylated oligosaccharides are transferred to protein in trypanosomatid protozoa, which lack DolP-Glc synthase, and depending on the strain, may also have a defect in lipid-oligosaccharide mannosylation [67, and reviewed in 681, or in mammalian mutants that synthesize primarily Man5GlcNAcz (lack of DolP-Man synthase) [44, 691 or MangGlcNAc2 (lack of glucosyltransferase) [70]. The dolichol moiety of lipid-intermediates is not homogeneous but a mixture of 14-20 isoprene units [71-731. For the yeast OST it was shown that the enzyme hardly discriminates specific isoprene chain lengths [ 741. Whether unsaturated polyprenols, preferred in eubacterial cell wall synthesis, will be accepted is not known. However, the formation of the DolPP-GlcNAc, i.e. the first step in lipid-linked precursor formation shows an absolute dependency for saturation of the w-isoprenoid unit, i.e for dolichol-type polyprenols [74],what seems to be generally true for other eukaryotic glycosyltransferases involved in lipid-linked sugar formation [75, 761.
48
4 Biochemistry and Molecular Biology
4.2.2 Acceptor Specificity of OST Insights into the structural requirements for the peptidyl substrate were obtained both by using synthetic peptides or by engineering novel sequons into recombinant proteins. An Asn-X-Thr/Ser tripeptide is a sufficient in vitro acceptor provided that both termini are blocked [77, 47-49]. Only rare exceptions from the consensus “sequon” Asn-X-Thr/Ser occur, e.g. glycosylation of Asn-X-Cys [ 78-80] and Asn-GlyGly-Thr [XI] have been described. In vitro, peptides with Asn-X-Thr are about 40fold better substrates for the OST than peptides containing an Asn-X-Ser sequon [82]. This holds also true in vivo, even though the preference for Thr is less pronounced [81, 831. Peptides with Pro in the position X do not function as acceptors [82, 841 and fail to inhibit co-translational glycosylation in cell free systems [8S-87]; also introduction of Pro into recombinant sequons blocks glycosylation [86, 881. Pro in the flanking region is inhibitory when present C-terminal to the sequon, whereas OST function is not influenced when Pro immediately preceeds the sequon [XI, 82, 85, 891. Besides Pro, also large hydrophobic amino acids (e.g. Trp) or negatively charged ones (e.g. Asp and Glu) in the position Ximpair glycosylation [86, 89, 901. Finally, the amino acid following a sequon was found to be an important determinant of glycosylation efficiency [89). Sequons near the C-terminus of a protein are less likely used by the transferase [XI, 91, 921, perhaps because prior or ongoing folding of the protein imposes physical constraints on glycosyl transfer. The minimum distance of an acceptor site from the lumenal end of a transmembrane segment was determined to be 12-14 residues, suggesting that the active site of the OST is positioned 30-40 A above the ER membrane surface [93]. N-glycosylation of multispan membrane proteins reveals that glycosylated sequons occur in the first extracytosolic loop, provided that it is greater than 30 residues in size and spaced at least 10 amino acids from the transmembrane domain [94]. It has been speculated that in polytopic membrane proteins, only one polypeptide segment can be optimally occupied by the OST during protein synthesis P41. Since about 10-30% of potential sequons are either not glycosylated at all, or are not efficiently glycosylated, additional, as yet not well defined factors. influence N-glycosylation in vivo [XI, 951. Protein folding is assumed to favour or hinder OST action [96-981, or may influence core glycosylation at a further sequon. Thus, deletion of the Am297 in gpSS of Friend spleen focus-forming virus leads to glycosylation of a downstream sequon that is normally not used [79, 96, 99, 1001. Conversely, glycosylation can influence the local structure of the glycosylation site [ 101-1 031 by altering the solvation properties, by participating in hydrogen bonding interactions with the polypeptide, or for sterical reasons. NMR studies on glycopeptides provided evidence that derivatization with N-acetylchitobiose induces conformational switching into a p-turn structure, similar to the structure found in many native proteins [101, 102, 1041. On the other hand it is clear also that other types of secondary structures fulfil the spatial requirement of having the glycan exposed at the surface of proteins [103, 81, 9.51, and the widely believed assumption that the glycosylated tripeptide sequence has to adopt a p-turn conformation no longer holds true (see also below).
4.2 Biochemistry of OST
49
4.2.3 Catalytic Mechanism of OST
Since N- and C-terminal capped tripeptides are the simplest substrates for Nglycosylation, local turn motifs, such as a p-turn or an Asx-turn (Figure 2) seem to be a sufficient secondary structural requirement, rather than an a-helix or P-sheet which would require longer peptides for complete formation. Using a series of conformationally constrained peptide substrates Imperiali et al. 130, 1051 suggest the Asx-turn as the favourable conformation and propose a plausible reaction mechanism involving carboxamide tautomerization in which hydrogen-bond contacts are formed between the p-carbonyl group of asparagine and the a-NH and P-OH group, respectively, of the hydroxyamino acid. This enhances a proton dissociation from the p-nitrogen generating an imidate yielding the reactive nucleophile required for glycosyl transfer (Figure 2, model A). An alternate mechanistic model by Bause et al. [106-108], which can also explain both specificity and enhanced amide nitrogen nucleophilicity, proposes that the pamide nitrogen is the hydrogen-bond donor generating an anionic species (Figure 2, model B). The essential threonine/serine hydroxy group would act in this case as the acceptor upon deprotonation by an enzyme-bound base. However, this mechanism requires a p-turn or loop conformation, what, as discussed above, seems to be rather unlikely. For a comprehensive discussion that addresses the catalytic mechanism see 130, 3 1, 109, 110, 1061. Independent of this, the unique ability of asparagine residues to adopt a specific loop structure may explain, why e.g. a Gln-X-Thr is not glycosylated, although this sequence is found in nature. Using photoprobes containing the Asn-X-Thr sequence, in which X wasp-benzoylphenylalanine, a protein could be labelled in yeast microsomes that was identified as Ostlp indicating that this subunit may recognize the peptide glycosylation site in nascent chains [ 11 11. Similarly, it was shown that an epoxyethylglycyl acceptor peptide irreversibly inactivates pig liver OST and simultaneously leads in the presence of radioactive DolPP-GlcNAcz to the labelling of the 66 kDa ribophorin I subunit (the homolog of Ostlp) and OST48 (the homolog of Wbplp) by covalent attachment via the epoxy-group [ 1081. The reason why two subunits become labelled is not clear at present. Perhaps it may indicate that the active site is placed at the interface between the two components. For yeast OST circumstantial evidence exists in favour of Wbplp being also a subunit involved in catalysis. Thus, the thiol-modifying reagent biotinoyl(aminoethane)-thiosulfonate was found to inactivate enzyme activity and to label Wbpl p. Since furthermore DolPP-GlcNAcz was able to protect the inactivation of the enzyme, it was proposed that WbpIp may contain a binding site for the lipidlinked oligosaccharide [21]. On the other hand, none of the three Cys residues in WbpIp is conserved in the corresponding eukaryotic OST48 sequences; hence a direct role of Cys residues in the catalysis is doubtful. The development of peptidyl OST inhibitors has also been reported [112]; they may contribute to the structural and functional analysis of the enzyme, or being helpful diagnostic tools to evaluate the role of protein glycosylation and glycoproteins in vivo.
50
4 Biochemistry and Moleculur Biology
A) Y
CH3
*d Enzyme
Model A
Model B
P-Turn
ASX-Turn
Figure 2. Proposed reaction mechanisms for oligosaccharyltransferase. (A) Model A by Imperiali et al. [39, 31, 1051 proposes an Asx-turn conformation for the peptide backbone. Hydrogen bonding interactions occur between the asparagine p-carbonyl group and both the backbone amide and hydroxyl group of threonine or serine. Enzyme-mediated deprotonation at the asparagine nitrogen by a basic residue in the active site induces tautomerization of the carboxdmide to an imidol species. The generated nucleophilic species could then react with the electrophilic lipid-linked oligosaccharide. Model B by Bause et a]. [106-108] favours the existence of a 0-turn or other loop structures stabilized by hydrogen bonds between the P-amide nitrogen and the hydroxyl group of the hydroxy amino acid leading to amide deprotonation. (B) Comparison of an Asx-turn and a p-turn conformation for the Ac-Asn-X-Thr-NHz tripeptide.
4.3 Isolation of OST C'orwpkexes)om Different Sources
51
4.2.4 Regulation of OST Activity There is little information available dealing with this aspect. Hormones are known to influence N-glycosylation of proteins. But in most cases the type of regulation is unclear. The observed enhancement could be indirect by stimulating lipidintermediates [ I 13, 1141 or due to accelerated protein synthesis rather than to an increase of OST action. A marked stimulation of OST activity (4.2-fold) was reported in porcine thyroid cells stimulated by thyrotropin 11 131. In human lymphocytes OST activity increased ten-fold by interleukin-2 mitogen activation and remained elevated during long-term culture in the presence of interleukin-2 1261. A different type of regulation of N-glycosylation was suggested to occur by OST transferring the oligosaccharide to water as acceptor, if appropriate protein synthesis is not available [ 115-1 17, 401. Moreover, also transcriptional regulation could take place. S W P l and also WBPl from yeast were reported to be regulated in a cell proliferation-dependent manner. Together with the ALG genes, they are down regulated when cells enter the Go-phase, and they are induced following growth stimulation in absence of de novo protein synthesis [ 1181.
4.3 Isolation of OST Complexes from Different Sources The first isolation of an active OST complex, composed of three subunits (ribophorin I and 11, and OST48) succeeded from dog pancreas cells [17]. The ribophorins had been characterized earlier as possible receptors for ribosomes and thought to be involved in protein translocation [ 119- 1211. Since OST activity could be depleted from detergent solubilized extract by anti-ribophorin I antibodies, it became clear that ribophorins are indeed constituents of OST (171. They are no longer believed to be involved in ribosome binding, but in accord with the cotranslational nature of N-glycosylation, the identification of ribophorins as part of the OST may indicate a close location of the enzyme to the protein translocation channel. A breakthrough in the understanding of composition and molecular biology of OST was achieved upon isolation of two essential genes from yeast, WBPl and S W P l , and the demonstration that they are required for N-glycosylation in vitro and in vivo [122, 1231. Subsequently, independent OST purifications have been reported from Succharornyces cerevisiue yielding enzymatically active complexes consisting of either four polypeptides: Ostlp (64/62 kDa), Wbplp (47 kDa), Ost3p (34 kDa), and Swplp (30 kDa) 119, 211, or five ([22] in addition Ost2p; 16 kDa), or of six subunits (1201 in addition Ost2p and Ost5p; 9.5 kDa). Apparently, Ost2p and Ost5p are dispensable for enzyme activity in vitro. In the meantime, yeast-specific genetic approaches, such as high copy number suppression and synthetic lethality, or searches of the yeast protein sequence database, have identified STT3, OST4 and OST6 as additional OST genes. Studies using epitope-tagged OST proteins or specific antibodies against various subunits [ 124, 1251, or investigating the complex by
52
4 Biochemistr,v and Molecular Biology
Table 1. Comparison of OST complex from different organisms. Yeast
Mol. wt. Essential Dog (k) pancreas
Ostlp
64/62
Stt3p Wbplp Ost3p Ost6p Swplp
-60 47 34 32 30
Ost2p Ost5p Ost4p
16 9.5 3.6
+ + -
Hen Chicken Pig oviduct liver liver
Ribophorin I 65-1 (66)
65-1
66
~
~
~
OST48 (48) ~
~
50 ~
50 ~
+
65-11 ~
65-11
65-1 ._
65-1 ~
48 (40*) 50 ~
~
Ribophorin I1 (63/64) DADl (12)
Human Human liver lymphocytes
6 1/63
...
-
65-11
50 ~
~
65-11
~
-x
The numbers designate the apparent molccular weight of the subunits as determined by gel electrophoresis. : ts, necessary for growth at elevated temperatures. *: An additional 40 kDa band of unknown origin was reported for pig liver.
blue native polyacrylamide electrophoresis [23] indicate that these subunits are indeed constituents of the complex and necessary for optimal glycosyl transfer. They seem to have been lost during purification (for further discussion see below). There is also evidence that some of the subunits are not easily detectable by protein staining methods or reveal an abnormal migration behaviour in SDS-PAGE [23, 1241. In Table 1 a compilation of OST complexes from different organisms and the homology between the various subunits is depicted. In most cases the genes have been cloned or partial sequence information is available to allow their classification. Compared to yeast, the mammalian enzymes seem to be composed of fewer subunits. In the light of the discussion above. one may predict that additional subunits may be detected that have escaped detection so far. In fact, it was subsequently found that the trimeric OST complex from dog pancreas [17], contains in addition DADl (defender of apoptotic death) [18] which is 40% identical to Ost2p from yeast. The isolation of “incomplete”, but active complexes indicates that the respective subunits may be sufficient to constitute a “catalytical core” unit.
4.4 Molecular Biology of OST From the nine yeast genes encoding OST subunits, five are essential for growth of yeast under laboratory conditions (Table 1). In Figure 3 the presumed membrane topology of the derived proteins is depicted. Due to the high conservation of the
4.4Molecuhr Biology Dog pancreas
of OST
53
Yeast
N=--=fl=N ER membrane
Ribophorin I
Ostlp Ost5p
Ribophorin II
SWPl P
OST48
WbPl P
DAD1
Ost2p
Stt3p
C Ost4p N
Ost3p Ost6p
? = N-linked oligosaccharide 0
= signal sequence
I
I
I
I
I
0
100
200
300
400
ER lumen
1 -
500 0
100 cytosol
Figure 3. Predicted membrane topology of the oligosaccharyltransferase subunits from yeast. The topology is based partially on direct or indirect experimental evidence (N-terminal sequencing, protease accessibility, glycosylation sites, HIS4 fusions) as discussed. The presentation is not to scale. For comparison, the corresponding honiologues of the OST complex from dog pancreas are depicted which have the same predicted topology (not shown).
pathway, it is not unanticipated that both primary sequences and also their membrane topology are conserved. In the following, guided by the known OST genes from yeast, corresponding homologues from higher eukaryotes and homologous sequences found in databases will be discussed along with their possible properties and functions.
54
4 Biochemistry and Molecular Biologj,
4.4.1 WBPl /OST48 WBPl (wheat germ agglutinin binding protein) was originally identified as a yeast gene encoding an essential ER type I glycoprotein with the apparent molecular mass of 47 kDa and it received its name on account of its lectin binding property [126]. Later it was shown that W B P l is required for OST activity in vitro and in vivo 11221 and represents a component of the enzyme 119, 20, 231. Depletion of Wbplp in response to a regulatory promotor, or conditional wbpl mutations lead to an underglycosylation of yeast glycoproteins or to a reduced oligosaccharide transfer to an acceptor hexapeptide 11221. The membrane topology of Wbplp was determined using the HIS4C fusion technique [126]. The protein contains a short cytoplasmically exposed carboxy-terminal domain with the ER-retrieval motif KKXX, which, however, is not absolutely required for proper Wbplp function and localization [ 1271. It is assumed that OST complex formation is primarily responsible for ER retention of Wbplp. A genetic interaction between the wbpl and kar2 mutations was found (KAR2 encodes BIP in yeast) 11281. W B P l has 25% identity to OST48 from dog pancreas 11291 and to the 48/50 kDa subunit of the OST complex isolated from pig 11071 and human [25]liver, hen oviduct [24] and human lymphocytes 1261. The homology among the mammalian sequences is higher than 90%. In contrast to Wbplp, the OST48 protein is not glycosylated [17, 1291. Homologous sequences to W B P l are also available from nematode, insect and Schizosacchavomyces pombe. Genomic OST48 clones from human and mouse were isolated and shown that the gene is organized into 11 exons expanding about 9 kb. Fluorescence in situ hybridization localizes the human gene to chromosome lp36.1 [ 1301.
4.4.2 SWPl/Ribophorin I1
Since overexpression of Wbplp did not result in increased OST activity, it was assumed that Wbplp is a non-limiting component of a larger complex [123]. This led to the identification of S W P l (suppressor of W B P ) by using a high copy number suppression approach. S W P l was isolated as an allele-specific suppressor of the temperature sensitive wbpl-2 mutation and encodes a 30 kDa, essential transmembrane protein that can be chemically crosslinked to Wbplp 11231. By immunodetection [19] and N-terminal sequencing 120, 211 Swplp was also identified as a component of the isolated complex. No sequence homology to other proteins was originally detected in searches of the databases [ 123). However, a direct comparison of Swplp with the C-terminal half of rat and human ribophorin 11, i.e. about the size of Swplp, reveals 46% similarity and 24% identity. Whether the function of the N-terminal half of ribophorin I1 is dispensable in Swplp, or whether other subunits of yeast OST serve this function, remains an open question. Characteristic of both Swplp and ribophorin I1 is the arrangement of three hydrophobic stretches at the C-terminus. Depletion of Swplp leads to underglycosylation of proteins in vivo and to reduced OST activity in vitro, similar to the lack of Wbplp. Ribophorin I1 as a component of other OST complexes has been documented (see Table 1).
4 . 4 Moleculur Biolnyj, of OST
55
4.4.3 OSTl/Ribophorin1 The OSTl gene is the yeast homologue of ribophorin I with 58% sequence similarity and 28°/0 identity and has been identified in all OST preparations so far (Table 1). Protein sequence information from the purified 64 kDa subunit [19, 1321 led to the identification of OSTl [131]; in an independent isolation of this gene it was termed N L T l (for H-Linked oligosaccharyl rransferase) [ 1321. Ostlp is an essential type I membrane glycoprotein with a cleavable signal peptide of 22 amino acids, a large lumenal domain and five potential glycosylation sites, maximally four of which are used. Ostlp is incompletely glycosylated in wild-type cells and migrates as 64/62/60 kDa glycoforms containing four, three or two core-oligosaccharides. Incomplete glycosylation is also true for ribophorin I. The fact that Ostlp and Wbplp (and also Stt3p, see below) are glycoproteins is fascinating but also puzzling and means that the mature complex must be able to process some of its own components. The most striking difference between yeast Ostlp and the canine ribophorin I is that the yeast protein has only a short 9 amino acid cytoplasmic domain at the C-terminus compared to the 150 residues in ribophorin I. Interestingly, a truncated version of ribophorin I, missing the cytoplasmic segment, was shown to be rapidly degraded in vivo (1331. This may indicate that this part is not necessary in yeast, or that its function is served by another protein. Defects in OSTI cause a pleiotropic underglycosylation of soluble and membrane-bound glycoproteins in temperature sensitive ostl mutants, and lead to a reduced OST activity in vitro that correlates with the glycosylation index in vivo [ 1311.
4.4.4 OST3/OST6 OST3 [ 131, 231 and OST6 [23] from yeast encode the 34 kDa and 32 kDa, respectively, subunits. They reveal only a weak sequence identity of 21%, but according to a hydropathy analysis they display a striking similar topology with four putative Cterminal membrane spanning segments (Figure 3). These proteins have not been identified yet in other OST preparations, but homologous proteins with the same membrane topology exist in Caenovhahditis elegans and man. The human gene, designated N33, was identified as a candidate tumor suppressor gene [ 1341 and has 21% identity to Ost3p/Ost6p. The relationship of OST3, OST6 or N33 to a neoplastic phenotype is obscure, but altered glycosylation of cell surface proteins is a well known feature of tumor cell lines [ 135, 1361. Neither OST3 nor OST6 gene is essential for yeast growth. A single disruption of OST3 [ 137, 231 or of OST6 [23] leads to only a minor defect in N-glycosylation both in vivo and in vitro. However, an Aost3Aost6 double mutant displays a synthetic phenotype leading to a severe underglycosylation with a similar pattern as that observed for mutants with a defect in the essential WBPI or OSTl genes [23]. Nevertheless an Aost3Aost6 double mutant is viable and hardly displays a temperature sensitive phenotype up to 37°C. Thus the degree of underglycosylation cannot be considered a direct predictor of a temperature sensitive growth, nor can it explain the essential role of N-glycosylatjon. Overexpression of Ost6p rescues most of the severe underglycosylation
56
4 Biochemistry and Molecular Biology
defect of proteins in the double mutant and also the milder defect in host3. On the other hand, each of the two genes seems to have also a specific function, since agents affecting cell wall biogenesis reveal different growth phenotypes in the respective null mutants. It was found that an Aost6, but not an host3 null mutant is sensitive to caffeine, Calcofluor White and SDS. In the case of SDS the host3 null mutant was even more resistant compared to an isogenic wild-type strain [23]. 4.4.5 OST5 This gene encodes the 9.5 kDa subunit of the yeast complex; homologous sequences are found in man, mouse and C. elegans, but the corresponding proteins have not been identified in OST preparations from higher eukaryotes. OST5 was identified in a genetic screen [ 1381 combining the observation that ost mutations, in combination with a deficiency in DolP-Glc biosynthesis (halg5), resulting in incomplete lipidoligosaccharide precursor, reveal a synthetically lethal growth phenotype [ 591. OST5 itself is not essential for growth, but its depletion results in a reduction in OST activity to about half that of wild-type levels. Experimental evidence for its identity with the 9.5 kDa subunit of the complex was obtained by comparison of the deduced and experimentally determined amino acid sequence of the N-terminus of the purified protein. Genetic analysis also shed light on its possible role in the OST complex [138]. It was observed that overexpression of OST5, but not of genes for other OST components, specifically suppresses the ostl-5 mutation. Since the Ostl p level is normal in Ost5p-depleted strains, and no instability of OST activity is observed in vitro, the small hydrophobic protein was suggested to provide a proper membrane requirement for optimal activity and optimal OST complex assembly. The latter point is experimentally underlined by the finding that an stt3-3Aost5 double mutant is inviable. Stt3p (see below) has been shown to influence OST assembly [66]. 4.4.6 OST4 Genetic screens for temperature-sensitive mutants with a defect in glycosyl transfer to an [ 1251]-labelledpeptide acceptor [ 1391 as well as for vanadate resistant mutants [140] led to the identification of a further gene, designated OST4. It encodes an unusually small protein of 36 amino acids [141]. host4 mutants grow poorly at 30 "C and are inviable at 37 "C, demonstrating that Ost4p is necessary for growth at elevated temperatures. OST activity is greatly diminished both in vitro and in vivo [ 1411. Hydropathy analysis leads to the prediction that Ost4p lies entirely in the hydrophobic core of the membrane. Ost4p was originally not detected in any of the purified OST complexes. But recent co-immunoprecipitation data, obtained with in vivo labelled OST subunits, suggest that it is indeed a component of the enzyme [124]. The specific function of Ost4p is not clear. A five-fold increased level of Kar2p (BIP) was noted even at the permissive temperature in Aost4 pointing to a defect in protein folding [141]. Kar2p induction, however, is not a general response
related to defects in OST function. Alternatively, Ost4p may facilitate or maintain OST assembly. OST4 mutants were reported to be subject to multicopy suppression by the MEGI gene, the nature and function of which is not known [ 1411. ESTs with sequence similarities to 0ST4 exist in man and mouse. 4.4.7 OST2/DAD1
The essential OST2 encodes the 16 kDa subunit of the yeast OST [142]. It is 40'Yo identical to DADl (dejender against upoptotic death), a highly conserved protein found in many higher eukaryotes, such as man, mouse, chicken, hamster, pig. Xenopus Zurvis, C. eleyans and several plant organisms. More recently, D A D l was identified now as the 12 kDa subunit of the OST from dog pancreas by demonstrating its copurification with the other subunits [ 181. Cross-linking studies [ 181 and a yeast two-hybrid assay [143] indicate a physical interaction with OST48. DADl seems to be required for the structural integrity of the mammalian enzyme. In a temperature sensitive cell line DADl was no longer detectable upon shift to the restrictive temperature, and the steady state levels of the other three OST subunits (OST48, ribophorin I and 11) were drastically reduced [ 1441 without affecting other ER components, e.g. the translocon. Experimental evidence suggests for Ost2p a membrane topology as shown in Figure 3. Ost2p and DADl lack an N-terminal cleavable signal sequence. A third predicted hydrophobic segment at the C-terminus is probably of insufficient length and hydrophobicity to span the membrane. In the case of the mammalian DAD1, both the N- and C-terminus could be released upon mild digestion with proteinase K [ 1451. Analysis of conditional ost2 strains demonstrates a pleiotropic underglycosylation of soluble and membrane-bound glycoproteins because of a defect in OST activity. Lipid-linked oligosaccharide assembly was normal in these strains [ 1421. D A D l was originally isolated as a cDNA complementing a t s BHK cell line that dies by apoptosis at its non permissive temperature [ 1461. The loss of DADl function probably leads to apoptosis indirectly by disturbing cellular functions, what occurs when the essential process of N-linked glycosylation is impaired. Thus, inhibition of N-glycosylation by tunicamycin was also reported to induce apoptosis of HL-60 cells [ 1471. However, in another report contrary to this result, tunicamycin addition did not lead to apoptosis [ 1451; the reason for this discrepancy remains obscure at the moment. Surprisingly, the region of highest sequence identity between Ost2p and DADl extends into the predicted membrane spanning segments [ 1421, and interestingly, the point mutations interfering with Ost2p function are also located in this region. Since Ost2p lacks a significant lumenal hydrophilic extension (Figure 3), and because OST activity is located in the lumen, it was speculated that the possible function of Ost2p is less likely to catalyze the OST reaction, but rather serves a structural role in the assembly of the complex [ 1421, as has been seen for DA D l (see above). Consistent with this is also the finding that Ost2p, like Swplp, can function as an allele-specific suppressor of wbpl-2 when overexpressed. This finding may indicate a physical and functional interaction among these three subunits. This is underlined by the observation that in vivo labelling of yeast cells and
58
4 Biochemistry und Moleculur Biology
immunoprecipitation of solubilized enzyme extract using an anti-Wbpl p antibody leads to coprecipitation of Wbpl, Swpl and Ost2p [161]. Why the lack of most of the essential Ost2p in the “tetrameric” OST complex leads to an active enzyme is not clear [19]. Perhaps Ost2p is only needed for catalytic activity in vivo, but not in vitro. DAD1 homologs in Arahidopsis thaliana [148] and rice [149] were reported to rescue the ts dad1 mutant of hamster cells from apoptotic death.
4.4.8 STT3 This gene encodes an essential transmembrane protein, which is the most conserved among proteins of the OST. Homologous sequences occur also in archaea. STT3 (gaurosporine and femperature sensitive) was originally identified in a screen for temperature-sensitive yeast mutants with an increased sensitivity towards the protein kinase C ( P K C I )inhibitor staurosporine [150] and later found to be involved in protein glycosylation [151]. PKCl has been shown to have a regulatory function in cell wall biogenesis of Saccharomyces cerevisiue, and pkcl mutations result in rapid cell lysis and an altered cell wall [152-1541. Since N-glycosylation has a key function in cell wall formation, it is not surprising that STT3 was detected in an independent screen for mutants synthetically lethal in combination with whpl [66] or with Aalg.5 (leading to incomplete, non-glucosylated lipid precursor oligosaccharide) [138].Yeast STT3 encodes a 78 kDa transmembrane protein containing a cleavable signal peptide, 12 putative transmembrane segments [ 1S O ] , three potential glycosylation sites (some of which seem to be used [ 1241) and a hydrophilic C-terminus covering about one third of the protein [66, 1501. In spite of its essential function in glycosyl transfer, Stt3p was not identified as a component in previous purified OST complexes. More recently, however, co-immunoprecipitation studies using epitopetagged Stt3p or Ost3p [ 124, 1251, or analysis of the complex by blue native electrophoresis [23] clearly demonstrate that Stt3p is indeed part of the complex. One of the reason it may have escaped detection in purified complexes is that Stt3p has an anomalous gel mobility and migrates as a diffuse 60 kDa band (the calculated molecular mass is 78 kDa) that is not well resolved from Ostlp, and stains poorly with Coomassie or silver [124, 125, 231. Protein sequence comparison reveals homologies of more than 50% identity between Stt3p and eukaryotic proteins from human, mouse, zebrafish, C. eleguns, Schizosaccharomyces pombe and Schistosoma mansoni. ORFs encoding homologous proteins with a lower identity (around 25%), but again with a similar transmembrane topology, exist also in archaea: in Methanococcus junaslzii, Archaeoglobus fulgidus, Pyrococcus horikoshii and Methanobucterium thermoautotrophicum. Most interestingly, the highest similarity between the proteins is the hydrophilic C-terminal domain, the area where all temperature-sensitive stt3 mutations have been located [ 12.51. N-linked glycosylation is not restricted to eukaryotes, but was demonstrated to occur also in archaea, even though the type of linkage (Asn-Glc-Hex,, and AsnGalNAc-Hex,) is different [6, 71. A mutation in the STT3 locus influences the substrate specificity of the OST complex [66]. In the stt3-3 mutant, transfer from truncated lipid-oligosaccharides
4.5 Strirc~turalOryanizution
of
the OST Complex
59
(DolPP-GlcNAc2 or DolPP-GlcNAc2Mans) is almost completely abolished, whereas glycosylation from the full length precursor DolPP-GlcNAc2Man~Glc~ is scarcely affected. Depletion of Stt3p causes also a loss of OST activity as well as a deficiency in the assembly of the complex [66]. The incorrectly-assembled, suboptimal complex may no longer be able to accept incomplete lipid-linked oligosaccharides as substrate. Moreover, genetic interactions between STT3 and other OST components, such as O S T 3 and OST5 were reported [138]. Whereas the double mutant stt3-3Aost3 is viable and results solely in a temperature sensitive phenotype at 37 "C, a combination of .stt3-3 and Aost5 is lethal.
4.5 Structural Organization of the OST Complex Although the sequence homology between the mammalian OST subunits and their yeast counterparts in most cases amounts to only 20-30% identity, biochemical and genetic evidence indicates that the composition and organisation of the complexes, as well as the interaction of the various subunits appear to be conserved between these phylogenetically distant organisms. As pointed out, in contrast to previous findings, all genetically defined OST genes from yeast can be detected as constituents of the enzyme complex, if it is analyzed by a quick and gentle method, such as blue native electrophoresis [23]. One may predict that components lacking so far in other eukaryotic OST complexes, will be identified in the future. Studies in yeast demonstrating genetic interactions among different OST genes, either by using a high copy number suppression approach or by constructing double mutants with a synthetic phenotype have led to the suggestion that OST subunits can be sorted into three groups: 1) Ostlp-Ost5p; 2) Wbplp-Swplp-Ost2p; and 3) Stt3p-Ost4p-Ost3p-Ost6p [ 124, 125, 231. Complementary biochemical studies (chemical cross-linking, co-immunoprecipitation) indicate a direct physical interaction between Wbplp, Swplp [123], and also with Ost2p [161], as well as between Stt3p, Ost3p and Ost4p [124] or between Ostlp and Wbplp [155]. Similarly, analogous to the interaction among Wbplp. Swplp and Ost2p, a physical interaction between the respective mammalian homologs OST48, ribophorin I1 and DAD 1 was demonstrated by using the yeast two-hybrid system, and also by biochemical means [ 1431. The interaction occurred between the short cytoplasmic tail of OST48 and the N-terminal region of DADl, whereas luminal domains were found to be responsible for the interaction between OST48 and ribophorin 11. OST48 interacted also with ribophorin I, but no direct interaction was observed between the two ribophorins suggesting that OST48 may serve as a bridge. Interestingly, no glycosylation of the ribophorins was necessary for interaction. At present it is not clear, however, whether the proposed subcomplexes comprise intermediates in the assembly of a fully functional complex, or whether they are autonomous in vivo pools that may assemble into OST complexes with slightly different composition and function. Recently, three complexes for protein translocation into the ER have been identified [156] that in part share subunit components and which can be recruited to
60
4 Biochemistry and Moleculuv Biology
complexes serving different functions (i.e. SRP-dependent and SRP-independent mode of translocation). This raises the intriguing possibility that slightly different OST complexes may exist, not yet detected by currently applied analytical methods. Now that the composition of one of the most complex enzymes in nature as well as some of the interactions between subunits have been defined, further work needs to concentrate on a number of issues. These include the specific functions of the various subunits of the complex in the glycosylation process, the regulation of the enzyme and the coupling of OST to lipid-linked oligosaccharide precursor formation, to protein translocation and to protein folding. One may also predict defects in this enzyme that may lead to abnormal glycosylation as it occurs in the so-called carbohydrate deficient glycoprotein syndrome, a severe human disorder with a multisystemic clinical picture [ 1 57- 1601.
Acknowledgments This work has been supported by the Deutsche Forschungsgemeinschaft (SFB 52 l/ A l ) and by the Fonds der Chemischen Industrie. References 1. R. Kornfeld, S. Kornfeld, Annu. Rev. Biochem. 1985, 54, 631-664. 2. A. Herscovics, P. Orlean, FASEB J. 1993, 7, 540-550. 3. L. Lehle, W. Tanner, 1995, in G1ycoprotein.s (Montreuil, J., Vliegenthart, J.F.G. and Schachter, H., eds). 29a, pp 475-509 Elsevier, Amsterdam. 4. W. Tanner, L. Lehle, Biochim. Biophys. Actu 1987, 906, 81-99. 5. H. Schachter, Glycobioloqy 1991, 1. 453-461. 6. J. Lechner, F. Wieland, Annu. Rev. Biocheni. 1989, 58, 173--194. 7. M. Sumper, F. T. Wieland, 1995, in Glycoproteins (Montreuil, J., Vliegenthart, J.F.G. and Schachter, H., eds), 29a, pp 455-473 Elsevier, Amsterdam. 8. P. Messner, U.B. Sleytr, Glycobioloqy 1991, 1, 545-551. 9. T. C . Hufdker, P. W. Robbins, J. Biol. C/iem. 1982, 257, 3203-3210. 10. E. Ioffe, P. Stanely, Proc. Nut1 Acud. Sci. U S A 1994, 91, 728-732. 1 1 . M. Metzler, A. Gertz, M. Sarkar, H. Schachter, J. W. Schrader, J. D. Marth, E M B O J. 1994, 13, 2056-2065. 12. S. Strahl-Bolsinger, M. Gentzsch, W. Tanner, Biochim. Biophys. Actu 1999, 1426, 297-307. 13. A. Varki, Glycobioloqy 1993, 3, 97-130. 14. H. A. Kaplan, J . K. Welply, W. J. Lennarz, Biochim. Biophys. Acta 1987, 906, 161-173. 15. C . B. Sharma, L. Lehle, W. Tanner, Eur. J. Biochem. 1981, 116, 101-108. 16. R. C . Das, E. C. Heath, Proc. Natl Acad. Sci. USA 1980, 77, 3811-3815. 17. D. J. Kelleher, G. Kreibich, R. Gilmore, Cell 1992, 69, 55-65. 18. D. J. Kelleher, R. Gilmore, Proc. Nut1 Acad. Sci. U S A 1997, 94, 4994 4999. 19. R. Knauer, L. Lehle, FEBSLett. 1994, 344, 83-86. 20. D. J. Kelleher, R. Gilmore, J. Bid. Chem. 1994, 269, 12908-12917. 21. R. Pathak, T. L. Hendrickson, B. Imperiali, Bioc/?emistry 1995, 34, 4179-4185. 22. R. Pdthdk, B. Imperiah, Arch. Biochem. Biophys. 1997, 338, 1-6. 23. R. Knauer, L. Lehle, J. Bid. Chem. 1999, 274, 17249-17256. 24. V. Kumar, S. Heinemann, J. Ozols, J. Bid. Chem. 1994, 269, 13451-13457. 25. V. Kumar, G . Korza, F. S. Heinemann, J. Ozols, Arch. Biochem. Biopliys. 1995, 320, 217-223.
R t jerences J
61
V. Kumar, F. S. Heinemann, J. Ozols, Biochem. Biophys. Res. Comrn. 1998. 247. 524-529. W. Breuer, E. Bause, Eur. J. Biochern. 1995, 228, 689-696. S. Silberstein, R. Gilmore, FASEB J. 1996, 10, 849-858. R. Knauer, L. Lehle, Biochim. Biophys. Actu 1999, 1426, 259-273. B. Imperiali, T. L. Hendrickson, Bioorg. M e d Cheni. 1995, 3. 1565-1578. B. Imperiali, Ace. Chem. Res. 1997, 30, 452-459. T. L. Hendrickson, B. Imperiali, Biochmi~~try 1995, 34, 9444-9450. R. B. Trimble, J. C. Byrd, F. Maley, J. Biol. Chrm.1980. 255, 11892 11895. M. J. Spiro, R. G. Spiro. V. D. Bhoyroo. J. B i d Chern. 1979, 254, 7668-7674. R . J. Chalifour. R. G. Spiro, J. Bid. Cliem. 1988, 263, 15673-15680. J. L. Franc, S. Bouchilloux, Biochim. Biopliys. Acta 1984, HOI), 166-170. R. D. Marshall, Annu. Reu. Biochem. 1972, 41. 673-702. S. J. Turco, B. Stetson, P. W. Robbins. Proc. Nut1 Acad. Sci. U S A 1977, 74, 441 1-4414. L. A. Murphy, R. G. Spiro, J. Bid. C'hem. 1981, 256, 7487-7494. K. R. Anumala, R. G. Spiro, J. Biol. Chcm 1983. 258, 15274-15282. K. W. Runge, T. C. Huffaker, P. W. Robbins, J. Bid. Chem. 1984, 259, 412-417. M. F. Verostek, P. H. Atkinson, R. B. Trimble, J. Biol. Chem. 1991. 266. 5547-5551. M. F. Verostek, P. H. Atkinson. R. B. Trimble, J. Bid. Chem.1993. 268, 12095-12103. J. Stoll, R. Cacan, A. Verbert. S.S. Krag, Arch. Biochem. Biophys. 1992, 299, 225-231. E. Alvarado. T. Nukada, T. Ogawa, C. E. Ballou, Biochemistry 1991, 30, 881-886. A . J. Petrescu, T. D. Butters, G . Reiukensmeier, S. Petrescu, F. M. Platt, R. A. Dwek, M. R. Wormald, EMBO J. 1997, 16, 4302--4310. 47. E. Bause, H. Hettkamp, FEBS Lett. 1979, 108, 341-34419. 48. E. Bause, L. Lehle, Eur. J. Biocheni. 1979, 101. 531-540. 49. G. W. Hart, K. Brew, G. G. A. Grant. R . A. Bradshaw, W. Lennarz, J. B i d Chcw. 1979, 254, 9747-9753. SO. T. C. Huffaker, P. W. Robbins, Proc. Nut1 Acad. Sci. USA 1983, KO, 7466-7470. 51. P. Burda, M. Aebi, Biochim. Biophys. act^ 1999, 1426, 239-257. 52. B. J. Jackson, C. D. Warren, B. Bugge, P. W. Robbins, Arch. Biochem. Biophys. 1989. 272, 203-209. 53. B. J. Jackson, M. A. Kukuruzinska, P. W. Robbins, Glycohiology 1993, 3, 357-365. 54. M. Aebi, J. Gassenhuber, H. Domdey, S. te-Heesen, Glycohiohgy 1996, 6 , 439-444. 5 5 . S. te Heesen, L. Lehle, A. Weissmann, M. Aebi, Eur. J. Bioclzem. 1994. 224, 71-79. 56. G. Reiss, S. te Heesen, J. Zimmerman, P. W. Robbins, M. Aebi, Glycohiology 1996, 6 , 493498. 57. K. W. Runge. P. W. Robbins, J. Biol. Cliern. 1986, 261, 15582-15590. 58. P. Burda. C. A. Jacob, J. Beinhauer, J. H. Hegemann, M. Aebi, Gl.vcohioloyy 1999, Y, 617625. 59. J. Stagljar, S. te Heesen, M. Aebi, Proc. Natl Acad. Sci. USA 1994, Y l , 5977-5981. 60. P. Burda, S. te Heesen, A. Brachat, A. Wach, A. Diisterhoft, M. Aebi, Proc. Nut1 Acad. Sci. USA 1996, Y3, 7160-7165. 61. C.A. Jakob, P. Burda, S. te Heesen, M. Aebi. J. Roth, Clycohioloy~1998, 8 , 155-164. 62. L. Ballou. P. Gopal. B. Krummel, M . Tammi, C.E. Ballou, Proc. Natl Acad. Sci. USA 1986, 83, 3081-3085. 63. M. D. Munoz, L. M . Hernandez, R. Basco, E. Andaluz, G . Larriba, Biochim. Biophys. Acta 1994, 1201, 361-366. 64. R. Cueva, M. D. Munoz, E. Andaluz, R. D. Basco, G. Larriba, Biochim. Biophys. Acta 1996, 1289, 336-342. 65. R Cueva, C. Cotano, G. Larriba, Yeust 1998, 14, 773-781. 66. R. Zufferey, R. Knauer, P. Burda, 1. Stagljar, S. te Heesen. L. Lehle, M. Aebi, EMBO J. 1995, 14, 4949--4960. 67. M. Bosch, S. Trombetta, U. Engstrom, A.J. Parodi, J. Biol. Chem. 1988, 263, 17360-17365. 68. A. J. Parodi, Glycobiology 1993, 3, 193-199. 69. S. Kornfeld, W. Gregory, A. Chapman, J. Biol. Chem. 1979, 254, 9176-9177. 70. G. J. Quellhorst jr, J. L. O'Rear, R. Cacan, A. Verbert, S. S. Krag, Glycohiology 1999, 9, 6572.
26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46.
62
4 Biochemistry and Moleculur Biology
71. P. Jung, W. Tanner, Eur. J. Biochern. 1973, 37, 1-6. 72. F. W. Hemming, 1974, in Biochen?istry of Lipids (Goodwin, T.W., ed), pp 39-97, Butterworth, London. 73. T. Chojnacki, G. Dallner, Biochemistry 1988, J. 251, 1-9. 74. G. Palamarczyk, L. Lehle, T. Mankowski, T. Chojnacki, W. Tanner, Eur. J. Biochem. 1980, 105, 517-523. 75. L. Jaenicke, K. Van Leyen, H.-U. Siegmund, Biol. Chem. H o p p Seyler 1991, 372, 10211026. 76. J. S. Rush, J. G. Shelling, N. S. Zingg, P. H. Ray, C. J. Waechter, J. Bid. Chem. 1993, 268, 13110-13117. 77. C. Ronin, S. Bouchilloux, C. Granier, J. van Rietschoten, FEBS Letters 1978, 96, 179-182. 78. J. P. Miletich, G. J. J. Broze, J. Bid. Chem. 1990, 265. 11397-11404. 79. B. W. Grinnell, J. D. Walls, B. Gerlitz, J. Bid. Chenz. 1991, 226, 9778-9785. 80. K. Titani, S. Kumar, K. Takio, L.H. Ericsson, R.D. Wade, K. Ashida, K.A. Walsch, M.W. Chopek, J.E. Sadler, K. Fujikawa, Biochemistry 1986, 25, 3171-3184. 81. Y. Gavel, G. von Heijne, Protein Eng. 1990,3, 433-442. 82. E. Bause, Biochenz. J. 1983, 209, 331-336. 83. L. Kasturi, J. R. Eshleman, W. H. Wunner, S. H. Shakin-Eshleman, J. Biol. Chem. 1995,270, 14756-14761. 84. L. Lehle, E. Bause, Biochim. Biophys. Actu 1984, 799. 246-251. 85. T. Roitsch, L. Lehle, Eur. J. Biochem. 1989, 181, 525-529. 86. S. H. Shakin-Eshleman, S. L. Spitalnik, L. Kasturi, J. Bid. Chem. 1996, 271, 6363-6366. 87. J. T. Y. Lau, J. K. Welply, P. Shenbagamurthi, F. Naider, W. Lennarz, J. Biol. Clzem. 1983, 258, 15255-15260. 88. D. T. Berg, B. W. Grinnell, Protein Sci. 1993, 2, 126-127. 89. J. L. Mellquist, S. L. Kasturi, S. H. Spitalnik, S. H. Shakin-Eshleman. Biochem. 1998, 37, 6833-6837. 90. L. Kasturi, H. Chen, S. H. Shakin-Eshleman, Biochenz. J. 1997, 323, 415-419. 91. K. Nakai, M. Kanehisa, J. Biochem. 1988, 104, 693-699. 92. S. H. Shakin-Eshleman, W. H. Wunner, S. L. Spitalnik, Biochemistry 1993, 32, 9465-9472. 93. I. Nilsson, G . von Heijne, J. Bid. Chem. 1993, 268, 5798-5801. 94. C. Landolt-Marticorena. R. A. F. Reithmeier, Biochem. J. 1994, 302, 253-260. 95. I. Mononen, E. Karjalainen, Biochim. Biophys Acta 1984, 788, 364-367. 96. N. J. Bulleid, R. S. Bassel-Duby, R. B. Freedman, J. F. Sambrook, M. J. Gething, Biochein. J. 1992,286, 275-280. 97. R. W. Doms, R. A. Lamb, J. K. Rose, A. Helenius, Viro/oqy 1993, 193, 545-562. 98. A. S. Vidal, G. Mottet, D. Kolakofsky, L. Roux, J. Virol. 1989, 63, 892-900. 99. L. W. McGinnes, T. G. Morrison, Virology 1994, 200, 470-483. 100. S. Rau, R. Geyer, R. W. Friedrich, J. Gen. Viro/. 1993, 74, 699-705. 101. A . H. Andreotti, D. Kahne, J. Am. Clzem. Soc. 1993, 115, 3352-3353. 102. S. E. O’Connor, B. Imperiali, Chemistry and Biology 1998, 5, 427-437. 103. A. Imberty, S. Perez, Protein Eng. 1995, 8, 699-709. 104. B. Imperiali, K. W. Rickert, Proc. Nut1 Acud. Sci. USA 1995, 92, 97-101. 105. B. Imperiali, K. L. Shannon, K. W. Rickert, J. Am. Chem. Soc. 1992, 114, 7942-7944. 106. E. Bause, G. Legler, Biochem. J. 1981, 195, 639-644. 107. E. Bause, W. Breuer, S. Peters, Biochem. J. 1995, 312, 979-985. 108. E. Bause, M. Wesemann, A. Bartoscheck, W. Breuer, Biochem. J. 1997, 322, 95-102. 109. T. Xu, J. K. Coward, Biochemistry 1997. 36, 14683-14689. 110. J. Lee, J. K. Coward, Biochemestry 1993, 32, 6794-6801. 111. Q. Yan, G. D. Prestwich, W. Lennarz, J. Bid. Chem. 1999,274, 5021-5025. 112. C. Kellenberger, T. L. Hendrickson, B. Imperiali, Biochemistry 1997, 41, 12554-12559. 113. S. Desruisseau, A. Valette, J.-L. Franc, 0. Chabaud, 0. Mol. CeL Endocrinoll996, 122, 223228. 114. M. Konrad, W. E. Merz, J. Biol. Chem. 1994,269, 8659-8666. 115. R. Cacan, C. Villers, M. Belard, A. Kaiden, S. S. Krag, A. Verbert, Glycohiology 1992, 2, 127-136.
Rcjerences
63
116. R. Cacan, S. Duvet, D. Kmiecik, 0. Labiau, A. M. Mir, A. Verbert, Biochimie 1998. 80, 5968. 117. M. J. Spiro, R. G. Spiro, J. B i d . CIZLW.1991, 266, 5311-5317. 118. K . Lennon. R. Pretel, J . Kesselheim, S. te Heesen, M. A. Kukuruzinska, Glycohioloyy 1995, 5, 633-642. 119. G. Kreibich. B. L. Ulrich, D. D. Sabatini, J. Cell Biol. 1978, 77, 464-487. 120. E. E. Marcantonio, A. Amar-Costesec, G. Kreibich. J. Cell Biol. 1984, 99, 2254-2259. 121. Y. Yu, D. D. Sabatini, G. Kreibich, J. Cdl Bid. 1990, I l l , 1335-1342. 122. S. te Heesen, B. Janetzky, L. Lehle, M. Aebi, EMBO J. 1992, 11, 2071-2075. 123. S. te Heesen, R. Knauer, L. Lehle. M. Aebi. EMBO J. 1993, 12, 279-284. 124. D. Karaoglu, D. J. Kelleher, R. Gilmore, J. Bid. C'hrm. 1997, 272, 32513-32520. 125. U. Spirig, M. Glavas, D. Bodmer, G. Reiss, P. Burda, V. Lippuner, S. te Heesen. M. Aebi, Mol. Gen. Genet. 1997, 256, 628-637. 126. S. te Heesen, R. Rauhut, R. Aebersold, J . Abelson, M. Aebi, M. W. Clark, Eur. J. Cell Biol. 1991, 56, 8-18. 127. E. C. Gaynor, S. te Heesen, T. R. Graham, M . Aebi. S. D. Emr. J. Cell Biol. 1994, 127, 653665. 128. S. te Heesen, M. Aebi, Eur. J. Biocheni. 1994, 222, 631-637. 129. S. Silberstein, D. J. Kelleher, R. Gilmore, J . Biol. CIiem. 1992, 267, 23658-23663. 130. T. Yamagata, T. Tsuru, M. Y . Monioi, K. Suwa, Y. Nozaki, T. Mukasa, H. Ohashi, Y. Fukushima, T. Momoi, Genomics 1997, 45, 535-540. 131. S. Silberstein, P. G. Collins, D. J. Kelleher, P. J. Rapiejko, R. Gilmore, J. Cell Biol. 1995, 128, 525-536. 132. R. Pathak, C. S. Parker, B. Imperiali. FEBS Lett. 1995, 362, 229-234. 133. Y. S. Tsao, N . E. Ivessa, M. Adesnik, D. D. Sabatini, G. Kreibich, J. Cell Bid. 1992, 116. 5767. 134. D. MacGrogan, A. Levy, G. S. Bova, W. B. Isaacs. R. Bookstein, Genomics 1996, 35, 55-65. 135. J. W. Dennis, 1992. in Cell Surfuce Curbohydruntes rind Development (Fukuda. M., ed.) pp. 161-194, CRC Press, London, UK. 136. A. Kobata, 1996, in Glycoproteins und Diseuse (Montreuil, J., Schachter, H., and Vliegenthart, J.F.G., eds.), New Comprehensive Biochemistry 30, pp. 21 1-228, Elsevier Science. Netherlands. 137. D. Karaoglu, D. J. Kelleher, R. Gilmore, J. Cell B i d . 1995, 130, 567-577. 138. G. Reiss, S. te Heesen, R. Gilmore, R. Zufferey, M Aebi, EMBO J. 1997, 16, 1164-1172. 139. J. Roos, R. Sternglanz, W. J. Lennarz, Proc. Nut1 Acad. Sci. U S A 1994, 91, 1485-1489. 140. N. Dean, 1995, 92, 1287-1291. 141. J. H. Chi, J. Roos, N. Dean, J. Bid. Clietn. 1996, 271, 3132-3140. 142. S. Silberstein, P. G. Collins, D. J. Kelleher, R. Gilmore, J. Cell Biol. 1995, 131, 371-383. 143. J. Fu, M. Ren, G. Kreibich, J. Biol. Chetn. 1997, 272, 29687729692, 144. A. Sanjay, J . Fu, G. Kreibich. J. Bid. Chern. 1998, 273, 26094-26099. 145. T. Makishima, T. Nakashima, K. Nagata-Kuno, K. Fukushima, H. Iida. M. Sakaguchi, Y. Ikehara, S. Komiyama. T. Nishimoto. Genes Cells 1997, 2 , 129-141. 146. T. Nakashima, T. Sekiguchi, A. Kuraoka, K. Fukushima, Y. Shibata, S. Komiyama, T. Nishimoto, Mol. Cell. Biol. 1993, 13, 6367 -6374. 147. D. Perez-Salva, F. Mollinedo, J. Cell. Physiol. 1995, 163, 523-531. 148. P. Gallois, T. Makishima, V. Hecht, B. Despres, M. Laudie, T. Nishimoto, R. Cooke. Plunt J. 1997,II, 1325-1331. 149. Y. Tanaka. T. Makishima, M. Sasabe, Y. Ichinose. T. Shiraishi, T. Nishimoto, T. Yamada, Plant Cell Physiol. 1997, 38, 379-383. 150. S. Yoshida, E. Ikeda, I. Uno. H. Mitsuzawa, Mol. Gen. Genet. 1992, 231, 337-344. 151. S. Yoshida. Y. Ohya, M. Goebl, A. Nakano. Y. Anraku, J. Biol. C/iem. 1994, 269, 11661171. 152. D. E. Levin, E. Bartlett-Heubusch, J. C d Bid. 1992, 116, 1221-1229. 153. G. Paravicini, M. Cooper, L. Friedli, D. J. Smith. J.-L. Carpentier, L. S. Klis, M. A. Payton, Mol. Cell. Biol. 1992, 12, 4896-4905. 154. T. Roemer, G. Paravicini, M. A. Payton, H. Bussey, J. Cell B i d . 1994, 127, 567-579.
64
4 Biochemistry and Molecular Biology
155. I. Stagljar, C. Korostensky, N. Johnson, S. te Heesen, Proc. Nut1 Acud. Sci. USA 1998, 95, 5187-51 92. 156. T. A. Rapoport, B. Jungnickel, U. Kutay, Annu. Rev. Biochem. 1996, 65, 271-303. 157. J. Jaeken, H. Carchon, H. Stibler. Clycobiology 1993, 3, 423-428. 158. J. Jaeken, H. Schachter, H. Carchon, P. De Lock. B. Coddeville, G. Spik, Arch. Dis. Child. 1994, 71, 123-127. 159. C. Korner, R. Knauer, U. Holzbach, F. Hanefeld, L. Lehle, K. von Figurd, Proc. Natl Acad. Sci. USA 1999, 95, 13200-13205. 160. P. Burda, L. Borsig, J. de Rijk-van Andel, R. Wevers, J. Jaeken, H. Carchon. E. F. Berger, M. Aebi, J. Clin. Innest. 1999, 102, 647-652. 161. Knauer, Lehle, unpublished.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
5 Processing Enzymes Involved in the Deglucosylation of N-Linked Oligosaccharides of Glycoproteins: Glucosidases I and I1 and Endomannosidase Robert G. Spiro
5.1 Introduction Subsequent to the discovery that dolichol-linked oligosaccharides are involved in the N-glycosylation of proteins in eukaryotic cells [ 11, it became apparent that the carbohydrate portion of these molecules contains peripheral residues of glucose linked to polymannose-di-N-acetylchitobiose[2]. Further structural investigations demonstrated that the glucose residues occur in a trisaccharide sequence [3] in which a-glycosidic bonds link the constituents to each other, as well as to the polymannose unit [4] as shown in Figure 1. It moreover became evident that these glucose constituents are essential for the cotranslational en bloc transfer of the oligosaccharide to asparagine in the Asn-XSer (Thr) constellations in the polypeptide chain [ 5 , 6, 71. Since glucose is generally not observed in mature N-linked oligosaccharides, their presence in newly glycosylated proteins prompted the suggestion that trimming enzymes which can remove these saccharides must be present as components of the intracellular processing apparatus [S]. Indeed, reports from a number of laboratories soon indicated that a-glucosidases which can cleave the glucose residues from the N-linked parent oligosaccharide occur in microsomal fractions from various sources [4, 9, 101. Subsequently it became apparent that two distinct processing a-glucosidases coexist in the endoplasmic reticulum (ER) which release the terminal a 1 1 2 and the two more internal wl43-linked glucose residues, respectively [ 1I], and were accordingly designated as glucosidase I and glucosidase 11 (Figure 1). Concurrently or subsequently, ER and Golgi situated a-mannosidases trim the N-linked oligosaccharide to ManSGlcNAcI [12] which in turn is acted upon by a number of glycosyltransferases to yield the large variety of complex carbohydrate units which have been observed in eukaryotes. In some instances, however, mannose excision is limited and untrimmed or incompletely trimmed polymannose oligosaccharides can be found in the mature glycoprotein, as in the polymannose oligosaccharides of thyroglobulin or the hybrid units of ovalbumin. In any case, glucose removal is a pre-
66
5 Processing Enzymes Involved in the Deglucosylution
-
a1-2
Man Glc
- --
k"
Glucosidase II
\
a1-2
Ma+l-8
ala+lan Mana-l;? Man
h !
Glucosidase I Glc
Glc
Glc
a13
/
Glc
Man 'GlcNAc
\
a13
a1-2
Man -Man
a1-2
-.+
01-4
--GlcNAc
A -Asn
413
Man
Endomannosidase
\
Glc (af-3) Man
Figure 1. Trimming enzymes involved in the deglucosylation of asparagine-linked oligosaccharides. The sites of action and the products released are shown.
requisite to the formation of the vast array of N-linked oligosaccharides which are present on membrane and secreted glycoproteins. In 1987 an endomannosidase was discovered [13] which can bring about deglucosylation by a quite distinct mechanism based upon the unusual capacity of this enzyme to effect an internal cleavage between the glucose-substituted mannose and the remainder of the polymannose oligosaccharide with the release of G l c a l j 3 M a n (Figure 1). Indeed, by its endoglycosidase action this processing enzyme differs from the other trimming enzymes which operate by a sequential release of monosaccharide residues. The wide distribution of the enzyme in members of the chordate phylum provides an alternate processing route which is particularly important in glucosidase-inhibited or glucosidase-deficient cells [ 141. It is the aim of this review to describe the enzymatic and molecular properties as well as the intracellular location of these three glucose-removal enzymes (see Table 1) as well as to describe their physiological role when acting in concert with the other processing enzymes in normal cells and in mutants. Moreover, this Chapter will evaluate some recent studies which indicate that monoglucosylated polymannose N-linked oligosaccharides may play an important role in the quality control of proteins due to their interaction with chaperones. The biological consequences of glucosidase inhibition in regard to protein degradation and viral replication will also be examined.
5.2 Glucosidase I The a-glucosidase I which is the first processing enzyme to act on the triglucosylated polymannose oligosaccharides by specifically cleaving the a1 t 2 linked glucose residue (Figure 1) has been observed in all eukaryotes examined, ranging from yeast to higher mammals as well as plants. Under physiological circumstances the enzyme acts with great speed and during pulse-chase radiolabeling of intact tissues or cells, triglucosylated N-linked oligosaccharides (Glc3Man9GlcNAcz) can
5.2 Glucosiduse I
67
Table 1. Properties of processing glycosidases involved in the removal of glucose from N-linked oligosaccharides of glycoproteins”. Property
Glucosidase I
Predominant action Subcellnlar location Subunit Mr (kDa) Cation requirement pH optimum Inhibitors‘ Deficient mutant‘ N-Linked carbohydrate N-Glycosylation consensus sequenceg Ph ylogenetic distribution Source of purified enzyme
None 6.5-7.0 DNJ, CST, N-Me & N-Bu-DNJ Lec 23 (CHO) glsl-1 (S. cerevisiae) Yes
Glucosidase I1
Endomannosidase
G2 I M Y + M ~
GI M9-StMX-4 IC, cis/medial Golgi 56
ER, 1C‘ u 100-123 p 58--80* None 6.5-7.0 DNJ, CST, MDL
None
7.0 Glcul+3DMJ
PHAR2.7 (mouse lymphoma) Yes
No
One
Two
None
Eukaryotes
Eukaryotes
Chordates’
Liver, mammary gland, mung bean, yeast
Liver, kidney, mammary gland T-cells, soybean, yeast Yes Pig and rat liver, mouse T-cells’ CHO cells Other glucohydrolases
Rat liver
Cloned Clone library
Yes Human hippocampus,
Expressed Sequence homology
COS- 1 None
(U)h
None
Yes Rat liver
E. coli None
For references to information in this table see text. The abbreviations for the predominant reaction are G, glucose; M, mannose. All oligosaccharides terminate in a di-N-acetylchitobiose sequence by which they are linked to Asn in the peptide chain. ‘ZC, intermediate compartment refers to the membranes interposed between the ER and Golgi compl ex. The values given refer to recent reports which indicate that the enzyme has an a catalytic peptide chain as well as a P-subunit [34, 351. Several other reports have not identified the latter polypeptide. Moreover, early studies underestimated the M, of the catalytic subunit due to proteolytic cleavage during isolation [37]. Abbreviations for inhibitors are DNJ, I-deoxynojirimycin; CST, castanospermine; N-Me and NBu, A‘-methyl and N-butyl-DNJ, respectively; M D L , 2,6-dideoxy-2,6-imino-7-O-(~-~-glucopyranosy1)-D-glycerol-L-glucoheptitol;DMJ, 1deoxymannojirimycin. The entry in parenthesis refers to the cell type in which the mutation has been observed. Refers to the Asn-X-Ser(Thr) sequence. Present on the a-subunit. i In addition to the various classes of the phylum Choru’utu, the enzyme has been observed in Mu/luscu [42]. ’The a-subunit has also been identified in the genome of yeast and man [34]. a
68
5 Processing Enzymes Involced in the Deglucosylation
only fleetingly be detected [15, 161. As measured in vitro, the enzyme acts optimally on oligosaccharides which are not processed by mannosidase action beyond the Glc3MangGlcNAc2 stage [ 111. The enzyme is believed to be an ER-situated integral membrane protein with a transmembrane anchor and a luminally oriented catalytic domain and a short cytosolic tail [ 17, 181. Glucosidase I has been purified from several tissues including calf and pig liver [ 19, 201, bovine mammary gland [21], mung bean seedlings [22] and Saccharomyces cereuisiae [23] utilizing various chromatographic procedures, among which the affinity matrix containing the N-carboxypentyl-1 -deoxynojirimycin proved to be the most useful [19-211. There is general agreement that the enzyme consists of a single polypeptide chain in the 85-97 kDa molecular mass range [ 17-22], which may associate into a tetramer [21]. Several reports indicate that the enzyme contains a single N-linked oligosaccharide, which on the basis of its endo-P-Nacetylglucosaminidase H (endo H) susceptibility, appears to be of the polymannose type [17, 18, 20, 221, as would be anticipated in an ER residential protein. Indeed, a single N-glycosylation site has been identified at Asn 655 in the cloned enzyme from human hippocampus, which became substituted with an endo H-labile oligosaccharide when expressed in COS-I cells [18]. A comparison of the glucosidase I cDNA from human hippocampus with the cDNA of other processing enzymes revealed no homology [18]. The enzyme has no metal requirements and can be effectively inhibited by a number of agents (Table 1) which however also block the action of glucosidase I1 to various extents [24]. The in vivo effect of the glucosidase inhibitors on glycoprotein processing is only partially effective due to the alternate deglucosylation pathway provided by endomannosidase (see below). Since the glucosidase I does not act on aryl a-glucosides, its assay has routinely relied on the use of radiolabeled Glc3Man9GlcNAc as substrate followed by quantitation of the cleavage products subsequent to their separation by various thin layer, paper, or column chromatographic procedures [17-23, 251. In these assays it is important to measure the GlczMan9GlcNAc product, as the excised monosaccharide could be a product of the combined action of glucosidase I and contaminating glucosidase 11. A spectrophotometric assay for glucosidase I has been reported in which Glcal + 2Glcal + ~ G ~ C - O - ( C H ~ ) ~ C O is OC used H ~as a substrate, but since this procedure only measures glucose release it also cannot be used when the additional presence of glucosidase I1 is suspected [26].
5.3 Glucosidase I1 The second a-glucosidase in the usual processing sequence has the capacity to cleave both a1+3 linked glucose residues despite the fact that these two inner residues are attached to glucose and mannose respectively (Figure 1). This dual action of the enzyme has been attributed to two distinct active sites [27]. Glucosidase 11 has strong specificity for the oligosaccharide with untrimmed mannose branches (Glc2-1MansGlcNAcz) [ 111. This interaction of the enzyme with the 6’-
69
5.3 Gliccosiduse II
Table 2. Comparison of the specificities of rat liver ghcosidase 11, endomannosidase and calreticulin”. Oligosaccharide
Glucosidase 11
Glcl MangGlcNAc Glcl ManzGlcNAc Glcl Man7GlcNAc Glcl ManbGlcNAc Glc, MansGlcNAc
100 21
9 5 3
Endomannosidase
Relative activity” I00 I20 150 ND‘ 220
Calreticulin
100 81
63 62 62
Glucosidase I1 values are taken from [ 1I], while the data for endoniannosidase is derived from [41] and that for calreticulin was obtained by [46]. GlqMan4GlcNAc was a good substrate for the endomannosidase but had no affinity for calreticulin. Relative glycosidase (glucosidase I1 and endomannosidase) and binding (calreticulin) activities. Not determined.
pentamannosyl branch of the substrate determines that excision of even one mannose residue results in a steep fall in activity (Table 2). In vivo the Glc2ManqGlcNAcz is a very transient intermediate, while substantial amounts of the monoglucosylated polymannose oligosaccharide accumulate [ 15, 161. Although this finding suggests that the innermost glucose is removed much slower than the more peripheral residue, this interpretation is complicated by the possibility that the UDP-g1ucose:glycoprotein glucosyltransferase can regenerate Glcl Mans GlcNAcz from the deglucosylated polymannose oligosaccharides [28]. Glucosidase I1 has been shown by immunoelectron microscopy of hepatocytes to be located in the ER and transitional elements between the ER and Golgi complex [29], which have more recently been ascribed to the intermediate compartment [30]. Furthermore, the enzyme has been detected in the nuclear envelope, but not in the Golgi apparatus [29, 301. In contrast to glucosidase I, it has become evident that the glucosidase I1 is a soluble luminal or loosely associated membrane enzyme [ 3 1, 321 and indeed its catalytic subunit as determined from its cDNA does not contain a transmembrane domain, nor has an ER retention signal been found [33-351. The enzyme has been detected in all eukaryotic cells which have been examined, including yeast and higher plants and has been purified from rat 134. 36, 371 and pig [33]liver, bovine mammary gland [38], pig kidney [31] and mouse T-cells [35]. Early studies on the purified enzyme from rat liver [36] as well as bovine mammary gland [38] reported that the catalytic subunit had a molecular mass of about 65 kDa and indicated that this occurred in the form of a tetramer. However, a subsequent study on rat liver indicated that the enzyme has an M, of 123 kDa which readily undergoes proteolytic cleavage to a 73 kDa fully active fragment [37]. The importance of including protease inhibitors during isolation of glucosidase 11 was emphasized in this investigation [37]. Indeed more recent reports of the enzyme isolated from pig kidney [31]. mung bean seedlings [39], mouse T lymphocytes [35] and rat [34] as well as pig [33] liver have assigned M, values of 100-1 16 kDa to the a-glucosidase 11 catalytic subunit. Furthermore, studies on rat liver [34] and mouse T lymphocytes
70
5 Processing Enzymes Involved in the Deglucosylution
1351 have indicated that the glucosidase I1 is a heterodimer in which the catalytic subunit (a) is associated with another polypeptide (subunit P) with M, values of 58 kDa in liver and 80 kDa in T-lymphocytes. It has been proposed that this noncatalytic subunit may promote interaction of the enzyme with other proteins, such as calnexin and calreticulin or that it is responsible for the ER localization of the enzyme [34, 351; the latter possibility is supported by the fact that the P subunit has an HDEL retention signal, which is a feature not found on the a-chain 134, 351. Glucosidase I1 like glucosidase I is believed to contain a single endo Hsusceptible and concanavalin A-reactive N-linked oligosaccharide [31-40]. From its pig liver cDNA a single N-glycosylation site has been identified close to the amino terminus of the enzyme [ 331, while two potential glycosylation consensus sequences have been reported to occur in the rat liver a-subunit, as inferred from its cDNA 1341. The enzyme has been cloned from rat liver [33, 341 and mouse T-lymphocytes [35] and these studies have indicated that the catalytic subunit has no transmembrane domain nor ER retention signals, which is consistent with a soluble protein. Although glucosidase I1 shows no homology to glucosidase I, it manifests a high degree of homology with other glucohydrolases, including lysosomal a-glucosidase, sucrase-isomaltase and the yeast family 3 1 glucosidases [33-351. Gene disruption experiments indicated that a S. cevevisiae gene is a functional homologue of mammalian glucosidase I1 [34]. The P-subunit of the liver glucosidase I1 showed no sequence homology to other known proteins [34, 3.51. In view of the broad homology of the catalytic subunit of glucosidase I1 it is not surprising that this enzyme, in contrast to glucosidase I, readily cleaves aryl a-glucosides and this property has been widely employed to assay this enzyme. However, this facile approach of measuring the enzyme has its pitfalls since the presence of non-processing a-glucosidases which are widely distributed could give misleading results unless a highly purified ER membrane is being assayed. The use of readily prepared radiolabeled Glcl MangGlcNAc as substrate [25, 381 can provide more definitive results. Glucosidase I1 has no cation requirements and a number of effective inhibitors of its activity are available (Table 1) but all of these agents also block glucosidase I action to various extents [24]. Of the various inhibitors currently available, castanospermine (CST) has proven to be highly effective in both in vitro and intact cell studies. In the latter case, as already mentioned, the glucosidase blockade is usually incomplete due to the presence of an alternate processing route provided by endomannosidase.
5.4 Endo-a-mannosidase The internal scission which endomannosidase brings about by cleaving the a1 4 2 linkage between the glucose substituted mannose and the remainder of the 3’trimannosyl branch of the N-linked oligosaccharides (Figure 1) gives it a unique place among the processing glycosidases. Although the primary physiological
5.4 End~-~-munnosiduse
71
action of the enzyme is to act on monoglucosylated oligosaccharides with the release of Glcul-t3Man, it also has the capacity to remove Glcal +3Glcal+3Man and Glcal+2Glcul+3Glca1+3Man from di- and triglucosylated polymannose species, respectively [41]. On the basis of differential and sucrose density centrifugation, it was determined that the endomannosidase of liver is present in the Golgi complex [ 131 and this has been recently confirmed by immunoelectron microscopy which demonstrated its localization in the cislmedial cisternae of this apparatus [ 301. The latter studies, moreover indicated that endomannosidase also occurs in the intermediate compartment but in contrast to the glucosidases it is not present in the ER [30]. Furthermore, it became apparent from confocal microscopy and double immunogold labeling of hepatocytes that although endomannosidase and glucosidase I1 are both present in the intermediate compartment, the two enzymes are spatially separated in these vesiculo-tubular elements [30]. The distribution of endomannosidase is limited to the phylum Chorduta, including placental and marsupial mammals, birds, reptiles, amphibians and fish, with the single exception of Molluscu, where it has been detected in three distinct classes [42]. This late evolutionary appearance of the enzyme. in contrast to the processing glucosidases, may reflect the more prominent biological role which complex oligosaccharides take on in higher organisms. The presence of an alternate processing pathway, in addition to the highly conserved glucosidase trimming route, would insure that no incompletely deglucosylated oligosaccharides are present on cell surfaces or secreted glycoproteins. Furthermore, as discussed below, the endomannosidase may be a component of a more sophisticated protein quality control machinery. The enzyme has been found in all mammalian cell lines so far assayed 125. 42-44] with the sole exception of Chinese hamster ovary cells (CHO) [25]. Purification of endomannosidase from rat liver Golgi membranes by affinity chromatography on a Glccll+3Man-O-(CH2)8CONH-Affi-Gel matrix yielded a polypeptide doublet (M, 60 kDa and 56 kDa) upon examination by SDS-PAGE in which the two components were present in approximately equal amounts [45]. Subsequent studies revealed on the basis of sequence analyses and immunoreactivity that the larger of the two components was calreticulin while all the enzyme activity was present in the 56 kDa component [46]. The copurification of the endomannosidase with the chaperone on a matrix containing the G l c a l i 3 M a n ligand suggested a biological relationship of the components which will be discussed (see below). The endomannosidase has recently been cloned from a rat liver cDNA library and the deduced open reading frame was found to encode a protein of 451 amino acids which corresponded to a molecular mass of 52 kDa [47]. Homology with other processing glycohydrolases or indeed any other known protein was not evident [47]. No N-glycosylation consensus sequence was identified and indeed, consistent with the latter, it was found that the electrophoretic mobility of the endomannosidase was not affected by N-glycanase treatment [47]. The enzyme was effectively expressed in Escherichiu coli yielding a protein which could be purified to homogeneity in high yield by affinity chromatography with a specific activity which was actually several fold higher than that obtained from rat liver Golgi membranes [47]. Indeed, since E. coli does not contain processing glycosidases, a lysate of these
72
5 Processing Enzymes Involved in the Deglucosylation
cells after transfection manifests endomannosidase as the sole enzyme with a capacity to trim N-linked oligosaccharides. Northern blotting revealed the presence of endomannosidase mRNA in all rat tissues examined [47]. Endomannosidase like the other deglucosylating enzymes has no cation requirements and moreover its activity is unaffected by any of the a-glucosidase or amannosidase inhibitors. By preparing a number of Glcal+3Man derivatives, several potent inhibitors of endomannosidase have been found [25] among which Glcal+3( 1-deoxy)mannojirimycin was the most effective ( I C ~= O 1.7 pM). While this inhibitor could block endomannosidase action in vitro as well as in some intact cell lines [25], in the latter case the possibility that the disaccharide could be cleaved by endogenous a-glucosidases necessitated the inclusion of CST in the medium. The assay of endomannosidase is routinely carried out in buffers other than Tris, which is highly inhibitory [41], with radiolabeled Glcl MangGlcNAc as substrate in the presence of glucosidase and mannosidase inhibitors [e.g., CST and 1-deoxymannojirimycin (DMJ)] followed by the thin layer chromatographic separation and quantitation of the released G l c a l i 3 M a n component [13, 25, 411; no inhibitors are of course required in the purified or E. coli expressed enzyme. Since concanavalin A-sepharose binds the polymannose product and substrate, but not the released disaccharide, assays can be carried out using the immobilized lectin, but the reliability of this procedure has not been found to be adequate when dealing with unexplored and unfractionated membrane systems. Recently it has been observed that the cloned enzyme can be used as an effective tool in assessing the glucosylation of radiolabeled glycoproteins or oligosaccharide lipids, since endomannosidase cleaves these constituents irrespective of whether their polymannose oligosaccharides are in the tri-, di-, or monoglucosylated state [69].
5.5 Concerted Action of Deglucosylation Enzymes Since the requirement of a triglucosyl sequence (Figure 1) on the donor oligosaccharide-lipid is a clear requirement for N-glycosylation in almost all eukaryotes, subsequent glucose removal becomes a necessity for the production of the vast array of complex oligosaccharide units which occur on these cells. The enzymes iniolved in these early trimming reactions consequently play an important role and indeed function efficiently, since under normal conditions glucose residues are not seen as constituents of N-linked oligosaccharides. It may even be conjectured that since glucose is not a constituent of either complex or polymannose N-linked carbohydrate units, it commends itself as a recognition signal for a variety of biological events. While the predominant deglucosylation route is believed to be mediated by the sequential action of glucosidase I and glucosidase 11, it has become apparent that trimming of mannose residues, instead of being relegated to the Golgi apparatus as it was initially believed, commences in the ER through the action of resident a1,2mannosidases [ 121 which at least in mammalian cells can extensively degrade the 6’-
5.5 Concerted Action of Deglucosylation Enzyrnes
73
pentamannosyl branch of the polymannose oligosaccharide. Since glucosidase I1 acts very poorly on monoglucosylated oligosaccharides which have been trimmed by a-mannosidase action (Table 2), these species would become suitable substrates for endomannosidase which in fact favors carbohydrate units with truncated mannose chains (Table 2). Indeed, in any case, endomannosidase which is situated distal to the location of the processing glucosidases would remove any glucose which had escaped scission by the latter enzymes. It has been calculated that in mouse lymphoma cells under normal conditions, about 16% of the total deglucosylated oligosaccharides are generated by endomannosidase action [48]. Numerous studies with cultured cells from various sources have indicated that in the presence of glucosidase inhibitors, such as CST, formation of complex N-linked oligosaccharides ranging from 10Y0to 700/0of normal (see [ 141) continued to occur. Since deglucosylation is an essential prelude to the formation of these mature carbohydrate units, the existence of an alternate glucosidase-independent processing pathway, for which endomannosidase is ideally suited, became evident. Indeed studies on HepG2 cells [ 141 indicated that in the presence of CST there was a release of glucosylated mannose oligosaccharides (primarily Glc3Man) which closely correlated on a molar basis with the number of N-linked complex oligosaccharides produced (Figure 2). Since the latter accounted for about 50% of the complex carbohydrate units produced under normal conditions, it is clear that the endomannosidase route can accommodate a substantial traffic [ 141. As anticipated
J COMPLEX
Figure 2. Pathways for glucose removal from N-linked triglucosylated polymannose oligosaccharides in normal, glucosidase-inhibited and glucosidase-deficient cells. Glucosidase I and I1 as well as endomannosidases can participate in the deglucosylation of normal cells. When the action of the two glucosidases is inhibited by an agent such as castanospermine ( C S T ) endomannosidase provides the only route for glucose removal. In the mouse lymphoma glucosidase 11-deficient mutant cell line (PHAR 2.7) endomannosidase circumvents the block subsequent to the release of the terminal glucose residue. Deglucosylation permits the formation of complex carbohydrate units. The abbreviations used are G, Glc; M , Man; and GN, GlcNAc.
74
5 Processing Enzymes Involved in the Deglucosylation
from the internal cleavage action of endomannosidase, Man9 GlcNAc was not present among the polymannose processing intermediates in the glucosidaseinhibited cells (Figure 2) and indeed in the presence of a mannosidase inhibitor, Mans GlcNAc and to a lesser extent Man~GlcNAcand MansGlcNAc, originating from Glq-1MangGlcNAc, Glq-1MangGlcNAc, and GIq-1Man~GlcNAc,were observed [14]. The endomannosidase pathway has now been identified in a number of cells and shown to lead during CST-induced glucosidase blockade to various endo H-resistant serum glycoproteins secreted by HepG2 cells [49] as well as the endo H-resistant G protein in several vesicular stomatitis virus (VSV)-infected cell lines [44]. It has been shown moreover that the addition of Glca1-3DMJ to CSTtreated cells inhibited the alternate pathway so that glycoproteins with complex endo H-resistant N-linked oligosaccharides ceased to be formed 1251. While it has been reported that the processing glucosidases can act on lipid-linked oligosaccharides in vitro 14, 501, evidence has emerged through studies in thyroid slices that these enzymes may mediate a glucosyltransferase-glucosidase shuttle in vivo which helps to control the level of dolichyl triglucosylated polymannose available for the N-glycosylation of proteins 1511.
5.6 Mutants A glucosidase 11-deficientmouse lymphoma cell line (PHAR2.7)has been described [ 521. Although the predominant N-linked oligosaccharides from these cells were in the diglucosylated form (GlczMan9GlcNAcz), a substantial portion (35-40%) were processed to complex oligosaccharides. It was demonstrated [ 5 3 , 541 that the endomannosidase pathway was responsible for circumventing the genetically determined glucosidase blockade with the release of GlczMan, as shown in Figure 2. As in the CST-inhibited cells, Mans GlcNAc was not present among the processing intermediates (Figure 2). A CHO cell line deficient in glucosidase I activity has been reported 1551. The observation that this mutant (Lec23) was highly resistant to wheat germ agglutinin and yet hypersensitive to concanavalin A suggested a block in the formation of N-linked complex oligosaccharides and indeed in radiolabeled VSV-infected cells, triglucosylated polymannose oligosaccharides were found to be the predominant carbohydrate attached to the G protein 1551. The addition of DMJ furthermore resulted in the presence exclusively of Glc3Man9GlcNAcz. Quite small amounts of complex oligosaccharides was reported to be present in these cells, but their occurrence is unlikely to be attributable to the alternate endomannosidase pathway as CHO cells are quite unique among cultured mammalian cells in not having detectable activity of this enzyme, as evaluated either in vitro or whole cell assays 125, 441. More specifically, endomannosidase assays of Lec23 postnuclear membranes were found to be negative and furthermore, subsequent to [ 3sS]methionine labeling, the G protein of VSV migrated to the same position on polyacrylamide gel electrophoresis as the one from CST-treated wild type cells; this G protein was sensitive to
5.7 Role of Monoylucosyluted N-Linked Oligosucchurides
75
in vitro treatments with either endo H or endomannosidase as would be anticipated if it occurred in an unprocessed state 1691. A glucosidase I deficient mutant has also been found in yeast [56]. Although this S. cevevisiue glsl-1 mutation results in the production of glycoproteins containing N-linked oligosaccharides with the intact triglucosyl sequence, the presence of these glucose residues does not appear to interfere with the formation of mature carbohydrate units. The defect in glucose excision in yeast does not result in the profound processing block which is noted in mammalian cells as in the former only the terminal mannose of the middle chain has to be removed to permit the addition of outer chains [57]. No significant effect on growth properties have been noted in either the glsl-1 or the Lec23 mutants [ 5 5 , 561.
5.7 Role of Monoglucosylated N-Linked Oligosaccharides and Glucose Trimming Enzymes in Regulating Quality Control of Glycoproteins In recent years important new insights into the biological role of glucose residues on N-linked oligosaccharides have emerged. The first evidence for a regulatory role of the glucose components on the fate of newly formed glycoproteins came from a number of in vivo studies with cultured liver cells indicating that inhibitors of glucosidase I and I1 action, which keep the triglucosyl sequence intact, delayed secretion of various glycoproteins and/or promoted their degradation in an ERassociated site [49, 58, 591. Indeed it was noted that the reduction in secretion of various serum glycoproteins by HepG2 cells in the presence of CST was directly related to their number of N-linked oligosaccharides [49]. A more definitive indication of the effect of glucose trimming inhibition on the degradation of glycoproteins was observed in the CMT-cKd1 cell line which produces unassembled major histocompatibility complex (MHC) class I molecules which never reach the Golgi compartment. Upon exposure of these cells to CST the MHC heavy chains underwent a rapid degradation during a 60-min chase in contrast to control cells in which they remained stable during this period [60]. These findings of the effect of CST on MHC class I molecules were also noted in BW thymoma cells [61] and moreover it was noted that the a-subunit of T-cell antigen receptor in these cells underwent accelerated degradation in the presence of the glucosidase inhibitor and indeed also in the mutant PHAR2.7 [62, 701. The molecular basis of the instability of proteins with an untrimmed triglucosyl sequence began to become apparent from studies which indicated that the ER-situated transmembrane chaperone, calnexin, selectively associates itself in a transient fashion with incompletely folded glycoproteins produced by HepG2 cells [63]. In a series of reports, Helenius and coworkers (for review see [64]) provided evidence that the association between an ER-situated chaperone, whether calnexin or calreticulin, and a glycoprotein represents at least initially a lectin-like interaction with monoglucosylated N-linked polymannose oligosaccharide (i.e., GlqMansGlcNAc) on the latter molecule. The transient nature of this interaction and its role in protein quality control was clarified by a model in which
76
5 Processing Enzymes Involved in the Deylucosylution
Calreticulin .......... ............... ................ ...................
.............. ..........
Fhdomannosidase
-------
A
E m Calreticulin
Folded
w
B
Figure 3. Schematic representation of the role by which trimming of monoglucosylated N-linked oligosaccharides can regulate the interaction of glycoproteins with calreticulin during quality control. In the model shown in scheme B, which is based on the studies of Helenius and coworkers [64] the ER-situated monoglucosylated N-linked oligosaccharide with as yet untrimmed mannose residues interacts with the chaperone and subsequently undergoes a cyclic deglucosylation by glucosidase 11 and reglucosylation by UDP-G1c:glycoprotein glucosyltransferase until folding of the polypeptide chain has been achieved, at which point the latter enzyme ceases to function. In scheme A , which is based on the work of Spiro et al. [46]. variable degrees of trimming of the 6’-pentamannosyl branch has occurred by ER and Golgi mannosidases so that the specificity of glucosidase IT precludes its action. This more processed glycoprotein-calreticulin complex then enters or is formed in the intermediate and Golgi compartments where endomannosidase is situated. This enzyme readily cleaves even extensively trimmed monoglucosylated oligosaccharides with the release of G l c a l i 3 M a n ( G - M ) and thereby dissociates the glycoprotein-chaperone complex. If the protein has properly folded it can continue to traverse the secretory channels, while if it remains incompletely folded it will undergo degradation. The closed squares).( represent mannose residues while the open squares (0) indicate mannose which in various degrees has been excised by ER and Golgi mannosidases.
glucosidase I1 works in tandem with the UDP-G1c:glycoprotein glucosyltransferase to bring about deglucosylation followed by reglucosylation until proper folding and/or oligomerization has taken place (Figure 3, scheme B). The termination of this cycle in this proposal is brought about by the observation that the ER-situated glucosyltransferase acts solely on denatured substrates [28]. Binding studies with radiolabeled oligosaccharides indicated that calnexin [65] and calreticulin [46] had a high specificity for monoglucosylated oligosaccharides; indeed, the finding that the tri- and di-glucosylated polymannose units completely failed to bind to these chaperones provided a basis for understanding the results of
5.8 Effect qf Glucosiclase Inhibitors on Viral Proliferation
77
the in vivo studies in which accelerated degradation of glycoproteins was noted in the presence of glucosidase inhibitors. Examination of a series of monoglucosylated oligosaccharides in which the mannose residues of the 6’-pentamannosyl branch had been trimmed indicated that the lectin-like binding activity of calreticulin was retained to a substantial extent (Table 2). Excision of the a1+6 linked mannose from GlcIMansGlcNAc however resulted in a complete loss of interaction with the chaperone [46]. Since it is known that glucosidase I1 acts very poorly on trimmed monoglucosylatcd oligosaccharides (Table 2) it would appear that the chaperone-glycoprotein disassociation mechanism depicted in Scheme B of Figure 3 would not be effective if excision of mannose residues from the 6’-pentamannosyl branch had taken place through the action of the ER mannosidases. Since endomannosidase, however. has the distinct capacity to cleave even extensively trimmed monoglucosylated carbohydrate units (Table 2) it has been proposed [46] that this enzyme could readily achieve this disassociation when the glycoprotein-chaperone complex reaches the intermediate compartment or &/medial Golgi location where this glycosidase is known to be located (Scheme A, Figure 3 ) . This model was prompted by the finding that endomannosidase and calreticulin can be copurified in a approximately 1 : 1 ratio from rat liver Golgi membranes by affinity chromatography on a G l c a l b 3Man-containing matrix [45]. Furthermore it has recently been shown by immunomicroscopy that calreticulin, while primarily present in the ER, is also situated in the intermediate compartment and the cislmedial Golgi stacks [30], which is consistent with its properties as a soluble chaperone and its known role in intracellular trafficking. Indeed it was demonstrated that endomannosidase and calreticulin colocalize in these more distal compartments [ 301. The proposed dissociative capacity of endomannosidase in the more distal location would make it possible for properly folded or oligomerized glycoproteins to continue their journey through the secretory channels while glycoproteins which had not reached their mature configuration would be susceptible to degradation with the release of their oligosaccharides into the cytosol [66].
5.8 Effect of Glucosidase Inhibitors on Viral Proliferation A number of reports have appeared which indicate that several glucosidase I and I1 blocking agents can function as inhibitors of human immunodeficiency virus (HIV) proliferation and syncytium formation (for review see [67]). These antiviral effects which were noted when the glucosidase inhibitors were added to infected cultured cells have however not been observed with some other viruses and this might be attributable to the unusually high degree of N-glycosylation which is a characteristic of the HIV gp 120 envelope glycoprotein [67]. Retention of the outer two glucose residues of N-linked oligosaccharides of viral envelope glycoproteins would interfere with their association with the lectin-like chaperones, calnexin and calreticulin, and this could lead to their enhanced degradation due to improper folding or oli-
78
5 Processing Enzymes Involved in the Deglucosylution
gomerization. Indeed it has been shown that a variety of envelope glycoproteins including HIV gp 160 bind to calreticulin and/or calnexin [64, 681. Since it has been reported that viral glycoproteins (e.g. the G protein of VSV) can be processed by the endomannosidase pathway in many cells [44], it can be conjectured that the maximum effect of a glucosidase blockade on viral replication should be evident in cells in which the glycoprotein under study is not a substrate for this alternate deglucosylation route or if an endomannosidase inhibitor (e.g. Glcal+3DMJ) is added to the infected cells in addition to the glucosidase blocking agent. Acknowledgments
The author’s studies were supported by Grants DK17325 and DK17477 from the National Institutes of Health. References 1. Behrens, N.H., Parodi, A.J., and Leloir, L.F. (1971) Proc. Nut/ Acud. Sci., U.S.A., 68, 28572860. 2. Spiro, R.G., Spiro, M.J., and Bhoyroo, V.D. (1976) J. Biol. Chern. 251, 6409-6419. 3. Li., E., Tabas, I., and Kornfeld, S. (1978) J. Biol. Chem. 253, 7762-7770. 4. Spiro, R. G., Spiro, M. J., and Bhoyroo. V. D. (1979) J. Bid. Chem. 254, 7659-7667. 5. Turco, S.J., Stetson, B., and Robbins, P.W. (1977) Proc. Nut1 Acud. Sci., U.S.A., 74, 44114414. 6. Spiro, M. J., Spiro, R. G., and Bhoyroo, V. D. (1979) J. Biol. Chem. 254, 7668-7674. 7. Murphy, L. A,, and Spiro, R. G. (1981) J. Biol. Chem. 256, 7487-7494. 8. Spiro, M. J., Spiro, R. G., and Bhoyroo, V. D. (1976) J. Biol. Chem. 251, 6400-6408. 9. Ugalde, R.A., Staneloni, R.J., and Leloir, L.F. (1979) Biochem. Biophys. Res. Commun. 91, 1 174-1 181. 10. Chen. W.W., and Lennarz, W.J. (1978) J. Bid. Chem. 253, 5780-5785. 11. Grinna, L. S., and Robbins, P.W. (1980) J. B i d Cllenz. 255, 2255-2258. 12. Daniel, P.F., Winchester, B., and Warren, C.D. (1994) Glycobiology 4, 551-566. 13. Lubas, W.A., and Spiro, R.G. (1987) J. Bid. Chem. 262, 3775-3781. 14. Moore, S.E.H., and Spiro, R.G. (1990) J. Biol. Chern. 265, 13104-13112. 15. Godelaine, D., Spiro, M. J., and Spiro, R. G . (1981) J. B i d Chem. 256, 10161-10168. 16. Hubbard, S.C., and Robbins, P.W. (1979) J. Biol. Chem. 254, 4568-4576. 17. Shailubhai, K., Pukazhenthi, B.S., Saxena, E.S., Varma, G.M., and Vijay, I.K. (1991) J. Biol. Chem. 266, 16587-16593. 18. Kalz-Fuller, B., Bieberich, E., and Bause, E. (1995) Eur. J. Bioclzern. 231, 344-351. 19. Hettkamp, H., Legler, G., and Bause, E. (1984) Eur. J. Biochem. 142, 85-90. 20. Bause, E., Schweden, J., Grass, A,, and Orthen, B. (1989) Eur. J. Biochem. 183, 661-669. 21. Shailubhai, K., Pratta, M.A., and Vijay, I.K. (1987) Biochem. J. 247, 555-562. 22. Zeng, Y.C., and Elbein, A.D. (1998) Arch. Biochem. Biophys. 355, 26-34. 23. Kilker, R.D., Jr., Saunier, B., Tkacz, J.S., and Herscovics: A. (1981) J. Biol. Chem. 256, 52995303. 24. Elbein, A.D. (1991) FASEB J. 5, 3055-3063. 25. Hiraizumi, S., Spohr, U., and Spiro, R.G. (1993) J. Biol. Chem. 268, 9927-9935. 26. Neverova, I., Scaman, C.H., Srivastava, O.P., Szweda, R., Vijay, I.K., and Palcic, M.M. (1994) Anal. Biochern. 222, 190-195. 27. Alonso, J.M., Santa-Cecilia, A,, and Calvo, P. (1991) Biochem. J. 278, 721-727. 28. Sousa, M.C., Ferrero-Garcia, M.A., and Parodi, A.J. (1992) Biochemistry 31, 97-105.
RLJ'erences
79
29. Lucocq, J.M., Brada, D.. and Roth, J. (1986) J. Cell Bid. 102, 2137-2146. 30. Zuber, C., Spiro, M.J., Guhl, B., Spiro, R.G., and Roth, J. Manuscript submitted for publication. 31. Brada, D., and Dubach, U.C. (1984) Eur. J. Biochem. 141, 149-156. 32. Strous, G.J, Van Kerkjof, P., Brok, R., Roth, J., and Brada, D. (1987) J. Bid. Chern. 262, 3620 ---362S. 33. Flura, T., Brada, D., Ziak, M. and Roth, J. (1997) Glycobiology 7, 617-624. 34. Trombetta, E.S.. Simons, J.F., and Helenius, A. (1996) J. Biol. Cliem. 271, 27509-27516, 35. Arendt, C.W., and Ostergaard, H.L. (1997) J. Biol. Chem. 272, 13117-13125. 36. Burns, D.M., and Touster, 0 . (1982) J. B i d . Clienz. 257, 9990- 10000. 37. Hino, Y., and Rothman, J.E. (1985) L3ioclieinistr.y 24, 800-805. 38. Saxena, S., Shailubhai, K., Dong-Yu, B., and Vijay, I.K. (1987) Biochem. J. 247, 563-570. 39. Kaushal. G.P., Pastuszak, I., Hatanaka, K.; and Elbein, A.D. (1990) J. Biol. Chem. 265, 16271-16279. 40. Saunier, B., Kilker, R.D., Jr., Tkacz, J.S., Quaroni, A,, and Herscovics, A. (1982) J. B i d . Cheni. 257, 14155-14161. 41. Lubas, W.A., and Spiro, R.G. (1988) J . Biol. Clirm. 263, 3990-3998. 42. Dairaku, K., and Spiro, R.G. (1997) G/~vcobiology7, 579-586. 43. Weng. S., and Spiro, R.G. (1993) J. Rid. Chem. 268, 25656-25663. 44. Karaivanova, V.K., Luan, P., and Spiro, R.G. (1998) Glycohiology 8, 725-730. 45. Hirdizumi, S., Spohr, U., and Spiro, R.G. (1994) J. Biol. Chem. 269, 469774700, 46. Spiro, R.G., Zhu, Q., Bhoyroo, V, SBling, H.-D. (1996) J. Biol. Cheni. 271, 11588-11594. 47. Spiro, M.J., Bhoyroo, V.D., and Spiro, R.G. (1997) J. Biol. Chem. 272, 29356-29363. 48. Weng, S., and Spiro, R.G. (1996) Glyc,ohzo/oyy 6, 861-868. 49. Rabouille, C., and Spiro, R.G. (1992) J. Biol. Chem. 267, 11573-11578. 50. Eking, J.J.. Chen, W.W., and Lennarz, W.J. (1 980) J. Biol. Chen7. 255, 2325--233I . 51. Spiro, M.J., and Spiro, R.G. (1991) J. Bid. C/iem. 266, 5311-5317. 52. Reitman, M.L., Trowbridge, I.S., and Kornfeld, S. (1982) J. Bid. Cliem. 257, 10357-10363, 53. Fujimoto, K., and Kornfeld, R. (1991) J. B i d . Clzem. 266, 3571-3578. 54. Moore, S.E.H., and Spiro, R.G. (1992) J. Biol. Chern. 267, 8443-8451. 55. Ray, M.K., Yang, J., Sundaram, S., and Stanley, P. (1991) J. Biol. Chen7. 266, 22818-22825. 56. Esmon. B., Esmon, P.C., and Schekman, R. (1984) J. Biol. Chem. 259, 10322-10327. 57. Herscovics, A,; and Orlean, P. (1993) FASEB J . 7, 540-550. 58. Lodish, H.F., and Kong, N. (1984) J. Cdl Bid. 98, 1720-1729. 59. Parent, J.B., Yeo, T.-K., Yeo, K.-T., and Olden, K. (1986) Mol. Ce//. L3iocht.m. 72, 21-33. 60. Moore, S.E.H., and Spiro, R.G. (1993) J. Riol. Chem. 268, 3809-3812. 61. Balow, J.P., Weissman, J.D., and Kearse, K.P. ( 1 995) J. Biol. Chem. 270, 29025-29029. 62. Kearse, K.P.. Williams, D.B., and Singer, A. (1994) E M B O J. 13, 3678-3686. 63. Ou, W.-J., Cameron, P.H.. Thomas, D.Y., and Bergeron, J.J. (1993) Nature 364, 771-776. 64. Helenius, A,, Trombetta, E.S., Hebert, D.N., and Simons, J.F. (1992) Tretid~sCell Biol. 7. 193200. 65. Ware, F.E., Vassilakos, A,, Peterson, P.A., Jackson, M.R., Lehrman, M.A., and Williams, D.B. (1995) J. Biol. Clzem. 270, 4697-4704. 66. Moore, S.E.H., and Spiro, R.G. (1994) J. Bid. C/iern. 269, 12715-12721. 67. Feizi, T., and Larkin, M. (1990) G/ywhio/ogy 1, 17-23. 68. Otteken, A,, and Moss, B. (1996) J. Bid. C h m . 271, 97-103. 10 (in press). 69. Spiro, M.J., and Spiro, R.G. (2000) Gl,ycohio/o(gj~ 75. Karaivanova, V.K., and Spiro, R.G. (2000) G/iwhio/ogy 10 (in press).
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
6 a-Mannosidases in Asparagine-linked Oligosaccharide Processing and Catabolism Kelley W. Moremen
6.1 Overview The maturation of Asn-linked oligosaccharides is initiated in the ER and continues in the Golgi complex by the trimming of the Glc3MangGlcNAcz oligosaccharide structure down to a Man3 core that is necessary for maturation to complex type oligosaccharides. Several distinct enzymes are required for the trimming steps including several glucosidases and several mannosidases. Mannosidase activities are also found in other compartments of the cell, including the cytosol and lysosomes, where they are involved in glycoprotein and oligosaccharide catabolism. A comparison of the biochemical characteristics and protein sequences of the mannosidases involved in glycoprotein maturation and catabolism has revealed that they can be divided into two families with several subgroups within each family. Two subgroups of the Class 1 mannosidases are found in the ER and Golgi where they are involved in early trimming events in oligosaccharide processing. One of the other Class 1 mannosidase subgroups are comprised of secreted fungal mannosidases of unknown function. The last of the Class 1 mannosidase subgroups is conserved from yeast to mammals, but members of thus subgroup also have no known function. The Class 2 mannosidases have members that are found in the ER, Golgi, cytosol, and lysosomes. Four subgroups have been identified with distinctive functions in glycoprotein biosynthesis or catabolism. One of the subgroups of Class 2 mannosidases has members conserved between eubacteria, archaea, and eukaryotes. This review summarizes the biochemical and sequence relationships between the members of the two mannosidase classes and their roles in glycoprotein biosynthesis and catabolism.
82
6 a-Mannosiduses in Asparugine-linked Oliyosucchuvide Processing
6.2 Introduction 6.2.1 Roles of N- and 0-Linked Glycans and Compartmentalization of Biosynthetic and Catabolic Reactions Protein-linked carbohydrate structures are among the most complex and diverse set of post-translational modifications on intracellular and secreted proteins. Large numbers of intracellular and extracellular proteins contain N-linked oligosaccharides including enzymes, cell surface receptors, secreted proteins, hormones, immunoglobulins, and viral antigens [I]. Although there are many examples of the contribution of N-glycans to the bioactivity, folding, localization, and immunogenicity of the attached polypeptide [2], the functional roles of individual oligosaccharide structures on a given glycoprotein are difficult to predict. At the cellular level, N - and O-glycan structures have been shown to contribute to several types of biological recognition events including cell adhesion during development, immune surveillance, inflammatory reactions, hormone action, viral infection, arthritis, and metastasis of oncogenically transformed cells [ 1, 3-51, The pathways leading to the synthesis and catabolism of glycoproteins are highly compartmentalized in eukaryotic cells [6-81. Biosynthesis of cellular and secreted glycoproteins occur in the membranes of the secretory pathway including the ER, the Golgi complex, secretory vesicles, and the transport vesicles between these compartments. Glycoprotein catabolism was originally considered to be the exclusive domain of the endosomal/lysosomal system [9], but recent data have indicated that a considerable amount of glycoprotein degradation also occurs in the cytoplasm as a result of a failure in glycoprotein folding in the ER [lo]. Glycoproteins that fail to fold properly are translocated from the lumen of the ER into the cytosol where the protein portion is degraded by the proteosome. The released oligosaccharides are partially cleaved in the cytosol and further degraded in the lysosome. Thus, the compartmentalization of the synthesis and catabolism of glycoproteins into discrete and non-overlapping regions of the cell allows an independent regulation of the two pathways. 6.2.2 Processing of Asn-Linked Oligosaccharides
The synthesis and maturation of N-linked oligosaccharides occurs in four stages in the membranes of the ER and Golgi complex and several of these stages are completely, or at least partially, conserved in all eukaryotic organisms. The first stage of the pathway involves the synthesis of the precursor oligosaccharide by the stepwise addition of monosaccharides to the polyisoprenoid lipid, dolichol (summarized in Chapter 3 in this volume), with the early reactions occurring on the cytoplasmic face of the ER and the later reactions occurring on the luminal face of the membrane [ 11, 121. The final Glc3MangGlcNAc;!-P-P-dolichol structure is conserved in all eukaryotic organisms, with the only known exceptions being the Trypanosoma-
6.2 Introduction
83
tids which have been shown to synthesize and transfer non-glucosylated lipid-linked oligosaccharide structures [ 13, 141. The second stage of oligosaccharide biosynthesis is the transfer of the lipid-linked precursor to the polypeptide through the action of a highly conserved multi-subunit oligosaccharide transferase complex (Figure 1, OST [ 151) that recognizes Asn-X-Ser/Thr sequons on newly synthesized polypeptides as they emerge on the luminal side of the ER membrane (summarized in Chapter 4 in this volume). The third stage of N-linked oligosaccharide maturation involves the trimming of the transferred oligosaccharide and invariably involves the removal of all three glucose residues (Glc I and Glc I1 in Figure 1) and a variable degree of mannose trimming [7, 16, 171. In all eukaryotic organisms examined thus far, with the possible exception of the fission yeast Schi~osaccharomycespombe [ 181, mannose trimming is initiated in the ER by the removal of a single mannose residue to produce a specific MangGlcNAcz isomer (ManxGlcNAcz isomer B in Figure 1) structure. In Saccharomyces cerevisiae, the single mannose removal is the sole mannose cleavage step [ 171, whereas oligosaccharide processing in vertebrate organisms results in the removal of six of the original nine mannose units [7]. The glucose trimming steps are summarized in Chapter 5 in this volume, while the trimming of mannose residues will be the subject of this review. The final stage of oligosaccharide maturation involves the branching and extension of the oligosaccharides by Golgi glycosyltransferases [ 19,201 (summarized in numerous Chapters in this volume). The final elaboration of N-glycans is the most varied stage in eukaryotic organisms and is the major cause of the extreme diversity in mature oligosaccharides between organisms, between different cells in the same organism, and between different glycoproteins in the same cell [20]. The conserved nature of the early steps in the pathway for the synthesis of Nglycans suggests an important role for the mature lipid-linked oligosaccharide and the trimmed intermediates in the efficient N-glycosylation and maturation in the ER. Sequence similarities have been noted between the enzymes involved in the synthesis of the lipid-linked precursor oligosaccharide in eukaryotes, the enzymes for glycoconjugate synthesis in archea, and the enzymes catalyzing lipopolysaccharide biosynthesis in eubacteria [ 111. Indeed, archea have also been found to synthesize glycoproteins containing Asn-linked oligosaccharides attached to Asn-XSer/Thr sequons that are generated as lipid-linked precursors [211. In eukaryotes, the full Glc3MangGlcNAc2 oligosaccharide structure is strongly preferred for efficient transfer to protein acceptor sites [ 111 suggesting that full glucosylation may act as a signal for recognition by the oligosaccharide transferase. Smaller oligosaccharide structures can be transferred to proteins in yeast [22] and mammalian cells [23], either as a result of mutations in the enzymatic machinery for the synthesis of the glucosylated oligosaccharide or as a potential alternative pathway for oligosaccharide transfer, as in mouse teratocarcinoma cells [24], but the efficiency of transfer is significantly compromised. The pathological consequences of genetic defects in the synthesis of the full-length lipid-linked oligosaccharide have been observed in human patients with type I CDGS [25] (see below), where incomplete lipid-linked oligosaccharide synthesis leads to an underglycosylation of cellular and secreted glycoproteins.
9
B1,4-Man
Legend
fi1,4-Gal o2.6-NeuNAc
I
I
Glycoprotein Catabolism
Cytosol
Man
Figure 1. The biosynthesis and catabolism of glycoproteins in mammalian cells. The glycosidases and glycosyltransferases involved in glycoprotein maturation and degradation are indicated by a bold solid arrow with the following enzyme abbreviations: OST, oligosaccharide transferase; Glc I, glucosidase I; Glc TI, glucosidase 11, Glc T, UDP-G1c:glycoprotein glucosyltransferase; ER Man I, ER mannosidase I; ER Man 11, ER mannosidase TI (the ER form of the cytosolic/ER mannosidase 11); Endo a-Man, endo a-mannosidase; Golgi Man IA/IB, Golgi niannosidases IA and IB; GnTI, GlcNAc transferase I; Golgi Man 11, Golgi mannosidase IT; GnTII, GlcNAc transferase 11; Golgi Man III/IIx (??), either a-mannosidase 111or Golgi mannosdiase IIx; Cytosolic Man, Cytosolic mannosidase (the cytosolic form of Cytosolic/ER mannosidase 11); Lysosomal Man, broad specificity lysosomal a-mannosidase; Lys ul.6-Man, lysosomal ul,6-mannosidase; Lys P-Man, lysosomal P-mannosidase. Positions where processing inhibitors can act to block enzyme reactions are indicated by a thin arrow with the following abbreviations: dMNJ, 1-deoxymannojlrimycin; Kif, kifunensine; Sw, swainsonine. The legend for the oligosaccharide structures displayed in the figure is indicated in the lower left. Solid boxes indicate the membrane boundaries of the ER, Golgi, and lysosomes as labeled in the figure. The plasma membrane in indicated by the bold dotted line.
Plasma Membrane
. I '
: 99
-: p @l,4-GlcNAc 9 ul,2-Glc : 4 o1.3-Glc
=-
..I
Glvcom-otein Biosvnthesis
6.2 Introduction
85
6.2.3 Early Trimming Events: importance for quality control glycoprotein degradation and anteriograde transport The transfer of the Glc3MangGlcNAcl structure to protein is followed by the rapid and immediate removal of the glucose residues from the oligosaccharide by glucosidases I and I1 (Figure 1) 17, 16, 171. A role for glucosylated oligosaccharides has been proposed as a part of the quality control process of glycoprotein folding in the ER 126-281. Proteins that are glycosylated as they are extruded into the lumen of the ER undergo a concerted folding process that is facilitated by the interaction with a variety of molecular chaperones including BiP, GRP94, calnexin, and calreticulin 1291. The latter two chaperonins have been shown to contain a lectin activity and initiate their interaction with unfolded glycoproteins by specifically binding to Glcl MangGlcNAcz oligosaccharides 1261. Subsequent interactions between the chaperonin and the polypeptide have been proposed to promote polypeptide folding. Correctly folded glycoproteins are released from calnexin or calreticulin through the action of glucosidase I, which removes the last glucose residue, and the glycoconjugate will then proceed through the secretory pathway to the Golgi complex and other parts of the cell. Proteins that have not folded correctly will remain transiently associated with the chaperonins until glucosidase I removes the last glucose residue. Unfolded glycoproteins in the ER containing MangGlcNAcz structures can then be acted upon by UDP-G1c:glycoprotein glucosyltransferase to replace the Glc residue to the same position and linkage where it was removed by glucosidase I1 130, 311, thus allowing the protein to re-bind to the chaperonins. Since the glucosyltransferase recognizes only unfolded glycoprotein intermediates, it has been proposed that the enzyme acts as the “sensor” of the folding status of the newly synthesized glycoproteins [ 3 I ] . The cycle of binding to the chaperonins, sugar cleavage by glucosidase 11, release from the chaperonins, sugar addition by the glucosyltransferase, and re-binding to the chaperonins can proceed through multiple rounds in an effort to effectively fold the newly synthesized glycoproteins [26]. Under normal conditions glycoproteins that fail to fold after a defined period of time are targeted for translocation into the cytosol via the Sec6lp translocon complex followed by degradation by the cytosolic proteosome [32, 331. While many of the components involved in glycoprotein folding have been identified, the components that recognize glycoproteins which have failed the folding process and target them for degradation are not well understood. It was originally proposed that mannose trimming could act as a timing step for the quality control cycle [ 341 by interrupting one or more steps in the glucosylation/de-glucosylation cycle and targeting glycoproteins with trimmed oligosaccharides for degradation. Several lines of evidence now indicate that mannose trimming, especially to a specific Man8GlcNAc2 structure (isomer B), plays an important role in targeting unfolded proteins for degradation. The role of a mannose cleavage step in targeting the degradation of a mutant form of carboxypeptidase Y in S. cerruisiur has been shown by blocking the formation of the MangGlcNAcz isomer B structure. When glucose or mannose trimming was abrogated through gene disruptions in the processing w-glucosidases or ER mannosidase I, or when the synthesis of the lipid-linked
86
6 a-Munnosidases in Asparagine-linked Oligosaccharide Processing
precursor was blocked by gene disruption of the oligosaccharide biosynthetic enzymes, misfolded carboxypeptidase Y accumulated in the ER [22, 351. In mammalian cells, the degradation of a misfolded variant form of a,-antitrypsin [36, 371 or the T cell receptor subunit CD3-6 [38] was blocked by treatment of the cells with DMJ indicating the involvement of ER mannosidases. Treatment of cells with kifunensine, a selective inhibitor of ER mannosidase I but not ER mannosidase 11, also blocked the degradation of a1 -antitrypsin implicating a requirement for the formation of the MangGlcNAc2 isomer B structure by ER mannosidase I in targeting glycoproteins for degradation [37]. It is noteworthy that ER mannosidase 1 is the last step in oligosaccharide processing that is fully conserved from yeast to mammals. In S. cerevisiue oligosaccharide structures are extended into mannan structures after the single mannose cleavage step by ER mannosidase I [17, 391. In mammals, trimming to the MansGlcNAq isomer B structure is followed by further mannose trimming and extension into complex type structures [7]. ER mannosidase I does not appear to be essential for growth in S. cerevisiue [40] or extension of mannan structures (411, but evidence described above indicates that it does appear to be involved in the timing of glycoprotein folding [22]. Inhibition of ER mannosidase 1 and subsequent Golgi trimming by treatment of mammalian cells in culture with kifunensine does not appear to cause alterations in cell morphology or growth, but does have an effect on the rate of degradation of unfolded glycoproteins in the ER [36, 371. Two models have been proposed for the recognition and degradation of unfolded glycoproteins containing the MansGlcNAcz isomer B structure. In one model, the MansGlcNAcl isomer B structure can be acted upon by the glucosyltransferase to make Glcl MansGlcNAcz, but the product may be cleaved less efficiently by glucosidase I1 [37]. This would lead to a prolonged interaction with calnexin/calreticulin and potential targeting to the Sec6lp translocon. An alternative model proposes that an, as of yet unidentified, lectin may recognize the MansGlcNAc2 isomer B structure in the context of an unfolded protein to target the glycoconjugate for translocation [22]. In each model discrimination between folded and unfolded substrates must be made and a low efficiency of recognition of the oligosaccharide on unfolded proteins would be predicted since transiently unfolded nascent polypeptides and correctly folded glycoproteins should not be targets for translocation and degradation. Thus, the conservation of early processing events in oligosaccharide maturation appears to contribute to the quality control recognition processes in the ER, and these processes are independent of the roles that the oligosaccharides may play in later compartments of the secretory pathway. A role for high mannose structures has also been proposed for facilitating glycoprotein transport through the secretory pathway. ERGIC-53, a protein that had previously been identified as a marker for the intermediate compartment between the ER and Golgi, has recently been found to act as a binding protein specific for high mannose oligosaccharides [42, 431. This protein has been hypothesized to mediate glycoprotein transport between the ER and Golgi complex through a direct interaction with high mannose oligosaccharides on newly synthesized glycoproteins [43]. A dominant mutant form of the protein that is retained in the ER has been expressed in HeLa cells and results in the mislocalization of cathepsin C, while other
6.2 Intvoduction
87
lysosomal and cell surface glycoprotein markers were unaffected [44]. A human genetic disease characterized by a combined deficiency in coagulation factors V and VIII has recently been shown to be linked to mutations that result in truncated or “null” expression of ERGIC-53 [45, 461. The levels of these coagulation factors are 5% and 30% of normal, respectively, in the serum of affected patients indicating that ERGIC-53 may be involved in the transport of a subset, but presumably not the majority, of glycoproteins through the secretory pathway.
6.2.4 Glycoprotein Catabolism: multiple routes for glycoprotein breakdown Lysosomes contain a wide range of proteolytic and glycosidic enzyme activities for the full degradation of glycoprotein substrates to monosaccharides and amino acids. At least three potential transport routes converge in the lysosome for the final degradation of glycoprotein oligosaccharides. Two routes, including classical uptake of glycoproteins by receptor mediated endocytosis and autophagy result in the delivery of substrates to lysosomes by vesicle-mediated fusion or engulfment 191. In contrast, soluble oligosaccharides can gain direct access to the lumen of the lysosome by transport from the cytoplasm. Free or protein-associated oligosaccharides can be released into the cytosol from the ER, cleaved to smaller structures, and transported directly into lysosomes via an ATP-dependent transporter in the lysosoma1 membrane [47, 481. Soluble oligosaccharides can be produced from a variety of sources in the ER. These sources include: 1) the release of Man5GlcNAc2-P from Man5GlcNAcz-P-P-Dol on the cytosolic face of the ER membrane by phosphodiesterases [lo]; 2) the release of Glc3-oMang-8GlcNAc2 by the hydrolysis of Glc3MansGlcNAc2-P-P-Do1, presumably through the transfer to water in an oligosaccharide transferase reaction 149, 501, and subsequent export of the oligosaccharide into the cytoplasmic compartment via an ATP- and calcium-requiring process [51]; and 3) the translocation of unfolded glycoproteins through the Sec61 translocon complex into the cytoplasm [33, 521 where they are acted upon by a cytosolic peptide-N-glycosidase 153, 541 to yield Mans-8GlcNAcl oligosaccharide structures [ 10, 551 and degradation of the protein component by the cytoplasmic proteosome. Evidence has also been presented for the release of oligosaccharides in the lumen of the ER by an endo-p-N-acetylglucosaminidasein thyroid microsomal membranes, but the in vivo requirements for substrate recognition are unknown 1491. Cleavage of the core of these soluble oligosaccharides to a single GlcNAc residue is accomplished by a cytosolic neutral chitobiase 156, 571 followed by the selective cleavage of the mannose residues to a distinctive Man5GlcNAcl isomer by a cytosolic neutral mannosidase [ 5 8 ] (see below). The sequential nature of the cleavage pathway is indicated by the requirement for cleavage to a GlcNAcl core prior to the mannose cleavage steps in the cytoplasm [58]. Thus, the routes for oligosaccharide catabolism can come from cellular uptake, autophagy, or through release of oligosaccharides from lipid or glycoprotein intermediates in the ER. The pathways for biosynthesis and degradation of glycoproteins diverge in the ER based on the folding-based decision of quality control. Folded glycoproteins which are no longer
88
6 a-Munnosiduses in Aspurugine-linked Oligosucchuride Processing
recognized by the glucosyltransferase, disengage from calnexin/calreticulin in the ER, and are packaged for anteriograde transport at exit sites in the ER for transport down the secretory pathway to the Golgi complex. Unfolded glycoproteins are cleaved to a Glcl-oMan&lcNAcz isomer B structure and, in a process that is still not understood, engage the Sec6lp complex for export from the ER into the cytoplasm for degradation.
6.2.5 Consequences of Genetic Defects in Oligosaccharide Biosynthesis and Catabolism The physiological importance of maintaining the balance of the biosynthetic and catabolic pathways can most effectively be seen in the variety of human genetic diseases where defects in the enzymes involved in either glycoprotein synthesis or degradation cause disruption of the respective pathway. In the heterogeneous collection of human genetic diseases characterized by the defective expression or targeting of lysosomal enzymes, termed lysosomal storage diseases, the enzyme defects cause an extreme accumulation of the normally degraded substrates [9]. The pathologic consequences of the oligosaccharide storage material are commonly a variety of neural, skeletal, and immune system defects that can result in death before the age of ten. In contrast to the severe clinical consequences of lysosomal storage diseases, genetic heterogeneity of the enzymes involved in glycoprotein biosynthesis can have varied consequences [20, 591. The enzymes that are involved in the maturation of N-linked and 0-linked glycans can generally be divided into two classes, those that are involved in the maturation of the oligosaccharide core and those that generate the terminal oligosaccharide capping structures. Genetic heterogeneity in the enzymes that generate the terminal capping reactions on N- and 0-glycans is surprisingly common in the human population, as evidenced by the variability in the carbohydrate-based blood group substances on cellular and secreted glycoconjugates [ 191. Animal models of genetic defects in some of the terminal transferases have been shown to cause a variety of immune system defects [60-621, yet these enzymes are not usually required for early development. Gene disruption in the enzymes catalyzing terminal capping reactions commonly results in the formation of alternate capping structures or compensation by other enzymes with a similar specificity. A hypothesis has been presented that the genetic and structural variation in terminal glycosylation reactions may reflect the genetic remnants of a selection process for resistance to natural pathogens by interrupting pathogen interaction with cell surface glycoconjugates [ 11. More severe pathological effects are commonly correlated with genetic defects in enzymes that are involved in the synthesis of the glycoprotein core. GlcNAc transferase I appears to be essential for mammalian development since mice lacking the enzyme die by day 10.5 in embryonic development [63, 641. A heterogeneous collection of genetic diseases termed carbohydrate-deficient glycoprotein syndrome (CDGS) [65] are caused by defects in either the synthesis of the lipid-linked precursor of N-linked oligosaccharides (type I CDGS[66]) or the early maturation of
6.3 Munnosiduses in GIj.coproteirz Processing und Catubolism
89
the glycoprotein core (GlcNAc transferase I1 deficiency in type I1 CDGS[67]). The outcome is a collection of multisystemic diseases with major nervous system involvement. In contrast, HEMPAS disease is a heterogeneous genetic disease characterized in some individuals by a genetic defect in Golgi mannosidase TI, a n enzyme that acts between GlcNAc transferases I and 11. Patients with this enzyme defect have relatively mild symptoms of anemia and mild secondary tissue effects [68, 691. Analysis of glycoprotein structures derived from patient samples [69] and tissues isolated from a mouse model for the Golgi mannosidase I1 deficiency [70] indicate that there is a previously undetected backup pathway for the trimming to the Man3 core. Multiple routes for oligosaccharide processing may also be available in the early glucose and mannose trimming steps. No naturally occurring genetic diseases have been identified involving the early oligosaccharide trimming steps indicating that each of these steps are either essential for development or that the overlapping specificities of alternate enzymes mask the pathological effects of an enzyme deficiency. There can also be an overlap in the processing of glucose and mannose residues between the exo-glucosidases (glucosidase I and 11), an endomannosidase activity that excises the terminal glucose residues along with an underlying mannose residue. and the collection of exo-mannosidases that lead to the trimming down to the MansGlcNAcr core [ 161. Thus, early steps in the synthesis, transfer, and trimming of N-linked oligosaccharides appear to be critical for normal development and physiology of vertebrate organisms, at least in the few cases where non-overlapping specificities can be demonstrated.
6.3 Mannosidases in Glycoprotein Processing and Catabolism The major goal of this review is to describe the most recent literature on the role of inannose cleavage reactions in oligosaccharide maturation and catabolism in mammalian systems. Reference will also be made to glycoprotein processing in other organisms when relevant. Several previous reviews have described the biochemistry and molecular biology of the processing and catabolic mannosidases in fungal and vertebrate systems [6, 7, 16, 17, 391 and additional relevant details and references may be found in these sources.
6.3.1 Classification of Mannosidases Mannosidases involved in glycoprotein maturation and catabolism have been divided into three broad classes based on their distinctive substrate specificities, responses to inhibitors, cation requirements, protein molecular weights, subcellular localizations, and enzyme mechanisms (Table 1) [7, 161. This classification, based on biochemical characteristics, has been reinforced by the sequence comparison of a growing number of family members (Figure 2). Two of the mannosidase classes are exo-glycosidases, cleaving mannose residues from the non-reducing terminus of the oligosaccharide. The third mannosidase class cleaves an internal glycosidic
al,2-Man
ER Man I1 al,2-Man
Golgi Man IA a1,2-Man
Golgi Man IB a1,3/1,6-Man
Golgi Ma nII ?
Golgi ManII"
DMJ, KIF None 6.5 Mouse, Human. Pig
Class 1
DMJ, DIM
pNP-Man
6.5 Rat
Class 2
DMJ, KIF
None
6.5-7.0 Human
Class 1
Caf2
Golgi
ER None
ER Ca+'
Class 1
6.5 Mouse, Human
None
DMJ, KTF
Golgi Ca+'
Class 2
5.6 Mouse, Human
pNP-Man
Sw, Manstat
Golgi None
Class 2
Human, Mouse
?
Lysosomal Man
Cytosol Co+' activates
116 kDa
Class 2
6.0 Rat
Class 2
4.5 Mouse, Human, Cat, Cow
pNP-Man
sw
Lysosomes None
111 kDa
retaining
Man~GlcNAc?" Manpl,4 GlcNAc
a1,2/1,3/1.6-Man al,2/1.3/1,6-Man
Cytosolic Man
DMJ, DIM, Sw(poor) pNP-Man pNP-Man
sw
Golgi None
MansGlcNAcl MansGlcNAcz ManSGlcNAcz* Man5GlcNAc2 * GlcNAcMan3- ? GlcNAcz inverting inverting (yeast) 82 kDad 76 kDa 13 kDa 73 kDa 131 kDa I29 kDa
al,Z-Man
ER Man I
"The nomenclature and abbreviations for the enzyme names as well as the references for the indicated characteristics are found in the text. Hypothesized in vivo limit digestion products. KIF, kifunensine; DMJ, deoxymannojirimycin; DIM, I ,4-imino-1,4-imino-D-mannitol; Sw, swainsonine, Manstat, mannostatin. Subunit Mr for ER mannosidase I1 is based on the SDS-PAGE mobility of the protein detected in an ER membrane fraction by Western blotting [ 1061. *Golgi Man IA cleaves the ManRB isomer to MansGlcNAcz while it cleaves MangGlcNAq to MansGlcNAc2 and with low efficiency to MansGlcNAcz. # Cytosolic Man cleaves ManTGlcNAc to a ManSGlcNAc isomer that is distinctive from the digestion product of Golgi Man IA or IB.
Synthetic substrates pH optimum Cloned mammalian cDNAs or genes Mdnnosidase Class
specificity Limit digestion product Glycosidase mechanism Subunit M, (not including glycosylation) Localization Metal requirement Inhibitors'
Linkage
Characteristics
Table I . Mammalian Processing and Catabolic a-Mannosidases"
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(BAA758171
Figure 2. Dendrograms for members of the two mannosidase classes. Full length members of the Class 1 and Class 2 mannosidase families that are present in the GenBank sequence database were identified by BLAST searches (194) and the sequences were edited to reflect the conserved core sequence in each family (-440-510 amino acid region for Class 1 mannosidases and -180 amino acid region for Class 2 mannosidases). Multiple sequence alignments were performed using the ClustalW program (195) and a neighbor-joining tree was generated within this program using 1000 bootstrap trials. The tree was displayed as a radial unrooted tree using the program Treeview (196). Sequence and species designations and their corresponding GenBank accession numbers (in parentheses) are indicated at the end of each branch. Those sequences that have quotation marks in the title indicate sequences that are proposed to have the corresponding activity based on sequence similarity to other members of the subgroup, but the catalytic activity has not been demonstrated by direct enzyme assays. Those sequences that have only gene designations, but no putative mannosidase designation in the title (as indicated by a "Man" in the title) are genes with unknown function. The outer oval with the indicated labels show the designations of the subgroups within the Class 1 and Class 2 mannosidase families based on the sequence similarity analysis.
ER
Gold Mannosidase I1
Unknown function
I
(Glycosylhydrolase Family 38)
(Glycosylhydrolase Family 47)
Epididy mal/sperm Mannosidase
Class 2 Mannosidases
Class 1 Mannosidases
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6 x-Mannosidases in Asparagine-linked Oligosaccharide Processing
linkage between two internal al,2-linked mannosyl residues, and is designated an endo-a-mannosidase [ 7 1, 721. Although the latter mannosidase class has only a single member, it is unique in that it is the sole enzyme involved in glycoprotein maturation that catalyzes an endoglycosidase reaction. The endo-a-mannosidase cleaves Glc+]Man&ilcNAcz structures by the removal of Glc3-lMan to yield a MangGlcNAcz isomer A structure (Figure 1) [71]. The enzyme is found predominately in chordate organisms, with the exception of several classes of molluscs [73], and is believed to play a role in ensuring the complete trimming of glucose residues from glycoproteins that have completed folding and have exited the ER prior to full excision of the terminal glucose residues [73]. A potential role of the endo-amannosidase in the dissociation of folded proteins from calnexin and calreticulin in the early Golgi complex has also been proposed [74]. Since the role of the endo-amannosidase is predominately for the removal of residual glucose residues, the discussion of the biochemistry and molecular biology of the enzyme is described in Chapter 5 , glucosidases in oligosaccharide processing, in this volume. The other two classes of mannosidases that will be the subject of the remainder of this review are exo-mannosidases that are involved in glycoprotein biosynthesis and catabolism. The original proposal for two exo-mannosidase classes was based on both the biochemical characteristics of the enzymes purified from natural sources and on sequence data that had been obtained on a handful of representative members of each of the two classes 171. Since that time, numerous additional related sequences have been obtained and additional biochemical characteristics have been determined for enzymes purified from either natural sources or as recombinant enzymes purified from heterologous expression systems. The Class 1 mannosidases are now distinguished by several characteristics (7, 161 including: 1) similarities in sequence including a conserved 440-5 10 amino acid catalytic domain; 2) specificity for cleaving a1,2-mannose linkages; 3 ) in most cases a requirement for Ca2+ for catalytic activity; 4) sensitivity to inhibition by the pyranose substrate mimics deoxymannojirimycin (DMJ) and kifunensine (KIF) [6]; and 5 ) cleavage of the glycosidic linkage by inversion of configuration of the released mannose residue [75, 761. This classification contrasts them with the more heterogeneous collection of processing and catabolic mannosidases, termed Class 2 mannosidases, that are present in the ER, Golgi, lysosomes, and cytosol of mammalian cells, but have additional homologs in a widely diverse array of organisms including eubacteria and archea (see below). A more restricted sub-region of sequence is conserved among the Class 2 mannosidases but this region contains an acidic amino acid that is known to be part of the active site [77]. Class 2 mannosidases are larger (1 10-135 kDa), do not require cations for catalytic activity, are sensitive to inhibition by the furanose transition state analogs swainsonine (Sw) and 1,4-dideoxy-1,4-imino-r>-mannitol (DIM) [6], and cleave glycosidic linkages by retention of anomeric configuration of the released monosaccharide [78]. The nomenclature for the multiple members of the mannosidase families can appear to be quite confusing, especially with multiple Class 1 and Class 2 enzymes in the ER and Golgi. Fortuitously, the nomenclature for the enzymes is in agreement with the nomenclature of the classes. Thus, ER mannosidase 1 and Golgi mannosidases IA and IB are Class 1 mannosidases. ER mannosidase 11, Golgi mannosidase 11, and Golgi mannosidase Ilx are Class 2
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mannosidases. Other Class 2 enzymes, including the lysosomal mannosidase and the cytosolic mannosidase, do not follow the same nomenclature, but so far they are the only mannosidases in each respective compartment. A web-based database (URL: afmb.cnrs-mrs.fr/-pedro/CAZY/ghf.html)has collected data on the characteristics of classes of glycosylhydrolases based primarily on sequence similarity and has also separated the two enzyme classes into different families. (Class 1 = family 47; Class 2 = family 38) [79-811. The remainder of this review will summarize the recent data on the members of the two mannosidase classes in an effort to emphasize their roles in glycoprotein biosynthesis and catabolism.
6.3.2 Class 1 Mannosidases: enzymes of the ER and Golgi The Class 1 mannosidases were originally defined based on the biochemical characteristics of a collection of mammalian Golgi processing mannosidases and an oligosaccharide processing mannosidase isolated from S. cerevisiae [7].We now know considerably more about the enzymes involved in early glycoprotein processing including their specificities and roles in individual processing steps. Comparison of the sequences of Class 1 mannosidases makes it possible to define subgroups within the members of the family (Figure 2). When the presently available full-length Class 1 protein sequences are compared, at least four subgroups can be identified and three of the four groups contain mammalian sequence homologs (Figure 2). Additional partial sequences can also be found in DNA sequence databases, including numerous additional mammalian EST sequences, but these sequences will not be considered as a part of this discussion.
ER mannosidase I subfamily As mentioned above, Class 1 mannosidases were first identified in S. cerevisiae and mammalian systems, but significant differences in substrate specificities were identified between the enzymes from the two sources [7]. The yeast enzyme was shown to cleave only a single mannose residue from MangGlcNAcz to form the Manx GlcNAcz isomer B structure, without further cleavage [82-85]. This organism does not process their oligosaccharides beyond MansGlcNAcz before extension into mannan structures [17, 391. The enzyme from S. cerrvisiae has been the most completely studied of the Class 1 mannosidases, both as an enzyme purified from natural sources and by characterization of the recombinant enzyme 140, 41, 76, 82-90]. Yeast ER mannosidase I is an ER resident transmembrane protein containing a lumenally oriented catalytic domain [88]. Like the other Class 1 mannosidases the yeast enzyme can not cleave aryl-a-mannosides, is inhibited by dMNJ and KIF, but not Sw, and requires Ca+2 for catalytic activity [ 17, 83, 841. Contrary to prior hypotheses, the yeast enzyme has recently been shown not to bind the cation via the putative EF-hand-like consensus sequence that was found in several of the Class 1 enzymes [ 871. Mutagenesis studies have also identified several acidic residues that are required for Ca+2 binding and catalytic activity [87]. The S. cerevisiae enzyme has been overexpressed in Pichia pastoris and the recombinant enzyme was the first
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6 r-Munnosiduses in Aspuragine-linked Oligosncchuride Processing
of the Class 1 enzymes in which the mannosidase reaction was shown to proceed with an inversion of the anomeric configuration of the released monosaccharide [76]. The recombinant yeast ER mannosidase I has been purified in milligram quantities, crystallized, and an X-ray diffraction data set has been obtained [90] indicating that this enzyme will most likely be the first glycoprotein processing hydrolase to have its structure determined. The initial characterization of the early processing mannosidases in mammalian systems was far more coniplicated than in yeast as a result of the multiple enzyme activities present in different subcellular compartments in the secretory pathway. Early studies on the processing mannosidases took advantage of the ability to identify glycoprotein processing intermediates by pulse labeling cultured cells with radioactive monosaccharide precursors, either in the presence of processing mannosidase inhibitors or following treatment with ionophores, such as CCCP, that blocked the transport of glycoproteins between the ER and Golgi [91-991. The early literature on the ER processing mannosidases is full of confusing and contradictory data indicating the presence of DMJ-sensitive and DMJ-resistant mannosidase activities producing MansGlcNAcl and smaller structures [91-93, 981. In addition, mannosidases were detected, purified, or cloned that had substrate specificities that did not match the substrate specificities predicted by the metabolic labeling studies in cultured cells. It is now clear that at least some of the prior confusion was the result of the presence of multiple unanticipated mannosidase activities in the ER. Recent data now indicate that there are at least three principal mannosidase activities in the ER of mammalian cells. The first mannosidase activity is the aforementioned endo-a-mannosidase that cleaves Glc3-l Man9GlcNAcz to MangGlcNAc2 isomer A [71, 72, 100-1041. The second activity, termed ER mannosidase I, cleaves MangGlcNAcl to Man8GlcNAcl isomer B, analogous to the processing mannosidase in S. cereuisiae [ 105-1071. A third activity, termed ER mannosidase 11, cleaves MansGlcNAcz to MangGlcNAc2 isomer C [ 106-1081. The latter enzyme is a Class 2 mannosidase, whereas ER mannosidase I is a Class 1 enzyme. In vitro enzyme assays using ER membrane preparations have been used to examine ER mannosidase I activity in rat liver. This activity was shown to be sensitive to inhibition by DMJ, KIF, and EDTA, but not Sw or DIM [106, 1071. Human ER mannosidase I has recently been cloned, expressed, and characterized and found to have a high degree of sequence similarity to yeast ER mannosidase I [ 1051. In addition, two putative open reading frames were identified in the Cuenorhubditis elegans genome that have a high degree of sequence similarity to the yeast and human enzymes indicating that they may be nematode orthologs [ 1051. Sequence analysis indicates that, like yeast ER mannosidase I, the human and C. elegans proteins have a single transmembrane domain near their NH2-terminus. The proposed cytoplasmic tails and the “stem regions” of the enzymes are quite variable in length and in sequence, but the COOH-terminal 440-504 amino acid region that contains the catalytic domain of the yeast enzyme is highly conserved. These four sequences constitute the presently known members of the ER mannosidase I subgroup of Class 1 mannosidases (Figure 2), although all organisms between
6.3 Munnosiduses in Gljicoprotein Processing unu' Cutuholisni
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yeast and mammals would be predicted to contain an equivalent of this Class 1 a-mannosidase sub-group. Recombinant human ER mannosidase I has been expressed and purified and the enzyme has many catalytic characteristics in common with the yeast enzyme, including substrate specificity, the requirement for Ca+2 for catalytic activity, and inhibition by DMJ and KIF, but not Sw [ 1051. A recombinant epitope-tagged form of the human enzyme is also localized to the ER of mammalian cells indicating a conservation of localization as well as function of this ER enzyme [105]. The position of ER mannosidase I as the last fully conserved step in glycoprotein processing between yeast and mammals argues strongly in favor of a critical role for this enzyme in order to maintain selective pressure during eukaryotic evolution. The proposal that the enzyme acts as the timing step in determining the failure of glycoprotein folding in ER and targeting the substrates for degradation [22, 371 is a reasonable hypothesis for this essential role. The mechanism and components involved in this timing step remain to be elucidated. Golgi mannosidase I sub-family The second subfamily of the Class I mannosidases includes enzymes that act predominately in the Golgi to cleave Mang-gGlcNAcz structures to MansGlcNAcz [7]. There are multiple members of this class that have been identified from mammalian, insect, and nematode origin (Figure 2), although the latter sequences are putative open reading frames based on sequence similarities alone. The nomenclature for the mammalian forms of the enzymes in this sub-class has been quite confusing. Sequence alignments indicate that there are at least two separate lineages of mammalian Golgi mannosidase I-like activities. One subgroup, designated Golgi mannosidase IA, has been cloned from human [109], mouse [110], rabbit [ 1 101, and pig [ 1111 sources. Members of the second sub-group are termed Golgi mannosidase IB and are described further below. The mouse cDNA encoding Golgi mannosidase IA has been expressed in mammalian cells [ 1101 and in P. pastoris [75] where it has been shown to encode an enzyme that can efficiently cleave Mans GlcNAc2 to MansGlcNAc2, but slowly cleaves the final ul,2-mannose residue to form Man5GlcNAcz. The released monosaccharide in the cleavage reaction is inverted in anomeric configuration indicating that the mammalian enzyme shares a common enzymatic mechanism with yeast ER mannosidase I [75]. Cleavage of MansGlcNAcz by the enzyme produces a distinct series of isomers and the w1,2mannose residue that was produced by the final cleavage step was found to be the sole mannose residue that is recognized and cleaved by the ER mannosidase I. These data indicate that there is a complementarity and nearly non-overlapping specificity in substrate recognition between ER mannosidase I and the Golgi processing mannosidases. Since ER mannosidase I is localized earlier in the secretory pathway it would be expected that the Golgi mannosidases would more commonly encounter substrate glycoproteins containing MangGlcNAcz isomer B structures. The difference in substrate recognition between ER mannosidase I and members of the Golgi mannosidase I subfamily is striking considering their sequence similarity
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6 a-Munnosiduses in Aspurugine-linked Oliyosuccharide Processing
as Class 1 mannosidases and their presumed similarity in protein structure. Recombinant murine Golgi mannosidase IA expressed in Pichiu has been purified in milligram quantities, crystalized, and X-ray diffraction data has been obtained [112]. Comparison of the structure of this enzyme with the structure of yeast ER mannosidase I will elucidate the structural features of the enzymes that specify their substrate recognition. The cDNAs encoding the human [ 1091 and pig [ 11 11 equivalents of Golgi mannosidase IA have also been isolated. These enzymes have previously been called Many-mannosidases, but this nomenclature remains confusing since several enzymes can cleave mannose residues from Man9GlcNAcz. Surprisingly, immunoEM studies using antibodies raised to the purified pig enzyme indicated that this enzyme is present in ER, not the Golgi, of pig hepatocytes [113]. Substrate specificity studies demonstrated that the pig and calf forms of this enzyme were able to cleave MangGlcNAc? to ManhGlcNAc2 [114]. The combination of an ER localization and the substrate specificity data led to a proposal that the enzyme was distinct from the Golgi-localized enzymes that were supposed to cleave down to MansGlcNAc2. With the cloning of the mouse, rabbit, pig, and human cDNAs it is now clear that they are all mammalian orthologs of the same enzyme activity, but with potentially varied localizations. The basis for the localization difference of the pig enzyme is presumably inherent to the primary sequence of the enzyme since parallel studies on the transfection and localization of the pig and human mannosidase cDNAs indicated an ER localization of the pig enzyme in contrast to a Golgi localization of the human enzyme [ 11I]. Mouse L-cells stably transfected with mouse Golgi mannosidase IA cDNA also resulted in a Golgi pattern of immunofluorescence [ 1 101. Cell and tissue specific variation in localizations of Golgi mannosidase IA within the Golgi and post-Golgi compartments of the secretory pathway have been previously seen in rat tissues [ 1151, and comparison of the localization of the porcine and human forms of Golgi mannosidase IA now indicates the possibility of species-specific variation in localization of these homologous enzymes in a given cell or tissue type. These data also indicate that the nomenclature distinguishing “ER” and “Golgi” mannosidases is misleading since there can be an overlap in localization. Despite the potential for confusion in nomenclature, the enzymes in the ER mannosidase I sub-family and the Golgi mannosidases I subfamily have historically employed this nomenclature and it will continue to be used here. Multiple forms of Golgi mannosidase I-like activity have been previously separated from rat liver Golgi membrane extracts by ion exchange chromatography and gel filtration [116, 1171. These forms were designated Golgi mannosidases IA and IB, and the enzymes were found to share many biochemical and enzymatic characteristics, including inhibition by DMJ and EDTA, and cross reactivity with antibodies raised to rat liver Golgi mannosidase IA. The two forms were distinguished by their chromatographic behavior and a low but detectable level of activity by Golgi mannosidase IB toward pNP-Man. In agreement with the presence of two enzymatic forms of Golgi mannosidase I-like enzymes in rat tissues, a second mammalian cDNA was isolated from a murine cDNA library that was distinct from Golgi mannosidase IA [ 1181. The protein encoded by this cDNA was des-
6.3 Mannosiduses in Glj~coj~roteiiz Processing und Cutaholisin
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ignated Golgi mannosidase IB by analogy to the two forms of mannosidase activity that were separated and isolated from rat liver Golgi membranes. It is not clear whether there is a direct correspondence between the enzymes separated from rat liver extracts and designated Golgi mannosidase IA and IB and the cDNAs with the same name. At the protein level murine Golgi mannosidases IA and IB are approximately 65% identical within the catalytic domain [ 1 181. The human cDNA and gene encoding Golgi mannosidase IB have also been isolated recently and the human protein is 94% identical to the murine Golgi mannosidase IB [119]. Expression of Golgi mannosidase IA and IB in Pichia has allowed a detailed comparison of their substrate specificities [75]. Both enzymes efficiently cleave MangGlcNAcl to ManhGlcNAc2 and slowly cleave the last al,2-mannose residue to yield ManjGlcNAcz. Differences were detected in the order of residues cleaved when MangGlcNAc2 was the substrate, but a similar order and rate of cleavage of the a1,2-mannose residues were detected when MansGlcNAc? was the substrate. An epitope-tagged form of Golgi mannosidase 1B was localized to the Golgi by immunofluorescence in transfected COS cells similar to murine Golgi mannosidase IA [ 1181. The transcripts encoding both Golgi mannosidase IA and IB were detected in Northern blots from most mouse [ 1181. rat [ 1 101, and human [ 1191 tissues, but there were significant differences in the ratios of the two transcripts. Differences in expression at the cellular level were also detected by immunofluorescence in the rat reproductive tract [ 1201. These data indicate that the similar Golgi mannosidase Ilike forms are most likely not redundant enzymes but that ancestral gene duplication and divergence has resulted in tissue-specific expression patterns. Fungal secreted mannosidases
Two fungal mannosidases have been purified and cloned that have sequence similarity to Class 1 mannosidases, but differ in that they both contain cleavable signal sequences, are found as secreted proteins, appear to not require Ca+' for catalytic activity, and have relatively lower pH optimums. The enzymes cloned from Aspergillus saitoi [ 121-1251 and Pennicilium citrinunz [ 126, 1271 appear to form a separate sub-group of the Class 1 mannosidases based on sequence comparisons (Figure 2). The enzymes have been shown to cleave al,2-mannose residues from high mannose substrates, and the detailed substrate specificity of the Aspergillus al,2-mannosidase has been recently determined [ 1241. Surprisingly, this fungal a-mannosidase has a substrate specificity that is quite similar to the mammalian Golgi mannosidases IA and IB and contains a bound Ca2+ despite prior data indicating that Ca2+ was not required for activity. Site-directed mutagenesis of the Aspergillus al,2-mannosidase identified several residues that are required for catalytic activity [ 1251. The Pennicilium enzyme has also been extensively characterized and cloned [126, 1271. Treatment of the Pennicilium enzyme with a carbodiimide reagent specific for acidic amino residues resulted in an inactivation of the enzyme and a single acidic amino acid residue was found to be protected by preincubation with DMJ [ 1271. The single residue is conserved among the 23 Class 1 mannosidases (Asp375 in the Penniciliunf enzyme) and indicates that this residue may be involved in the active site.
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6 a-Munnosiduses in Asparugine-linked Oligosacchuride Processing
New genes with unknown functions Sequence similarity searches have also identified a broad collection of mannosidase I-like genes whose functions are not clear. Members from this sub-group include putative open reading frames from S. cerevisiae (two distinct gene products), Schizosaccharomyces pombe, C. eleyans (three distinct gene products), Drosophilu melunogaster; and Homo supiens (at least one gene product). Members of this subgroup are unique among the Class 1 mannosidases in that they contain a sequence extension on the COOH-terminal end of the protein that is not conserved among the other Class 1 mannosidases and has no similarity to other known sequences. In most cases the sequence of the extension is not very well conserved even among members of this subgroup. Members of the subgroup generally contain an NH2terminal hydrophobic sequence, but it is not clear whether these sequence are membrane spanning domains or cleavable signal sequences. One member of this collection (Saccharomyces cerevisiae Y LR057w) is predicted to be an integral membrane protein and contains an extended insertion within the center of the conserved Class 1 mannosidase catalytic domain sequence with a corresponding deletion of part of the conserved sequence. Since no predicted processing mannosidase activities are unaccounted for among the other members of the Class 1 enzymes it is not clear what function these proteins play in vivo. Their identification in the genomes of organisms ranging from yeast to mammals indicate that they most likely play an important role in some biochemical process in the cell, but as of yet none of the members of this subgroup have been shown to contain a catalytic activity.
6.3.3 Class 2 Mannosidases: enzymes of the cytosol, ER, Golgi, and lysosomes The abilities of mammalian Class 2 mannosidases to cleave the synthetic aryl-amannoside substrates, including pNP-a-D-mannoside or 4-methylumbelliferyla-D-mannoside, were instrumental in the early identification, characterization, and distinction of these enzymes. These early studies examined the pH optimum, subcellular fractionation, and effects of cations and EDTA on the rate of substrate cleavage [ 128-1301. It was determined that mammalian tissues contained at least three easily distinguished activities that could act on the synthetic substrate [ 1301. The lysosomal mannosidase was distinguished by a low pH optimum (pH -4.5), stimulation by Zn+2, and co-fractionation with lysosomal enzyme markers. The soluble or cytosolic mannosidase was distinguished by a more neutral pH optimum , fractionation with the cytosolic fraction. The (pH -6.5), stimulation by C O + ~and Golgi mannosidase was distinguished by an intermediate pH optimum (pH - 5 4 , lack of an effect by cations, and fractionation with Golgi membranes. Each of these enzymes has subsequently been purified and cloned from multiple mammalian sources, and sequence comparisons have confirmed the distinctions made on biochemical grounds (Figure 2). Enzymes that fall within the category of Class 2 mannosidases are distinguished from Class 1 mannosidases by similarities in size of their catalytic region (110116 kDa). Although the sizes of the proposed “catalytic domains” of the Class 2
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mannosidases are large, the portion of this sequence that is most highly conserved among the family members is more restricted. Sequence comparisons used to assemble the dendrogram in Fig. 2 employed an -182 amino acid region that is nested to the NHz-terminal end of the catalytic region. Among the 24 independent full-length sequences that were identified in the sequence databases, phylogenetic analysis indicates that the enzymes are clustered into four subgroups, with three of the sub-groups corresponding to the mammalian enzymes that were originally defined based on biochemical and cell fractionation characteristics. Golgi mannosidase I1 The identification of a pNP-mannoside-cleaving mannosidase in the Golgi complex and its distinction from enzymes in the soluble fraction or lysosomes was one of the first indications that cleavage of oligosaccharide intermediates during glycoprotein maturation might be catalyzed by enzymes in the Golgi complex [128]. Metabolic radiolabeling studies indicated that glucose and mannose cleavage were a necessary prerequisite for extension into complex type structures [131, 1321 and the pNP-mannose cleaving activity in the Golgi complex was the most likely candidate for the processing reactions. It was only years later that it was discovered that several mannosidase activities were required for the oligosaccharide trimming steps and the enzyme that catalyzed the pNP-mannoside cleavage in vitro was capable of selectively cleaving only specific al,3- and al,6-mannose residues from the MangGlcNAcz processing intermediate [ 1 161. The natural substrate specificity was surprisingly restricted considering the ability of the enzyme to cleave the synthetic substrate. Only the two non-reducing terminal a1,3- and al,6-mannose residues on GlcNAcManSGlcNAcz could be cleaved by the enzyme and no direct cleavage of Man5GlcNAcz was detected. The substrate specificity indicated that there was an overlap in the trimming and elongation phases of the oligosaccharide biosynthetic pathway with Golgi mannosidase I activities cleaving to a MansGlcNAc2 structure followed by the action of GlcNAc transferase I and subsequent action by the enzyme designated Golgi mannosidase I1 to form GlcNAcMan3GlcNAcz [ 1331351. Enzymes with similar substrate specificities to mammalian Golgi mannosidase I1 have been identified in mung beans [136] and insect cell lines [137]. In contrast, oligosaccharide trimming in Dictyostelium discoideum appears to not require the prior action of GlcNAc transferase I [138, 1391. A Sw-sensitive direct cleavage of MansGlcNAcz to smaller structures was found in this organism. The human [140] and mouse [141] forms of Golgi mannosidase I1 have been cloned and were shown to be type I1 transmembrane proteins with a small (5 amino acid) NHz-terminal cytoplasmic tail, a single transmembrane domain, a region of sequence on the luminal side of the membrane that is dispensable for catalytic activity (-80 amino acids, generally termed the “stem domain”), and a large luminal catalytic domain (-1017 amino acids) [ 1411. This structural topology is common with other Golgi and ER processing mannosidases and glycosyltransferases involved in glycoprotein extension [7, 1421. It is noteworthy that only the luminal catalytic domain, excluding the cytoplasmic tail, transmembrane domain, and stem domain, has sequence similarity to the other Class 2 mannosidases. Clearly the process that
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6 a-Munnosiduses in Asparugine-linked Oligosuccharide Processing
has led to the sequence duplication and divergence between Golgi mannosidase 11, the lysosomal mannosidase, and the cytosolic/ER mannosidase I1 also led to the acquisition of additional NHz-terminal sequences that were unique to the Golgi enzyme. Evidence has been presented that the transmembrane domains and flanking sequences of these Golgi type I1 transmembrane processing activities may be involved in the retention of the enzymes in the Golgi complex [ 1431. cDNAs or genes encoding sequence homologs of the mammalian Golgi mannosidase I1 sequences have been identified in insect systems (Spodoptera [ 1441 and two independent Drosophilu sequences, one of which is published [ 1451) and C. elegans (2 independent sequences) (Figure 2). To this date only the cDNA from Spodoptera has been shown to possess activity toward pNP-mannoside [ 1441, but the natural substrate specificities of all of these additional gene products remain to be determined. A second cDNA and gene that are similar, but not identical, to Golgi mannosidase I1 have also been isolated from human [ 1401 and mouse sources. The corresponding enzyme has been designated Golgi mannosidase IIx [ 1401. Overexpression of the cDNA in COS cells resulted in a 2.5-fold increase in activity toward pNP-mannose, but further characterization of the oligosaccharide substrate specificity of the enzyme has not yet been published. The pattern of transcript expression for Golgi mannosidase I1 and Ilx in human tissues are overlapping, but there are significant differences in transcript abundance in several tissues. These differences in tissue-specific expression patterns are reminiscent of the differences in expression of the two Golgi processing mannosidases that act earlier in the pathway, Golgi mannosidases IA and IB. A deficiency in Golgi mannosidase I1 has been implicated in a human genetic disease characterized by ineffective erythropoiesis, bone marrow erythroid multinuclearity, and secondary tissue siderosis [ 691. The autosomal recessive disorder, termed congenetal dyserythropoietic anemia type I1 or HEMPAS, results from an incomplete processing of Asn-linked glycoproteins, including two erythrocyte membrane glycoproteins, called bands 3 and 4.5. These two glycoproteins are normally processed to complex type oligosaccharides with extended polylactosamine structures. In HEMPAS, the oligosaccharides on these glycoproteins are processed to hybrid type structures and the polylactosamine structures that are normally found on bands 3 and 4.5 are found on glycolipids instead. Patients with this disease suffer from life-long anemia, although recent literature suggests that the disease is often asymptomatic and may be more common than previously expected [ 1461. The genetic basis of the disease is complex. Biochemical analysis of the oligosaccharides accumulated on erythrocytes from patients indicates an enzyme defect in either the GlcNAc transferase I1 or the Golgi mannosidase I1 step [69]. Consistent with this hypothesis, two patients were found to have defects at the Golgi mannosidase I1 locus [68]. In contrast, other patients from Italy appear not to show linkage to GlcNAc transferase 11, Golgi mannosidase 11, or Golgi mannosidase IIx, but, instead, show a genetic linkage to a locus on chromosome 20q11.2 [ 147, 1481. A mouse model for HEMPAS disease was created by the inactivation of the Golgi mannosidase I1 gene and many of the characteristics of the human disease were reproduced [70]. The mice show anemia, splenomegaly, and immature erythrocytes or reticulocytes in the peripheral blood. Lectin blots and cell sorting
6.3 Munrzosidases in Glycoprotein Processing and Cutaholisnz
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indicated an absence of complex type structures on erythrocytes in the Golgi mannosidase I1 null mice. In contrast, oligosaccharide structural analysis in splenocytes or fibroblasts and enzyme assays from a variety of mouse tissues indicated the presence of an alternate processing activity that could bypass the Golgi mannosidase 11 step, but with lowered efficiency in most tissues. This activity was termed a-mannosidase I11 [70], but it remains to be resolved whether this bypass activity represents Golgi mannosidase IIx or an alternate activity that was detected in the tissue extracts that directly cleaves MansGlcNAc2 to Man3GlcNAc2 without the prior activity of GlcNAc transferase I. This latter activity was found to be stimulated by C O + and ~ weakly inhibited by Sw and had many of the catalytic features of an enzyme previously characterized from BHK cells and rat liver [ 14915 I]. The latter enzyme has been purified from rat liver microsomes and can cleave Man9-4GlcNAc2 to Man3GlcNAc2, is weakly inhibited by Sw, and is stimulated by Cof2 [150]. The cDNA or gene encoding this enzyme has not yet been isolated so comparison of its sequence with the other Class 2 mannosidases has not been performed. Future studies on the gene disruption of Golgi mannosidase 1Ix and the proposed a-mannosidase 111 activity that acts directly on MansGlcNAc2 should help to resolve the contributions of each of these enzymes in glycoprotein processing in mammalian tissues.
Lysosomal mannosidase Catabolism of mannose terminal oligosaccharide structures occurs predominately in lysosomes, with the additional contribution of cytosolic mannose trimming of oligosaccharides released from glycoproteins and glycolipids in the ER [SO]. Oligosaccharides from glycoproteins that enter the lysosomal compartment through endocytosis are cleaved from the non-reducing terminus by exo-mannosidases and from the reducing terminus by a glycosylasparaginase and, in some species, by a chitobiase, to result in the complete hydrolysis of oligosaccharide structures to monosaccharides [ 1521. Cytosolic oligosaccharides are cleaved by a distinct cytosolically-localized chitobiase [ 1531 and a cytosolic mannosidase [ 1541 (see below) to a distinct isomer of Man5GlcNAc before transport across the lysosomal membrane [48] for further degradation into monosaccharides. The cleavage of the a-linked mannose residues in lysosomes is largely accomplished by a broad specificity lysosoma1 mannosidase that is capable of cleaving crl,2/a I ,3/al ,6-mannose linkages [1551. The substrate specificity and order of mannose removal has been well described for the broad specificity enzyme and in most instances unique isomers are produced in the cleavage series [6]. The enzyme is capable of readily cleaving 7 of the 8 a-linked mannose residues on MangGlcNAc2, but cleaves the last a1,6mannose with relatively low efficiency. The lysosomal mannosidase has been purified from a number of species [ 130, 156-1601 and the subunit molecular weights vary significantly between species. The human enzyme can be separated into two forms by ion exchange chromatography, termed A and B, but significant evidence indicates that the two enzymes are differently modified versions of the same gene product [156]. Pulse-chase studies have indicated that the initial translation product of the human gene is a 110 kDa pre-
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6 ti-Mannosidases in Asparagine-linked Oligosaccharide Processing
cursor that is converted to subunits of 62, 58, and 26 kDa by proteolysis, presumably in the lysosome [ 1611. Fragments of different sizes have also been isolated from human tissues or cells indicating significant heterogeneity in processing of the initial precursor polypeptide [ 1601. The native apparent molecular mass of the lysosomal mannosidase A is 280 kDa and mannosidase B is 260 kDa [ 1561. The cDNAs and genes encoding the broad specificity mannosidase have been cloned from a variety of mammalian species including human [160, 162, 1631, mouse [164, 1651, cat [158], and cow [159], and the genes encoding slime mold (Dictyosteliurn discoideurn) [ 1661, protozoan parasite (Trypanosoma cvuzi) [ 1671, plant (Arabidopsis thalaina), and nematode (C. elegans) enzymes have also been identified. The latter two genes were identified in recent genome sequencing projects and functional demonstration of the encoded protein has not yet been performed. The D. discoideum enzyme was the first of the lysosomal mannosidases to be cloned and the developmental expression of the protein has been extensively studied [ 168, 1691. All of the genes encode a protein of similar size (-900-1000 amino acids) and those enzymes that have been studied have a similar broad specificity toward oligosaccharide substrates. The genomic organization of the nematode, plant, and mammalian genes are quite complex containing 19-28 introns [159, 163, 1651. Many of the intron positions are conserved among the diverse set of organisms with an extensive similarity in intron positions among the mammalian species. The complex gene organization in the higher organisms is in contrast with the single intron found in the Dictyostelium a-mannosidase gene [166] and the absence of introns in the T. cruzi gene [ 1671. In some species a second lysosomal a-mannosidase activity is expressed that is capable of cleaving the final al,6-mannose residue linked to the p-mannose core [6, 170, 1711, but this activity will not cleave other a-mannosyl residues on MangGlcNAcz-1. Cleavage of the core a1,6-mannose by this selective a1,6mannosidase appears to require the prior activity of the glycosylasparaginase and chitobiase since the enzyme strongly prefers oligosaccharides with a single GlcNAc at the reducing terminus [172]. Similar to other Class 2 mannosidases, including the broad specificity lysosomal mannosidase and Golgi mannosidase 11, the ul,6mannosidase is inhibited by Sw and DIM, but in contrast the enzyme acts poorly toward aryl-a-mannoside substrates [6]. The detection of the a1,6-mannosidase activity in a restricted number of mammalian species shows a surprising concordance with the species that express the lysosomal chitobiase [6, 1731. The chitobiase gene has been shown to be present both in species that express chitobiase activity, including humans, rodents, rabbits, chickens and slime mold, as well as in species that do not express detectable activity, including cats and cattle. In the latter species it appears that translation is suppressed [ 1731. Similarly, ul,6-mannosidase activity was detected in tissues or cell lines from rats and humans, but not cats or cattle [6]. It has been hypothesized that the cooperative specificity of the lysosomal a1.6mannosidase activity after chitobiase cleavage of the substrate has provided an additional route for oligosaccharide catabolism in some species, but that these enzymes are not required for glycoprotein catabolism. In this manner the enzymes are proposed to act as “accessory glycosidases” to aid in the efficient cleavage of the oligosaccharide core. For a detailed discussion of the relationship between the
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al,6-mannosidase and the chitobiase see the review in [6]. Confirmation that the al,6-mannosidase is a Class 2 enzyme and comparison of the mannosidase and chitobiase coordinate expression will await the cloning of the cDNA or gene encoding the al,6-mannosidase. A human lysosomal storage disease characterized by a defect in the broad specificity lysosomal mannosidase. a-mannosidosis, results in varied clinical features including hepatomegaly, recurrent infections, skeletal dysplasia, deafness, severe mental deterioration, facial coarsening, eye opacities, and commonly death before ten years of age (174). A milder form of the disease has been described in some patients where hearing loss and mental retardation is evident from early childhood, but the onset of the other clinical features may be varied or absent. Analysis of residual enzyme activities in cells from patients with severe and milder forms of the disease indicated that the clinical severity was directly related to the degree of enzyme impairment [ 1751. Patients accumulate linear and branched oligosaccharides consistent with the catabolic pathways described in Figure 2 [176]. The oligosaccharides are predominately linear Man4-l GlcNAcl structures consistent with catabolic intermediates derived from cytosolic oligosaccharide catabolism and cleavage by the lysosomal al,6-mannosidase. Larger branched oligosaccharides are also found, but these oligosaccharides represent a lesser percentage of the total accumulated structures. The genetic defect in patients with a-mannosidosis generally do not appear to involve the alterations in the transcription or translation of the mannosidase polypeptide [162]. but recent data indicate that a human mutations can include splicing, missense, or nonsense mutations, as well as small insertions or deletions [177-1791. Animal models for the enzyme deficiency have also been identified in Persian cats [ 1581 and in Angus and Galloway cattle [ 1591. The defect in the feline disease results from a 4 bp deletion resulting in a frame shift at codon 583 and a premature termination at codon 645 [158]. Each of the bovine mutations involve different missense mutations causing amino acid substitutions that influence the either the level of protein expression or enzyme stability [ 1591. Epididymal/sperm mannosidase a-Mannosidase activities have been identified in rat, mouse, and pig sperm or epididymal tissues that have characteristics of Class 2 mannosidases [ 180-1841. The rat sperm a-mannosidase has activity toward al,2-, a1,3- and a1,6-mannose linkages and can cleave Man9-4GlcNAc2 to Manc3GlcNAc2 [ 1801. The enzyme could also act on GlcNAcMan5GlcNAc2, releasing at least one mannose residue. The sperm enzyme does not cross-react with the antibody to the ER/cytosolic a-mannosidase or lysosomal mannosidases and evidence has been published that the enzyme is first expressed as a plasma membrane protein in testicular germ cells and subsequently on the external face of the plasma membrane in mature sperm where it has been hypothesized to take part in sperm-egg interactions [180, 1841. An enzyme with similar biochemical characteristics has been purified from porcine epididymal fluid and the cDNA encoding the latter enzyme has been cloned [185]. The pig enzyme was found to be synthesized and secreted by the principal cells between the distal
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6 a-Mannosidases in Asparagine-linked Oligosaccharide Processing
caput and proximal corpus of the epididymus with low expression in other parts of the epididymus and testicular tissue [186]. In contrast, a sequence homolog from mouse tissues was found to be expressed primarily in type A spermatogonia (stages IX-XI) and transcripts continue to be detectable through the formation of type B spermatogonia (stage VI) [ 1861. The relationship between the secreted porcine epididymal mannosidase and the plasma membrane associated rat sperm mannosidase remains to be established. Comparison of the polypeptide sequence encoded by the pig and mouse cDNAs indicate that these enzymes are Class 2 mannosidases, but significant distinctions from the other Class 2 enzymes result in their placement as a separate group distinct from the lysosomal, Golgi, or cytosolic mannosidases. The purified pig enzyme could cleave pNP-mannose in contrast to the inability of the rat sperm plasma membrane enzyme to cleave this substrate [185]. In contrast both enzymes could cleave oligosaccharide substrates [ 180, 183, 185, 1861. The effects of alkaloid inhibitors such as Sw, DIM, DMJ, and KIF have not been reported for either of the enzymes.
Heterogeneous cluster of mannosidase homologs among eukarya, eubacteria, and archaea The final subgroup of the Class 2 mannosidases are the most complex and unique in that they have sequence homologs among eubacteria and archaebacteria in addition to the eukaryotic family members. The first representative member of this subgroup that was identified was the vacuolar mannosidase from Saccharomyces cerevisiue [187,1881. This enzyme is localized to the internal face of the vacuolar membrane where it was originally proposed to play a role in glycoconjugate catabolism in the fungal vacuole. Suggestive evidence in support of this role was indicated by the reduction in the rate of turnover of metabolically labeled glycoconjugates in a yeast strain disrupted in the vacuolar mannosidase gene [lSS]. No other phenotype was detected in the disrupted strain. Subsequent sequencing of the yeast gene indicated that the encoded polypeptide contains no transmembrane domain or NH2-terminal signal sequence for entrance into the secretory pathway at the ER membrane [187]. Pulse-chase studies using the wild type mannosidase gene or fusions with genes encoding other reporter proteins resulted in the targeting of the protein into the vacuole in a manner that apparently involves entry into the secretory pathway within the Golgi complex or in some compartment downstream from the ER [189]. Yeast transpprt mutants (see mutants) that block transport within the ER or between the ER and Golgi had no effect on vacuolar targeting while some mutants that interrupt transport within the Golgi blocked delivery of the enzyme to vacuoles. None of the N-glycosylation sites on the enzyme or sites on a mannosidase-invertase fusion were glycosylated despite appropriate targeting of the proteins to the vacuole. These data indicate that the yeast a-mannosidase was delivered to the vacuole in a novel pathway separate from the secretory pathway. The gene encoding a putative vacuolar a-mannosidase activity was also isolated from the filamentous fungi, Aspergillus nidulans (now Emericella nidulans) [190]. The translation of this coding region has a high degree of sequence similarity to the S. cerevisiae vacuolar mannosidase (Figure 2) and gene disruption also had no discernable phenotype. A mammalian mannosidase has also been purified and cloned that has a high
6.3 Munnosiduses in Glycoprotein Processing and Cutuholism
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degree of sequence similarity to the yeast vacuolar enzyme [ 191, 1921. In contrast to the yeast enzyme, the mammalian enzyme is found predominately in the cytosol [ 1921. The mannosidase is a tetramer of 1 16 kDa subunits that can cleave pNPa-mannose and oligosaccharide substrates and is inhibited by Sw and DIM, but not DMJ [106]. The oligosaccharide substrate specificity is unusual in that it requires a single GlcNAc at the reducing terminus of the oligosaccharide substrate and selectively cleaves specific a1,2-, al,3-, and al,6-linkages to produce a unique MansGlcNAc2 isomer (see Figure 2) [55, 581. This specificity indicates that the enzyme plays a role in generating an intermediate digestion product in the catabolism of oligosaccharides in the cytosol before their delivery to lysosomes for completion of the degradation process. The cDNA encoding the enzyme has been isolated from a rat cDNA library and was found to encode a polypeptide of 1040 amino acids containing no transmembrane domain or NH2-terminal signal sequence [ 1911. Transfection of COS cells with an expression construct containing the rat cDNA resulted in a 400-fold overexpression of enzyme activity. Northern blots indicated that transcripts encoding the cytosolic mannosidase were ubiquitously expressed in adult rat tissues. Initial studies on the processing mannosidase activities in the ER in the early 1980s indicated the presence of an oligosaccharide processing enzyme that cleaved MangGlcNAc2 to MansGlcNAc2 [91-941. These studies eventually led to the identification of ER mannosidase I [ 105, 1061. In an early attempt to purify ER mannosidase I, an ER-associated a-mannosidase activity was identified that cleaved pNP-mannose and was relatively less sensitive to inhibition by DMJ than Golgi mannosidase IA [91-93]. Surprisingly, the characteristics of the ER-associated activity were strikingly similar to the cytosolic a-mannosidase. When the cDNA encoding the cytosolic mannosidase was cloned, over-expression of the enzyme in COS cells also resulted in an over-expression of the ER-associated a-mannosidase activity [ 19I]. Although the substrate specificity of the cytosolic a-mannosidase did not match the activity indicated for ER mannosidase I, the hypothesis was presented that the cDNA encoded an a-mannosidase that was translocated into the ER by an unknown process and that the enzyme associated with the ER membrane had an altered substrate specificity. Recent work now indicates that at least some of the original hypothesis was correct. Studies by the Spiro lab have demonstrated that the cytosolic a-mannosidase is immunologically-related to an a-mannosidase activity in the ER, termed ER mannosidase I1 [ 1061. Western Blots have demonstrated that the ER-associated activity is smaller (-82 kDa) than the cytosolic form (-105 kDa) and antibodies directed to the COOH-terminus of the cytosolic form reacted with the ER form while antibodies to the NH2-terminus did not. These data suggest that the ER form is derived from the cytosolic form by a translocation process that involves proteolytic cleavage. The cytosolic and ER forms of the enzyme share common characteristics of inhibition by DIM, DMJ, weak inhibition by Sw, and lack of inhibition by KIF [ 106, 1081. Both enzymes produce the MansGlcNAc isomer C from MangGlcNAc, but the cytosolic a-mannosidase appears to efficiently cleave the ManxGlcNAc structure further to MansGlcNAc, especially in the presence of the cation activator, Co2+.The ER enzyme appears to have lost the capacity for activation by Co'+ and is able to cleave past MansGlcNAcz to Man;iGlcNAcz only after prolonged incu-
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6 a-Mannosiduses in Aspurugine-linked Oligosaccharide Processing
bation. Comparison of the ER mannosidase I1 enzyme activity with other oligosaccharide processing activities, including ER mannosidase I, endomannosidase, and glucosidase 11, in various cell lines indicated that the ER mannosidase I1 is present at significant levels in all of the cell types and that the ratio of ER mannosidase I1 to ER mannosidase I could vary as much as ten-fold [ 1071. These data suggest that the product of the cytosolic/ER mannosidase I1 gene may play a separate role in each of the two cellular compartments. The ER form may catalyze an alternate pathway for early mannose trimming while the cytosolic form plays a role in oligosaccharide catabolism. In addition to the two fungal mannosidases and the mammalian cytosolic/ER mannosidase I1 sequences, three other sequences with similarity to Class 2 mannosidases were identified in database searches. Two of these sequences were most similar to each other, but were more distantly related to the fungal vacuolar and mammalian cytosolic/ER mannosidase I1 sequences. One of the genes was from a unicellular cyanobacterium, Synechocystis strain PCC6803, a eubacterium, and the second was from a hyper-thermophilic archaebacterium, Pyrococcus horikoslzii OT3. The third sequence, from the eubacteria Mycobacterium tuberculosis, appeared to be more similar to the fungal vacuolar a-mannosidases, the mammalian cytosolic/ER mannosidase 11, and the cyanobacterium and archaebacterium sequence homologs than the other Class 2 mannosidases. The degree of sequence similarity of the Mycobacterium putative a-mannosidase sequence to all at the other Class 2 mannosidases was more remote and the coding region was missing -40 amino acids of the most highly homologous sequence of the Class 2 enzymes. These data call into question whether the gene encodes a protein with a-mannosidase activity. Other mannosidase enzyme activities have been identified in mammalian systems linkages. Noteworthy among that are capable of cleaving al,2-/a1,3-/a1,6-mannose these enzymes is a catalytic activity partially purified from rat brain microsomes that can cleave Mang-4GlcNAc2 to Man3GlcNAc2 [193]. The enzyme is not inhibited by Sw or dMNJ, and has marginal activity toward pNP-mannose. Surprisingly, this enzyme cross-reacts with an antibody to the rat cytosolic/ER mannosidase I1 indicating that the enzyme is likely to be a Class 2 mannosidase. The enzyme activity termed mannosidase I11 (see discussion above) also has hallmarks of a Class 2 mannosidase. This enzyme has been found in rat liver microsomes [149%151]and in tissues from mice that have a disruption in the Golgi mannosidase I1 gene locus [70]. cDNAs or genes encoding the rat brain enzyme or a-mannosidase I11 have not been isolated, so the relationship to other Class 2 enzymes remains to be resolved.
6.4 Conclusions and Future Prospects Several mannosidases have been identified that are involved in glycoprotein biosynthetic reactions and in glycoprotein catabolism. Comparisons of biochemical characteristics and sequence similarities have allowed the exo-mannosidases to be divided into two classes. Within each of the classes are subgroups that have been
References
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defined based on greater similarities in sequences, unique substrate specificities, and subcellular localizations. Among the Class 1 mannosidases two subgroups have been identified that are involved in early glycoprotein trimming steps, yet their substrate specificities indicate that they have a distinct recognition of non-reducing terminal al,2-mannose residues on Man9 GlcNAq oligosaccharides. The structure determination of members of these two subgroups in the near future should provide a considerable insight into the molecular basis of their substrate recognition. A separate subgroup of Class 1 mannosidases is defined by the secreted enzymes from Aspergillus saitoi and Pennicilium citrinum. The roles of these enzymes in glycoprotein maturation or catabolism are unclear. The fourth subgroup of the Class l mannosidases is a novel subgroup that contains a region of sequence that is conserved among the Class 1 mannosidases and a second region extending from the COOH-terminus that has no obvious similarity to any known protein. Although members of this subclass are conserved from yeast to mammals, the function of the proteins remain obscure. Defining the role of this novel class of mannosidase-like proteins will be a major goal for the future. The Class 2 mannosidases have a diverse set of members, including mammalian enzymes that are involved on glycoprotein maturation in the Golgi complex as well as enzymes involved in glycoprotein catabolism in the cytosol and lysosomes. The roles of the Golgi and lysosomal subgroups of enzymes appear to be well defined, but the presence of additional activities in both compartments are indicated by the detection of an ctl,6-mannosidase in lysosomes and a-mannosidase I11 in the secretory pathway catalyzing a proposed alternate pathway for glycoprotein processing. The unusual features of dual localization of the mammalian cytosolic/ER mannosidase I1 and its fungal vacuolar homologs indicate that further study will be required to determine the mechanism of transport of these enzymes into their respective membrane bound compartments. In addition, the structural features of this broad class of enzymes that allow their discrimination in substrate specificity and localization should provide additional questions for investigation. The detection of genes encoding Class 2 mannosidase homologs in eubacteria and archeabacteria indicates that this subclass of mannosidases has a highly ancestral origin and could possibly represent the ancestral progenetor gene from which the other Class 2 mannosidases were derived by gene duplication and divergence. Confirmation of this hypothesis will require the identification and sequencing additional family members from other organisms and determination of their substrate specificities.
Acknowledgments
The work described from the author’s laboratory is supported by U.S. Public Health Service Grants GM-47533 and RR-05351 References I . Varki, A,, Biological Roles Of Oligosaccharides-All Of The Theories Are Correct. Glycohio/ogJ). 1993, 3, 97 - 130.
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6 a-Munnosiduses in Asparugine-linked Oligosucchuvide Processing
2. Cumming, D. A., Glycosylation of recombinant protein therapeutics: control and functional implication, Glycohiology, 1991, 1, 115-130. 3. Brandley, B. K., S. J. Swiedler, and P. W. Robbins, Carbohydrate ligands of the LEC cell adhesion molecules, Cell, 1990, 63, 861-863. 4. Feizi, T., Demonstration by monoclonal antibodies that carbohydrate structures of glycoproteins and glycolipids are onco-developmental antigens, Nature, 1985, 314, 53-57. 5. Feizi, G., and R. A. Childs, Carbohydrate structures of glycoproteins and glycolipids as differentiation antigens, tumour-associated antigens and components of receptor systems, TIBS, 1985, 24-29. 6. Daniel, P. F., B. Winchester, and C. D. Warren, Mammalian a-mannosidases--multiple forms but a common purpose?, Glycohiology, 1994, 4, 551-566. 7. Moremen, K. W., R. B. Trimble, and A. Herscovics, Glycosidases of the asparagine-linked oligosaccharide processing pathway, Glycobiology, 1994, 4, 1 13-125. 8. Kornfeld, R., and S. Kornfeld, Assembly of asparagine-linked oligosaccharides, Ann. Rel;. Biochem., 1985, 54, 631 -664. 9. Neufeld, E. F., Lysosomal storage diseases, Ann. Rev. Biochem., 1991, 60, 257-280. 10. Cacan, R., and A. Verhert, Free and N-Linked Ohgomannosides as Markers of the Quality Control of Newly Synthesized Glycoproteins, Biochem. Biophys. Res. Commun., 1999,258, I 5. 11, Burda, P., and M. Aebi, The dolichol pathway of N-linked glycosylation, Biochim. Biophys. Acta, 1999, 1426, 239-257. 12. Hirschberg, C. B., and M. D. Snider, Topography of glycosylation in the rough endoplasmic reticulum and Golgi apparatus, Annu. Rev. Biochem., 1987, 56, 63-87. 13. Parodi, A. J., Reglucosylation of glycoproteins and quality control of glycoprotein folding in the endoplasmic reticulum of yeast cells, Biochim. Biophys. Acta, 1999, 1426, 287-295. 14. Parodi, A. J., N-glycosylation in trypanosomatid protozoa, Glycobiology, 1993, 3, 193-199. 15. Knauer, R., and L. Lehle, The oligosaccharyltransferase complex from yeast, Biochim. Biophys. Acta, 1999, 1426, 259-273. 16. Herscovics, A,, Glycosidases of the asparagine-linked oligosaccharide processing pathway, 1999, Pages 13-35, in Comprehensive Natural Products Chemistry, B. M. Pinto, ed, Elsevier, New York. 17. Herscovics, A., Processing glycosidases of Saccharomyces cerezjisiae, Biochim.Biophys. Actu, 1999, 1426, 275-285. 18. Ziegler, F. D., T. R. Gemmill, and R. B. Trimble, Glycoprotein synthesis in yeast. Early events in N-linked oligosaccharide processing in Seliiro.saccharomyn);cesponibe, J. Biol. Chem., 1994,269, 12527-12535. 19. Brockhausen, I.; J. Schutzbach, and W. Kuhns, Glycoproteins and their relationship to human disease, Actu Anat., 1998, 161, 36-78. 20. Varki, A., Factors controlling the glycosylation potential of the Golgi apparatus, Trends Cell Biol., 1998, 8, 34-40. 21. Lechner, J., and F. Wieland, Structure and biosynthesis of prokaryotic glycoproteins, Annu. Rev. Biochem., 1989, 58, 173-194. 22. Jakob, C. A,, P. Burda, J. Roth, and M. Aebi, Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyce.~ceretlisiue is determined by a specific oligosaccharide structure, J. Cell Biol., 1998, 142, 1223-1233. 23. Quellhorst. G. J., Jr., J. L. O’Rear, R. Cacan, A. Verbert, and S. S. Krag, Nonglucosylated oligosaccharides are transferred to protein in MIS-5 Chinese hamster ovary cells, Glycobiology, 1999, 9, 65-72. 24. Romero, P. A,, and A. Herscovics, Transfer of nonglucosylated oligosaccharide from lipid to protein in a mammalian cell, J. Biol. Chem., 1986, 261, 15936- 15940. 25. Jaeken, J., G. Matthijs, R. Barone, and H. Carchon, Carbohydrate deficient glycoprotein (CDG) syndrome type I. J. Med. Genet., 1997, 34, 73-76. 26. Trombetta, E. S., and A. Helenius, Lectins as chaperones in glycoprotein folding, Curr. Opin. Struct. Biol., 1998, 8, 587-592. 27. Helenius, A., How N-linked oligosaccharides affect glycoprotein folding in the endoplasmic reticulum, Mol. Biol. Cell., 1994, 5, 253-265.
References
109
28. Williams, D. B., The Merck Frosst Award Lecture 1994/LA conference Merck Frosst 1994, Calnexin: a molecular chaperone with a taste for carbohydrate, Biochem. Cell B i d , 1995, 73, 123-132. 29. Hammond, C., and A. Helenius, Quality control in the secretory pathway, Curr. Upin. Cell B i d . 1995, 7, 523-529. 30. Sousa, M. C., M. A. Ferrero-Garcia. and A. J. Parodi, Recognition of the oligosaccharide and protein moieties of glycoproteins by the UDP-G1c:glycoprotein glucosyltransferase, Biochemistry, 1992, 31, 97-105. 31 Sousa, M. C., and A. J. Parodi, The interaction of the UDP-GLC:glycoprotein glucosyltransferase with the acceptor glycoprotein, Cell Mol. Biol., 1996, 42, 609- 6 16. 32 Wiertz, E. J., T. R. Jones, L. Sun, M. Bogyo, H. J. Geuze, and H. L. Ploegh, The human cytomegalovirus US1 1 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol, Cell, 1996, 84, 769- 779. 33 Wiertz, E. J., D. Tortorella, M. Bogyo, J. Yu, W. Mothes, T. R. Jones, T. A. Rapoport, and H. L. Ploegh, Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction, Nuture, 1996, 384, 432-438. 34 Hammond, C., I. Braakman, and A. Helenius, Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control, Proc. Nutl Accid. Sci. U. S. A , , 1994, 91, 913-917. 35 Knop, M., N. Hauser, and D. H. Wolf, N-Glycosylation affects endoplasmic reticulum degradation of a mutated derivative of carboxypeptidase yscY in yeast, Yeast, 1996, 12, 12291238. 36 Liu, Y., P. Choudhury, C. M. Cabral, and R. N. Sifers, lntracellular disposal of incompletely folded human alphal-antitrypsin involves release from calnexin and post-translational trimming of asparagine-linked oligosaccharides, J. Bid. Chem., 1997,272, 7946-795 1. 31 Liu, Y., P. Choudhury, C. M. Cabral, and R. N. Sifers, Oligosaccharide modification in the early secretory pathway directs the selection of a misfolded glycoprotein for degradation by the proteasome, J. Biol. Chem., 1999,274, 5861-5867. 38. Yang, M., S. Omura, J. S. Bonifacino, and A. M. Weissman, Novel aspects ofdegradation of T cell receptor subunits from the endoplasmic reticulum (ER) in T cells: importance of oligosaccharide processing, ubiquitination, and proteasome-dependent removal from ER membranes, J. E.xp. Med., 1998, 187, 835-846. 39. Herscovics, A,, and P. Orlean, Glycoprotein biosynthesis in yeast, FASEB J . , 1993, 7, 540550. 40. Camirand, A., A. Heysen, B. Grondin, and A. Herscovics, Glycoprotein biosynthesis in Sacckuromyces cereuisiae. Isolation and characterization of the gene encoding a specific processing a-mannosidase, J. Biol. Chem., 1991. 266, 15120-15127. 41. Puccia, R., B. Grondin, and A. Herscovics, Disruption of the processing a-mannosidase gene does not prevent outer chain synthesis in Succhuromyces cerevisiae, Biochem. J., 1993, 290, 21-26. 42. Arar, C., V. Carpentier, J. P. Le Caer, M. Monsigny, A. Legrand, and A. C. Roche, ERGIC53, a membrane protein of the endoplasmic reticulum-Golgi intermediate compartment, is identical to MR60, an intracellular mannose-specific lectin of myelomonocytic cells, J. Bid. Chem., 1995,270, 3551-3553. 43. Itin, C., A. C. Roche, M. Monsigny, and H. P. Hauri, ERGIC-53 is a functional mannoseselective and calcium-dependent human homologue of leguminous lectins: Mol. Biol. Cell, 1996, 7,483-493. 44. Vollenweider, F., F. Kappeler, C. Itin, and H. P. Hauri, Mistargeting of the lectin ERGIC-53 to the cndoplasmic reticulum of HeLa cells impairs the secretion of a lysosomal enzyme, J. Cell B i d , 1998, 142, 377-389. 45 Neerman-Arbez, M., K. M. Johnson, M. A . Morris, J. H. McVey. F. Peyvandi, W. C. Nichols, D. Ginsburg, C. Rossier, S. E. Antonarakis, and E. G. Tuddenham, Molecular analysis of the ERGIC-53 gene in 35 families with combined factor V-factor VIII deficiency, Blood, 1999, 93, 2253-2260. 46 Nichols. W. C.. V. H. Terry, M. A. Wheatlev, A. Y a w . A. Zivelin. N . Ciavarella. C. Stefanile, T. Matsushita, H. Saito, N. B. de Bosch,A . Ruiz-Saez, A. Torres, A. R. Thompson, D. I.
1 10
6 a-Munnosiduses in Aspuragine-linked Oligosucchuride Processing
Feinstein, G. C. White, C. Negrier, C. Vinciguerra, M. Aktan, R. J. Kaufman, D. Ginsburg, and U. Seligsohn, ERGIC-53 gene structure and mutation analysis in 19 combined factors V and VIII deficiency families, Blood. 1999, 93, 2261-2266. 47. Saint-Pol, A,, C. Banvy, P. Codogno, and S. E. Moore, Transfer of free polymannose-type oligosaccharides from the cytosol to lysosomes in cultured human hepatocellular carcinoma HepG2 cells, J. Cell Biol., 1997, 136, 45-59. 48. Saint-Pol, A,, P. Codogno, and S. E. Moore, Cytosol-to-lysosome transport of free polymannose-type oligosaccharides. Kinetic and specificity studies using rat liver lysosomes, J. Bid. Chem., 1999,274, 13547-13555. 49. Anumula, K., and R. Spiro, Release of glucose-containing polymannose oligosaccharides during glycoprotein biosynthesis. Studies with thyroid microsomal enzymes and slices., J. Biol. Chem., 1983,258, 15274-15282. SO. Cacan, R., S. Duvet, D. Kmiecik, 0. Labiau, A. M. Mir, and A. Verbert, ‘Glyco-deglyco’ processes during the synthesis of N-glycoproteins, Biochimie, 1998, 80, 59-68. 51. Moore, S. E., Transport of free polymannose-type oligosaccharides from the endoplasmic reticulum into the cytosol is inhibited by mannosides and requires a thapsigargin-sensitive calcium store, Glycohioloyy, 1998, 8 , 373-381. 52. Hughes, E. A,, C. Hammond, and P. Cresswell, Misfolded major histocompatibility complex class I heavy chains are translocated into the cytoplasm and degraded by the proteasome, Proc. Nut1 Acud. Sci. U. S. A . , 1997, Y4, 1896-1901. 53. Suzuki, T., K. Kitajima, S. Inoue, and Y. Inoue, N-glycosylation/deglycosylation as a mechanism for the post-translational modification/remodification of proteins, Glycoconj. J., 1995, 12, 183-193. 54. Suzuki, T., H. Park, K. Kitajima, and W. J. Lennarz, Peptides glycosylated in the endoplasmic reticulum of yeast are subsequently deglycosylated by a soluble peptide: N-glycanase activity, J. Bid. Chem., 1998, 273, 21526-21530. 5 5 . Duvet, S., 0. Labiau, A. M. Mir, D. Kmiecik, S. S. Krag, A. Verbert, and R. Cacan, Cytosolic deglycosylation process of newly synthesized glycoproteins generates oligomannosides possessing one GlcNAc residue at the reducing end, Biochem. J., 1998, 335, 389--396. 56. Pierce, R. J., G. Spik, and J. Montreuil, Cytosolic location of an endo-N-acetyl-b-D-glucosaminidase activity in rat liver and kidney, Biochem. J., 1979, 180, 673-676. 57. Pierce, R. J., G. Spik, and J. Montreuil, Demonstration and cytosolic location of an endo-Nacetyl-0-D-glucosaminidase activity towards an asialo-N-acetyl-lactosaminic-type substrate in rat liver, Biochem. J . , 1980, 185, 261-264. 58. Grard, T., V. Herman, A. Saint-Pol, D. Kmiecik, 0. Labiau, A. M. Mir, C. Alonso, A. Verbert, R. Cacan, and J. C. Michalski, Oligomannosides or oligosaccharide-lipids as potential substrates for rat liver cytosolic a-D-mannosidase, Biochem. J., 1996, 316, 787-792. 59. Marth, J., Will the transgenic mouse serve as a Rosetta Stone to glycoconjugate function?, Glycoconj. J . , 1994, 11, 3-8. 60. Hennet, T., D. Chui, J. C. Paulson, and J. D. Marth, Immune regulation by the ST6Gal sialyltransferase, Proc. Nut1 Acud. Sci. U. S. A , , 1998, YS, 4504-4509. 61. Maly) P., A. Thall, B. Petryniak, C. E. Rogers, P. L. Smith, R. M. Marks, R. J. Kelly, K. M. Gersten, G. Cheng, T. L. Saunders, S. A. Camper, R. T. Camphausen, F. X. Sullivan, Y. Isogai, 0. Hindsgaul, U. H. von Andrian, and J. B. Lowe, The a(l,3)fucosyltransferase FucTVII controls leukocyte traflicking through an essential role in L-, E-, and P-selectin ligand biosynthesis, Cell, 1996, 86, 643-653. 62. Ellies, L. G., S. Tsuboi, B. Petryniak, J. B. Lowe, M. Fukuda, and J. D. Marth, Core 2 oligosaccharide biosynthesis distinguishes between selectin ligands essential for leukocyte homing and inflammation, Immunity, 1998, 9, 881-890. 63. Metzler, M., A. Gertz, M. Sarkar, H. Schachter, J. Schrader, and J. Marth, Complex asparagine-linked oligosaccharides are required for morphogenic events during post-implantation development, EMBO J., 1994, 13, 2056-2065. 64. Campbell, R., M. Metzler, M. Granovsky, J. Dennis, and J. Marth, Complex asparaginelinked oligosaccharides in Mgatl-null embryos., Glycobiology, 1995, 5, 535-543. 65. Jaeken, J., H. Carchon, and H. Stibler, The carbohydrate-deficient glycoprotein syndromes: pre-Golgi and Golgi disorders?, Glycobiology, 1993, 3, 423-438.
References
11 1
66. Burda, P., L. Borsig, J. de Rijk-van Andel, R. Wevers, J. Jaeken, H. Carchon, E. G. Berger, and M. Aebi, A novel carbohydrate-deficient glycoprotein syndrome characterized by a deficiency in glucosylation of the dolichol-linked oligosaccharide, J. Clin. Invest., 1998, 102, 647652. 67. Jaeken, J., H. Schachter, H. Carchon, P. De Cock, B. Coddeville, and G. Spik, Carbohydrate deficient glycoprotein syndrome type 11: a deficiency in Golgi localised N-acetyl-glucosaminyltransferase 11, Arch. Dis. Child., 1994, 71, 123~-127. 68. Fukuda, M. N., K. A. Masri, A. Dell, L. Luzzatto, and K. W. Moremen, Incomplete synthesis of N-glycans in congenital dyserythropoietic anemia type I1 caused by a defect in the gene encoding a-mannosidase 11, Proc. Nut1 Acud. Sci. U. S. A , , 1990, 87, 7443-7447. 69. Fukuda, M. N., HEMPAS disease: genetic defect of glycosylation, Glycobioloqy, 1990,1,9-15. 70. Chui, D., M. Oh-Eda, Y. F. Liao, K. Panneerselvam, A. Lal, K. W. Marek, H. H. Freeze, K. W. Moremen, M. N. Fukuda, and J. D. Marth, a-Mannosidase-I1 deficiency results in dyserythropoiesis and unveils an alternate pathway in oligosaccharide biosynthesis, Cell, 1997, 90, 157- 167. 71. Lubas, W., and R. Spiro, Golgi endo-a-D-mannosidase from rat liver, a novel N-linked carbohydrate unit processing enzyme, J. Biol. Cliem., 1987,262, 3775- 378 1. 72. Lubas, W., and R. Spiro, Evaluation of the role of rat liver Golgi endo-a-D-mannosidase in processing N-linked oligosaccharides, J. Bid. Chem., 1988,263, 3990-3998. survey of endomannosidase indicates late evolution73. Dairaku. K.. and R. SDiro. Phvlogenetic . ary appearance of this N-linked oligosaccharide processing enzyme, Glycohioloqy, 1997, 7, 579-586. 74. Spiro, R., Q. Zhu, V. Bhoyroo, and H. Soling, Definition of the lectin-like properties of the molecular chaperone, calreticulin, and demonstration of its copurification with endomannosidase from rat liver Golgi, J. Bid. Chem., 1996, 271, 11588-1 1594. 75. Lal, A,, P. Pang, S. Kalelkar, P. A. Romero, A. Herscovics, and K. W. Moremen, Substrate specificities of recombinant murine Golgi al,2-mannosidases IA and IB and comparison with endoplasmic reticulum and Golgi processing al,2-mannosidases, Glycohioloqy, 1998, 8, 98 1995. 76. Lipari, F., B. J. Gour-Salin, and A. Herscovics, The Succhuromyces cereuisiue processing u1,2mannosidase is an inverting glycosidase, Biochern. Biophys. Res. Commun., 1995, 209, 322326. 77. Howard, S., S. He, and S. G. Withers, Identification of the active site nucleophile in jack bean a-mannosidase using 5-fluoro-P-L-gulosyl fluoride, J. Bid. Cliem., 1998, 273, 2067-72. 78. Howard, S., C. Braun, J. McCarter, K. W. Moremen, Y. F. Liao, and S. G. Withers, Human lysosomal and jack bean a-mannosidases are retaining glycosidases, Biochem. Biophys. Rex Commun., 1997,238, 896-898. 79. Henrissat, B., and A. Bairoch, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J., 1996, 316, 695-696. 80. Henrissat, B., and G. Davies, Structural and sequence-based classification of glycoside hydrolases, Cum Opin. Struct. Biol., 1997, 7, 637-644. 8 1. Henrissat, B., Glycosidase families, Biochem. Soc. Trans., 1998, 26, 153-1 56. 82. Byrd, J. C., A. L. Tarentino, F. Maley, P. H. Atkinson, and R. B. Trimble, Glycoprotein synthesis in yeast. Identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing, J. Biol. Chem.. 1982.257, 14657714666, 83. Jelinek-Kelly, S., T. Akiyama, B. Saunier. J. S. Tkacz, and A. Herscovics, Characterization of a specific a-mannosidase involved in oligosaccharide processing in Succhuromyces cereuisiue, J. Bid. Cliem., 1985, 260, 2253-2257. 84. Jelinek-Kelly, S., and A. Herscovics, Glycoprotein biosynthesis in Succhuromyces crrevisiue. Purification of the a-mannosidase which removes one specific mannose residue from Man9GlcNAc, J. Biol. Chem., 1988,263, 14757-14763. 85. Ziegler, F. D., and R. B. Trimble, Glycoprotein biosynthesis in yeast: purification and characterization of the endoplasmic reticulum Mans processing a-mannosidase, Glycobioloqy, 1991, 1, 605-614. 86. Lipari, F., and A. Herscovics, Production, purification and characterization of recombinant yeast processing al,2-mannosidase, Glycohioloqy, 1994, 4, 697-702.
1 12
6 a-Mannosidases in Asparagine-linked Oligosaccharide Processing
87. Lipari, F., and A. Herscovics, Calcium binding to the class I a-1,2-mannosidase from Succhuromyces cerevisiue occurs outside the EF hand motif, Biochemistry, 1999, 38, 1 1 11-1118. 88. Grondin, B., and A. Herscovics, Topology of ER processing a-mannosidase of Sacclzuromyces cerevisiae, Glycohioloyy, 1992, 2, 369-312. 89. Burke, J., F. Lipari, S. Igdoura, and A. Herscovics, The Succhuromyces cerehiae processing al,2-mannosidase is localized in the endoplasmic reticulum, independently of known retrieval motifs, Eur. J. Cell Bid.. 1996, 70, 298-305. 90. Dole, K., F. Lipari, A. Herscovics, and P. L. Howell, Crystallization and preliminary X-ray analysis of the class 1 a I ,2-mannosidase from Succhuromyces cereoisiae, J. Struct. Biol., 1997, 120, 69-72. 91. Bischoff, J., and R. Kornfeld, Evidence for an a-mannosidase in endoplasmic reticulum of rat liver, J. Bid. Chem., 1983, 258, 7907-7910. 92. Bischoff, J., and R. Kornfeld, The effect of I-deoxymannojirimycin on rat liver amannosidases, Biocliem. Biophys. Rex Commun., 1984, 125, 324-33 1. 93. Bischoff, J., L. Liscum, and R. Kornfeld, The use of 1-deoxymannojirimycin to evaluate the role of various u-mannosidases in oligosaccharide processing in intact cells, J. Biol. Chem., 1986,261,4766-4774. 94. Godelaine. D., M. J. Spiro, and R. G. Spiro. Processing of the carbohydrate units of thyroglobulin, J. Bid. Chem., 1981,256, 10161-10168. 95. Hickman, S.. J. L. Theodorakis, J. M. Greco, and P. H. Brown, Processing of MOPC 315 immunoglobulin A oligosaccharides: evidence for endoplasmic reticulum and trans Golgi al,2-mannosidase activity, J. Cell Biol. 1984, 98, 407-416. 96. Atkinson, P. H., and J. T. Lee, Co-translational excision of a-glucose and a-mannose in nascent vesicular stomatitis virus G protein, J. Cell Bid., 1984, 98, 2245-2249. 97. Rosenfeld, M. G., E. E. Marcantonio, J. Hakimi, V. M. Ort, P. H. Atkinson, D. Sabatini, and G. Kreibich, Biosynthesis and processing of ribophorins in the endoplasmic reticulum, J. Cell Biol., 1984, 99, 1076-1082. 98. Rizzolo, L. J., and R. Kornfeld, Post-translational protein modification in the endoplasmic reticulum. Demonstration of fatty acylase and deoxymannojirimycin-sensitive a-mannosidase activities, J. Biol. Chem., 1988, 263, 9520-9525. 99. Brown, P. H., and S. Hickman, Oligosaccharide processing at individual glycosylation sites on MOPC 104E immunoglobulin M. Differences in al,2-linked mannose processing, J. Bid. Chern., 1986,261, 2575--2582. 100. Moore, S., and R. Spiro, Demonstration that Golgi endo-a-D-mannosidase provides a glucosidase-independent pathway for the formation of complex N-linked oligosaccharides of glycoproteins, J. Biol. Chem., 1990, 265, 13104-13112. 101. Moore, S., and R. Spiro, Characterization of the endomannosidase pathway for the processing of N-linked oligosaccharides in glucosidase 11-deficient and parent mouse lymphoma cells, J. Biol. Chern., 1992, 267, 8443-8451. 102. Hiraizumi, S., U. Spohr, and R. Spiro, Characterization of endomannosidase inhibitors and evaluation of their effect on N-linked oligosaccharide processing during glycoprotein biosynthesis, J. Biol. Chem., 1993, 268, 9921-9935. 103. Spiro, M.; V. Bhoyroo, and R. Spiro, Molecular cloning and expression of rat liver endo-amannosidase, an N-linked oligosaccharide processing enzyme, J. Biol. Chem., 1997, 272, 29356-29363. 104. Karaivanova, V., P. Luan, and R. Spiro, Processing of viral envelope glycoprotein by the endomannosidase pathway: evaluation of host cell specificity, Glycobiology, 1998, 8, 725-730. 105. Gonzalez, D. S., K. Karaveg, A. S. Vandersall-Nairn, A. Lal, and K. W. Moremen, Identification, expression, and characterization of a cDNA encoding human ER mannosidase I, the enzyme that catalyzes the first mannose trimming step in mammalian Asn-linked oligosaccharide biosynthesis, J. Biol. Chem., 1999, 274, 21375-21386. 106. Weng, S., and R. Spiro, Endoplasmic reticulum kifunensine-resistant a-mannosidase is enzymatically and immunologically related to the cytosolic u-mannosidase, Arch. Biochem. Biophys., 1996,325, 113-123. ~
References
1 13
107. Weng, S . , and R. Spiro, Demonstration that a kifunensine-resistant a-mannosidase with a unique processing action on N-linked oligosaccharides occurs in rat liver endoplasmic reticulum and various cultured cells, J. B i d . Chenz., 1993, 268, 25656-25663. 108. Weng, S., and R. Spiro, Evaluation of the early processing routes of N-linked oligosaccharides of glycoproteins through the characterization of MangGlcNAcz isomers: evidence that endomannosidase functions in vivo in the absence of a glucosidase blockade, Glycohioloyy, 1996, 6, 861-868. 109. Bause. E., E. Bieberich, A. Rolfs, C. Volker, and B. Schmidt, Molecular cloning and primary structure of Man9-mannosidase from human kidney, Eur. J. Biochem., 1993, 21 7, 535-540. 110. Lal, A., J. S. Schutzbach, W. T. Forsee, P. J . Neame, and K. W. Moremen, Isolation and expression of murine and rabbit cDNAs encoding an al,2-mannosidase involved in the processing of asparagine-linked oligosaccharides, J. Bid. Chem., 1994,269, 9872-9881. 1 1 1 . Bieberich, E., K. Treml, C. Volker, A. Rolfs, B. Kalz-Fuller, and E. Bause, Man9mannosidase from pig liver i s a type-I1 mcmbrane protein that resides in the endoplasmic reticulum. cDNA cloning and expression of the enzyme in COS I cells, Eur. J. Biochem., 1997, 246, 68 1-689. 112. Valee, F., A. Lal, K. W. Moremen, and P. L. Howell, Purification, crystallization and preliminary X-ray crystallographic analysis of recombinant murine Golgi mannosidase IA, a class 1 a-mannosidase involved in Asn-linked oligosaccharide maturation, Actu Cryst., 1999, 0 5 5 , 571-573. 1 1 3. Roth, J., D. Brada, P. M. Lackie, J. Schweden, and E. Bause, Oligosaccharide trimming Man9-mannosidase i s a resident ER protein and exhibits a more restricted and local distribution than glucosidase 11, Eur. J . Cell Biol.. 1990, 53, 131 14 1. 114. Bausc, E., W. Breuer, J. Schweden, R. Roeser, and R. Geyer, Erect of substrate structure on the activity of Man9-mannosidase from pig liver involved in N-linked oligosaccharide processing, Eur. J. Biochem., 1992,208, 451-457. 115. Velasco, A,, L. Hendricks, K. W. Moremen. D. R. P. Tulsiani, 0. Touster, and M. G. Farquhar; Cell type-dependent variations in the subcellular distribution of a-mannosidase I and 11, J. Cell Biol., 1993, 122, 39-51. 116. Tulsiani, D. R., S. C. Hubbard, P. W. Robbins, and 0. Touster, a-D-Mannosidases of rat liver Golgi membranes. Mannosidase I1 is the GlcNAcMANs-cleaving enzyme in glycoprotein biosynthesis and mannosidases Ia and IB are the enzymes converting Man9 precursors to Mans intermediates, J. B i d . C/zem., 1982, 257, 3660-3668. 117. Tulsiani, D. R., and 0. Touster, The purification and characterization of mannosidase IA from rat liver Golgi membranes, J. Bid. Chem., 1988, 263, 5408-5417. 1 18. Herscovics, A,, J. Schneikert, A. Athanassiadis, and K. W. Moremen, Isolation of a mouse Golgi mannosidase cDNA, a member of a gene family conserved from yeast to mammals, J. B i d . Chew?.,1994, 269, 9864-9871. 119. Tremblay, L. O., N. Campbell Dyke. and A. Herscovics, Molecular cloning, chromosomal mapping and tissue-specific expression of a novel human ul,2-mannosidase gene involved in N-glycan maturation, Glycohioloyy, 1998. A', 585 -595. 120. Igdoural, S. A,, A. Herscovics. A. Lal. K. W. Moremen, C. R. Morales, and L. Hermo, aMannosidases involved in N-glycan processing show cell specificity and distinct subcompartmentalization within the Golgi apparatus of cells in the testis and epididymus. Euv. J. Cell B i d , 1999, in press. 121. Ichishima, E., M. Arai, Y. Shigematsu, H. Kuinagai, and R. Sumida-Tanaka, Purification of an acidic a-D-mannosidase from Asprryillus suitoi and specific cleavage of 1;2-a-Dmannosidic linkage in yeast mannan, Biocliirn. Biophys. Actu, 1981, 658, 45-53. 122. Yamashita, K., E. Ichishima, M. Arai, and A. Kobata, An alpha-mannosidase purified from Aspergillus saitoi i s specific for al,2 linkages, Biocheni. Bioph-vs. Res. Comrnun., 1980, 96: 1335 1342. 123. Inoue, T., T. Yoshida, and E. Ichishima, Molecular cloning and nucleotide sequence of the l u ~ and expression of the gene in yeast 1.2-a-D-mannosidase gene, msdS, from A ~ p r r ~ j i l .suitoi cells, Biochini. Biophys. Actci, 1995, 1253, 141- 5. 124. Ichishima, E., N. Taya, M. Ikeguchi, Y. Chiba, M. Nakamura, C. Kawabata, T. Inoue, K. Takahashi, T. Minetoki, K. Ozeki, C. Kumagai. K. Gomi, T. Yoshida, and T. Nakajima, -
114
6 a-Mannosiduses in Asparagine-linked Oligosaccharide Processing
Molecular and enzymic properties of recombinant 1,2-a-mannosidase from Asperqillus saitoi overexpressed in Aspergillus oryzue cells, Biochem. J.. 1999, 339, 589-597. 125. Fujita, A,, T. Yoshida, and E. Ichishima, Five crucial carboxyl residues of 1,2-a-mannosidase from Aspergillus saitoi ( A . phoenicis), a food microorganism, are identified by site-directed mutagenesis, Biochem. Biophys. Rrs. Commun., 1997,238, 719-783. 126. Yoshida, T., and E. Ichishima, Molecular cloning and nucleotide sequence of the genomic DNA for 1.2-a-D-mannosidase gene, msdC from Penicillium citrinum, Biochim. Biophys. Acta, 1995, 1263, 159-162. 127. Yoshida, T., K. Maeda, M. Kobayashi, and E. Ichishima, Chemical modification of Penicillium 1,2-a-D-rnannosidase by water- soluble carbodi-imide: identification of a catalytically important aspartic acid residue, Biochem. J., 1994, 303, 97-103. 128. Dewald, B., and 0. Touster, A new a-D-mannosidase occurring in Golgi membranes, J. B i d . Chem., 1973,248, 7223-7233. 129. Tulsiani, D. R., D. J. Opheim, and 0. Touster, Purification and characterization of u-Dmannosidase from rat liver golgi membranes, J. Biol. Chem., 1977, 252, 3227-3233. 130. Opheim, D. J., and 0. Touster, Lysosomal a-D-mannosidase of rat liver. Purification and comparison with the golgi and cytosolic a-D-mannosidases, J. Biol. Chenz., 1978, 253, 10171023. 131. Spiro, R. G., M. J. Spiro, and A. M. Adamany, Enzymic assembly of the carbohydrate units of glycoproteins and the role of lipid intermediates, Biochem. Soc. Symp., 1974, 37755. 132. Tabas, I., S. Schlesinger, and S. Kornfeld, Processing of high mannose oligosaccharides to form complex type oligosaccharides on the newly synthesized polypeptides of the vesicular stomatitis virus G protein and the IgG heavy chain, J. Biol. Chem., 1978,253, 716-722. 133. Harpaz, N., and H. Schachter, Control of glycoprotein synthesis. Bovine colostrum UDP-Nacety1glucosamine:alpha-D-mannosidep 2-N-acetylglucosaminyltransferase I. Separation from UDP-N-acety1glucosamine:a-D-mannoside p 2-N-acetylglucosaminyltransferase 11, partial purification, and substrate specificity, J. Biol. Chem., 1980, 255, 4885-4893. 134. Harpaz, N., and H. Schachter, Control of glycoprotein synthesis. Processing of asparaginelinked oligosaccharides by one or more rat liver Golgi a-D-mannosidases dependent on the prior action of UDP-N-acetylglucosamine: a-D-mannoside p 2-N-acetylglucosaminyltransferase I, J. Biol. Chrm., 1980, 255, 489444902, 135. Tabas, I., and S. Kornfeld, The synthesis of complex-type oligosaccharides. 111. Identification of an a-D-mannosidase activity involved in a late stage of processing of complex-type oligosaccharides, J. Biol. Chem., 1978, 253, 7779-1186. 136. Kaushal, G. P., T. Szumilo, I. Pastuszak, and A. D. Elbein, Purification to homogeneity and properties of mannosidase I1 from mung bean seedlings, Biochemistry, 1990, 29, 2168-21 76. 137. Altmann, F., and L. Marz, Processing of asparagine-linked oligosaccharides in insect cells: evidence for a-mannosidase 11, Glycocoi~.J . , 1995, 12, 150-155. 138. Sharkey, D. J., and R. Kornfeld, Developmental regulation of processing a-mannosidases and “intersecting” N-acetylglucosaminyltransferase in Dictyostrlium discoideum, J. Biol. Chem., 1991,266, 18477-18484. 139. Sharkey, D. J., and R. Kornfeld, Developmental regulation of asparagine-linked oligosaccharide synthesis in Dictyostelium discoideum, J. Biol. Chem., 1991, 266, 18485- 18497. 140. Misago, M., Y. Liao, S. Kudo, S. Eto, M. Mattei, K. Moremen, and M. Fukuda, Molecular cloning and expression of cDNAs encoding human a-mannosidase I1 and a previously unrecognized a-mannosidase IIx isozyme, Proc. Nut1 Acud. Sci. U.S. A , , 1995, 92, 11766-1 1770. 141. Moremen, K. W., and P. W. Robbins, Isolation, characterization, and expression of cDNAs encoding murine a-mannosidase 11, a Golgi enzyme that controls conversion of high mannose to complex N-glycans, J. Cell B i d , 1991, 115, 1521 1534. 142. Paulson, J. C., and K. J. Colley, Glycosyltransferases: Structure, localization, and control of cell type-specific glycosylation, J. B i d . Chem., 1989, 264, 17615-17618. 143. Colley, K. J., Golgi localization of glycosyltransfera~es:more questions than answers, Glycobiology, 1997, 7, 1-13. 144. Jarvis, D. L., D. A. Bohlmeyer, Y. F. Liao, K. K. Lomax, R. K. Merkle, C. Weinkauf, and K. W. Moremen, Isolation and characterization of a class I1 a-mannosidase cDNA from lepidopteran insect cells, Glycohioloqy, 1997, 7, 1 13-127. ~~
References
1 15
145. Foster, J. M., B. Yudkin, A. E. Lockyer, and D. B. Roberts, Cloning and sequence analysis of GmII, a Drosophila melanogaster homologue of the cDNA encoding murine Golgi amannosidase 11, Gene, 1995, 154, 183- 186. 146. Iolascon, A., G. D’Agostaro, S. Perrotta, P. Izzo. R. Tavano, and B. Mirdglia del Giudice, Congenital dyserythropoietic anemia type 11: molecular basis and clinical aspects, Haematologira, 1996, 81, 543-559. 147. Iolascon, A., E. Miraglia del Giudice, S. Perrotta, M. Granatiero, L. Zelante, and P. Gasparini, Exclusion of three candidate genes as determinants of congenital dyserythropoietic anemia type I1 (CDA-II), Blood, 1997, YO, 4197-4200. 148. Gasparini, P., E. Miraglia del Giudice, J. Delaunay, A. Totaro, M. Granatiero, S. Melchionda, L. Zelante, and A. Iolascon, Localization of the congenital dyserythropoietic anemia I1 locus to chromosome 20q I 1.2 by genomewide search, Am. J. Hum. Genet., 1997, 61, 1 112 11 16. 149. Monis, E., P. Bonay, and R. C. Hughes, Characterization of a mannosidase acting on a1-3and al-6-linked mannose residues of oligoniannosidic intermediates of glycoprotein processing, Eur. J. Biochem., 1987, 168, 287 294. 150. Bonay, P., and R. C. Hughes, Purification and characterization of a novel broad-specificity (al-2, al-3 and a-1-6) mannosidase from rat liver, Eur. J. Biochem., 1991, 197, 229-238. 151. Bonay, P., J. Roth, and R. C. Hughes, Subcellular distribution in rat liver of a novel broadspecificity (al-2: al-3 and al-6) mannosidase active on oligomannose glycans, Eur. J. Biochem., 1992,205. 399-407. 152. Aronson, N. N . , Jr., and M. J. Kuranda, Lysosomal degradation of Asn-linked glycoproteins, FASEB J., 1989, 3. 2615-2622. 153. Cacan, R., C. Dengremont, 0. Labiau, D. Kmiecik, A. M. Mir, and A. Verbert, Occurrence of a cytosolic neutral chitobiase activity involved in oligomannoside degradation: a study with Madin-Darby bovine kidney (MDBK) cells, Bioclrem. J . , 1996, 313, 597-602. 154. Haeuw, J. F., G. Strecker, J. M. Wieruszeski, J. Montreuil, and J. C. Michalski, Substrate specificity of rat liver cytosolic a-D-mannosidase. Novel degradative pathway for oligomannosidic type glycans, Eur. J. Biochen?., 1991, 202, 1257-1 268. 155. a1 Daher, S., R. de Gasperi, P. Daniel, N. Hall, C. D. Warren, and B. Winchester, The substrate-specificity of human lysosomal alpha-D-mannosidase in relation to genetic amannosidosis, Biochem. J., 1991, 277, 743-75 I. 156. Cheng, S. H., S. Malcolm, S. Pemble, and B. Winchester, Purification and comparison of the structures of human liver acidic a-D-mannosidases A and B, Biochem. J . , 1986, 233, 6572. 157. Okumura, T., and I. Yamashina, Further purification and characterization of a-mannosidase from hog kidney, J. Biochem., 1973, 73, 131-138. 158. Berg, T., 0. K. Tollersrud, S. U. Walkley, D. Siegel, and 0. Nilssen, Purification of feline lysosomal a-mannosidase, determination of its cDNA sequence and identification of a mutation causing a-mannosidosis in Persian cats. Biochem. J., 1997, 328, 863-870. 159. Tollersrud, 0. K., T. Berg, P. Healy, G. Evjen, U. Ramachandran, and 0. Nilssen, Purification of bovine lysosomal a-mannosidase, characterization of its gene and determination of two mutations that cause a- mannosidosis, Euu. J. Biochem., 1997, 246, 410-419. 160. Nilssen, O., T. Berg, H. M. Riise, U. Ramachandran, G. Evjen, G . M. Hansen, D. Malm, L. Tranebjaerg, and 0. K. Tollersrud, a-Mannosidosis: functional cloning of the lysosomal amannosidase cDNA and identification of a mutation in two affected siblings, Hum. Mol. Genet., 1997. 6, 717--726. 161. Pohlmann, R., A. Hasilik, S. Cheng, S . Pemble, B. Winchester, and K. von Figura, Synthesis of lysosomal a-mannosidase in normal and mannosidosis fibroblasts, Biochem. Biophys. Rex Commun., 1983, 115, 1083-1089. 162. Liao, Y.-F., A. Lal, and K. W. Moremen, Cloning, Expression, Purification, and Characterization of the Human Broad Specificity Lysosomal Acid a-Mannosidase, J. Biol. Chem.; 1996, 271, 28348--28358. 163. Riise, H. M., T. Berg, 0. Nilssen, G. Romeo, 0. K. Tollersrud, and I. Ceccherini, Genomic structure of the human lysosomal a-mannosidase gene (MANB), Genomics, 1997, 42, 200207. ~
1 16
6 a-Mannosiduses in Asparugine-linked Oligosuccharide Processing
164. Merkle, R. K., Y. Zhang, P. J. Ruest, A. Lal, Y. F. Liao, and K. W. Moremen, Cloning, expression, purification, and characterization of the murine lysosomal acid a-mannosidase. Biochim. Biophys. Actu, 1997, 1336, 132- 146. 165. Gonzalez, D. S., Y. Kagawa, and K. W. Moremen, Isolation and characterization of the gene encoding the mouse broad specificity lysosomal a-mannosidase, Biochim. Biophys. Acta, 1999, 1445, 177-183. 166. Schatzle, J.; J. Bush, and J. Cardelli, Molecular cloning and characterization of the structural gene coding for the developmentally regulated lysosomal enzyme, a-mannosidase, in Dictyostelium discoideum, J. Bid. Chem., 1992, 267, 4000-4007. 167. Vandersall-Nairn, A. S., R. K. Merkle, K. O’Brien, T. N. Oeltmann, and K. W. Moremen, Cloning, expression, purification, and characterization of the acid a-mannosidase from Trypunosoma cvuri, Glycohiuloyy, 1998, 8, 1183-1 194. 168. Schatzle, J., J. Bush, S. Dharmawardhane, R. A. Firtel, R. H. Gomer, and J. Cardelli, Characterization of the signal transduction pathways and cis-acting DNA sequence responsible for the transcriptional induction during growth and development of the lysosomal a-mannosidase gene in Dictyostelium discoideum, J. Biol. Chem., 1993, 268, 19632- 19639. 169. Schatzle, J., A. Rathi, M. Clarke, and J. A. Cardelli, Developmental regulation of the amannosidase gene in Dictyostelium cliscoideum: control is at the level of transcription and is affected by cell density, Mol. Cell. Biol., 1991. 11, 3339-3347. 170. Daniel, P. F.. J. E. Evans, R. De Gasperi, B. Winchester, and C. D. Warren, A human lysosomal a(1-6)-mannosidase active on the branched trimannosyl core of complex glycans, Glycohiology, 1992, 2, 321-336. 171. De Gasperi, R., P. F. Daniel, and C. D. Warren, A human lysosomal a-mannosidase specific for the core of complex glycans, J. Bid. Chenz.. 1992, 267. 9706 9712. 172. Haeuw, J. F., T. Grard, C. Alonso, G. Strecker, and J. C. Michalski, The core-specific lysosomal a( I -6)-mannosidase activity depends on aspartamidohydrolase activity. Biochem. J. 1994,297,463-466. 173. Fisher. K. J . , and N. N. Aronson, Jr., Cloning and expression of the cDNA sequence encoding the lysosomal glycosidase di-N-acetylchitobiase, J Biol Chem, 1992, 267, 19607-19616. 174. Autio, S., T. Louhimo, and M. Helenius, The clinical course of mannosidosis, Ann. Clin. Rex; 1982, 14, 93-97. 175. Bennet, J. K., P. P. Dembure, and L. J. Elsas, Clinical and biochemical analysis of two families with type I and type I1 mannosidosis, Am. J. Med Genet., 1995, 55, 21 -26. 176. Yamashita, K.. Y. Tachibana, K. Mihara, S. Okada. H. Yabuuchi, and A. Kobata. Urinary oligosaccharides of mannosidosis, J. Biol. Chem., 1980. 255. 5126-5133. 177. Nilssen, O., T. Berg, H. M. Riise, U. Ramachandran, G. Evjen, G. M. Hansen, D. Malm, L. Tranebjaerg, and 0. K. Tollersrud, a-Mannosidosis: functional cloning of the lysosomal amannosidase cDNA and identification of a mutation in two affected siblings, Hum. Mol. Genet., 1997, 6 , 717-726. 178. Gotoda, Y., N. Wakamatsu, H. Kawai, Y. Nishida, and T. Matsumoto, Missense and nonsense mutations in the lysosomal a-mannosidase gene (MANB) in severe and mild forms of amannosidosis, Am. J. Hum. Genet.. 1998, 63, 1015-1024. 179. Berg, T., 11. M. Riise, G. M. Hansen, D. Malm, L. Tranebjaerg, 0. K. Tollersrud, and 0. Nilssen, Spectrum of mutations in a-mannosidosis, Am. J. Hum. Genet., 1999, 64, 77-88. 180. Tulsiani, D. R., M. D. Skudlarek, and M. C. Orgebin-Crist, Novel a-D-mannosidase of rat sperm plasma membranes: characterization and potential role in sperm-egg interactions, J. Cell Biol., 1989, 109. 1257-1267. 181. Pereira, B. M., A. Abou-Haila, and D. R. Tulsiani, Rat sperm surface mannosidase is first expressed on the plasma membrane of testicular germ cells, Biol. Reprod., 1998, 59, 128% 1295. 182. Tulsiani, D. R., S. K. NagDas, M. D. Skudlarek, and M. C. Orgebin-Crist, Rat sperm plasma membrane mannosidase: localization and evidence for proteolytic processing during epididymal maturation, Dev. Biol., 1995; 167, 584-595. 183. Tulsiani. D. R.; M. D. Skudlarek, S. K. Nagdas, and M. C. Orgebin-Crist, Purification and characterization of rat epididymai-fluid a-D-mannosidase: similarities to sperm plasmamembrane a-D-mannosidase, Biochern. J . , 1993, 290, 427-436.
184. Cornwall, G. A,, D. R. Tulsiani, and M. C. Orgebin-Crist, Inhibition of the mouse sperm surface u-D-mannosidase inhibits sperm-egg binding in vitro, Bid. Reprod,, 1991, 44, 91 3921. 185. Okamura, N., M. Tamba. H. J. Liao. S. Onoe, Y. Sugita, F. Dacheux, and J. L. Dacheux, Cloning of complementary DNA encoding a 135-kilodalton protein secreted from porcine corpus epididymis and its identification as an epididymis- specific u-mannosidase, Mol. Reprod. Dev., 1995, 42, 141-148. 186. Hiramoto, S., M. Tambd, S. Kiuchi, Y. Z. Jin, S. Bannai, Y. Sugita, F. Dacheux, J . L. Dacheux, M. Yoshidd, and N. Okamura, Stage-specific expression of a mouse homologue of the porcine 135kDa a-D-mannosidase (MAN2B2) in type A sperniatogonia, Biochcw. Bioplzys. Rex Cornniun., 1997, 241, 439-445. 187. Yoshihisa, T., and Y. Anrdku, Nucleotide sequence of AMSI, the structure gene of vacuolar a-mannosidase of Sncchuromyces cer iue, Biochcm. Biiylzys. Rex Cornmun., 1989, 163, 908-915. 188. Kuranda, M. J., and P. W. Robbins, Cloning and heterologous expression of glycosidase genes from Saccliarornycc~scerevisine, Proc.. Nut1 Acud. Sci. U. S. A , , 1987, 84. 2585-2589. 189. Yoshihisa, T., and Y. Anraku, A novel pathway of import of u-mannosidase, a marker enzyme of vacuolar membrane, in Succlzurornji~esciwuisiue, J. Biol. Chem. 1990, 265, 2241 822425. 190. Eades, C. J., A. M. Gilbert, C. D. Goodman, and W. E. Hintz, Identification and analysis of a class 2 a-mannosidase from Aspergillus t~id~ilurz.~., Glycobioloyy, 1998, 8, 17-33. 191. Bischoff, J., K. Moremen, and H. F. Lodish, Isolation, characterization, and expression of cDNA encoding a rat liver endoplasmic reticulum a-mannosidase, J. Bid. Chem., 1990, 265, 17110-17117. 192. Shoup, V., and 0. Touster, Purification and characterization of the a-D-mannosidase of rat liver cytosol, J Biol Clzeni, 1976. 251, 384553852. 193. Tulsiani, D. R., and 0. Touster, Characterization of a novel a-D-mannosidase from rat brain microsomes, J. Biol. Chem., 1985,260. I308 I- 13087. 194. Altschul, S. F., T. L. Madden, A. A. Schaffer. J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids R e x , 1997, 25, 3389-3402. 195. Thompson, J. D., D. G. Higgins, and T. J. Gibson, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids R e x , 1994, 22, 4673 -4680. 196. Page, R. D. M., TREEVIEW: An application to display phylogenetic trees on personal computers, Computer Applic. Biosci., 1996, 12, 351-358.
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
7 The Role of UDP-Glcyglycoprotein Glucosyltransferase as a Sensor of Glycoprotein Conformations Armundo J. Purodi
7.1 Introduction N-glycoproteins are first glycosylated in the lumen of the endoplasmic reticulum (ER) upon transfer of an oligosaccharide (Glc3MangGlcNAc2 in most cells) from a dolichol-P-P derivative to Asn residues in nascent polypeptide chains [ 11. Dolichol is a polyprenol lipid having variable number of isoprene residues (18-2 1 in mammalian, 17-20 in plant, 15-18 in fungal and 10- 13 in protozoan cells). The first isoprene is always saturated. It is worth mentioning that Glc3MangGlcNAcl is transferred about 20-fold more efficiently by the oligosaccharyltransaferase than MangGlcNAc2. Mutations that affect formation of fully glucosylated oligosaccharides result in glycoprotein underglycosylation. Processing of protein-linked oligosaccharides in the ER is initiated immediately after transfer. Glucosidase I removes the external glucose unit and glucosidase I1 (GII) excises the two remaining glucoses. Specific mannosidases may remove up to two mannose units in the mammalian cell ER [l]. An additional ER processing reaction is that catalyzed by the UDP-Glcyglycoprotein glucosyltransferase (GT). This enzyme adds a single glucose unit to glucose-free, high mannose-type protein-linked oligosaccharides [2]. GI1 deglucosylates not only the monoglucosylated oligosaccharides generated by partial deglucosylation of the transferred oligosaccharides but also those formed by GT as they have identical structures [2]. Further oligosaccharide processing may occur in the Golgi apparatus. Protein N-glycosylation and oligosaccharide processing reactions occurring in the ER of mammalian cells as well as the reaction catalyzed by G T and the structure of Glc~MangGlcNAc2are depicted in Figure 1. This chapter deals with some peculiar features of GT specificity as well as with the role of this enzyme in sensing glycoprotein conformations.
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7 The Role o j UDP-Glcyglycoprotein Glucosyltransferase
B
GsMgGNA2-P-P-0
1 G3MgGNA2-Pr
1 G2MgGNA2-Pr
1 G1MgGNA2-Pr
4 4
G,MgGNA2-Pr _L MgGNA2-Pr
C G1M8GNA2-Pr
Man,.,GlcNAc,-Pr
+ UDP-Glc G1M7GNA2-Pr
__
M8GNA2-Pr
1
L M7GNA2-Pr
Figure 1. Glycosylation of glycoproteins in the mammalian cell ER. A: the structure of Glc3Man&ilcNAcz; B: Processing of oligosaccharides in the mammalian ER; C: the reaction catalyzed by GT. D stands for dolichol, Pr for protein, G for glucose, M for mannose and G N for N-acetylglucosamine.
7.2 General Properties GT has been detected in microsomes of mammalian, insect, protozoan, plant, and fungal cells [2, 31. The GT was purified to homogeneity from rat liver, Drosophila melanoyaster and Schizosaccharomyces pombe [ 3-51. The enzyme uses UDP-Glc as glycosyl donor and not ADP-Glc, TDP-Glc or UDP-Gal [4, 51. GT is a rather large (about 160 kDa) soluble protein of the lumen of the ER, and requires Ca2+ ions in millimolar concentrations for activity [4, 51. This last result is consistent with the subcellular location of the enzyme because Ca2+ concentrations in the ER may reach the relatively high values necessary for GT activity. Concentrations of the divalent cation in the cytosol are about three orders of magnitude lower than those in the ER. Further, Ca2+ concentrations in this organelle are regulated by the action of an ion-motif ATPase that pumps calcium into the lumen, and by inositol 1,4,5-triphosphate, that induces release of Ca2+ into the cytosol. It may be speculated, therefore, that variations of Ca2+concentrations in the lumen may affect GT activity.
7.3 GT Recognizes Glycoprotein Conjormations
121
7.3 GT Recognizes Glycoprotein Conformations A striking property of G T purified from either mammalian, insect or S. pomhe cells is that in cell-free assays it glucosylates denatured (misfolded) glycoproteins, but not properly folded ones [2, 61. It was this property that allowed the assay and purification of the enzyme. As is the case with native glycoproteins, glycopeptides containing only one (Asn) or a few amino acids linked to Man7-9GlcNAc2 were not glucosylated by purified G T [6]. This result indicated that the effect of denaturation was not simply to make the oligosaccharides accessible to the transferring enzyme, but rather to allow interaction of protein domains, hidden in native conformations, with GT. The interiors of the water-soluble proteins in their native states are predominantly composed of hydrophobic amino acids whereas the hydrophilic side chains are at the exterior, where they interact with water. Denatured states of a protein have, in general, more hydrophobic side chains at their exterior than native ones do. It could be, therefore, that the effect of denaturation is to create a hydrophobic environment in the vicinity of the oligosaccharide that is recognized by the GT. It was found that under physiological conditions of pH and salt concentration, the G T binds very efficiently to hydrophobic but not hydrophilic peptides, and that the binding of hydrophobic peptides can be inhibited by denatured but not native glycoproteins [7]. Additional experiments showed that GT recognizes not only protein domains exposed in denatured conformations, but also the innermost GlcNAc unit of the oligosaccharide, that is, the residue that is left linked to the protein moiety following treatment with endo-P-N-acetylglucosaminidaseH (endo H) [7].In many native glycoproteins the innermost GlcNAc interacts with neighboring amino acid residues and is not accessible to macromolecular probes such as endo H [8]. In such cases, cleavage of oligosaccharides by the endoglycosidase requires denaturation of the glycoprotein [9]. It may be speculated that proper folding of most glycoproteins would hinder recognition of the innermost GlcNAc unit by the G T and thus prevent glucosylation. Several experiments showed that both recognition elements-the protein domains (probably hydrophobic amino acid patches) and the oligosaccharides-had to be covalently linked [7].This is an important restriction. As there are numerous unfolded, partially folded, and misfolded proteins and glycoproteins in the ER lumen, then if the protein domains recognized by G T and the oligosaccharides were not required to be covalently linked it might be expected that domains exposed in improperly folded species would induce glucosylation of glycoproteins already in their native conformations, as long as the innermost GlcNAc units in the latter species were accessible to GT. G T appeared to be an extremely sensitive sensor of the tertiary structure of glycoproteins, as it was able to recognize two forms of a neoglycoprotein (that is, a glycoprotein formed by a chemical coupling of a protein and an oligosaccharide) having the same secondary but slightly different tertiary structures: a glycopeptide (MangGlcNAcz-Asn) was linked to a Cys residue in position 70 of a full length
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7 The Role of UDP-Glcyglycoprotein Glucosyltransferase
staphylococcal nuclease or of a truncated version of the same protein lacking the last 14 amino acid residues at the carboxyl terminus. The full length neoglycoprotein was not glucosylated by GT unless previously denatured upon a treatment with 8 M urea. On the other hand, the truncated neoglycoprotein was glucosylated by GT even without any denaturation treatment. Both forms of the neoglycoprotein (full length, not able to be glucosylated and truncated, able to be glucosylated) had the same secondary structure as judged by far UV spectra but they slightly differed in their tertiary structures as the full length species had the same specific nuclease activity as the native enzyme, whereas the truncated neoglycoprotein only showed one third of that activity. In addition, the truncated but not the full length neoglycoprotein was degraded by trypsin under controlled conditions [7]. The excellence of G T as a sensor of tertiary structures of glycoproteins was also revealed by the fact that the folding status of glycoproteins was paralleled by its ability to be glucosylated by the enzyme: soybean agglutinin was denatured with 6 M guanidine hydrochloride, diluted and allowed to renature under controlled conditions. The decrease in glucose acceptor capacity of this glycoprotein when incubated with GT plus UDP-Glc closely paralleled its renaturation, thus confirming the above mentioned conclusion [7].
7.4 The Primary Structure of the UDP-Glcyglycoprotein Glucosyltransferase The primary sequences of G T from two different species (D.melanogaster and S. pombe, GenBank accession numbers U20554 and U384 17, respectively) are known [3, 101. Based initially on partial amino acid sequences obtained from the pure rat liver G T and using current nucleic acid sequencing techniques we have recently derived the entire sequence of the enzyme from a rat liver cDNA open reading frame (a collaboration with D. Y. Thomas from the Department of Anatomy and Cell Biology, McGill University, Montreal). The sequence of an open reading frame (gene F48E3.3’; GenBank U28735) of the Caenorhabditis elegans genome probably corresponds to the gene encoding the G T due to its high level of identity with the other three sequences. The four sequences encode predicted proteins that are similar in size, and which have retrieval sequences typical of soluble ER proteins at their carboxyl terminal ends. All four proteins also have several consensus N glycosylation sites, consistent with the fact that the mammalian and fungal enzymes interact with concanavalin A [4, 51. All four enzymes show extremely high sequence homology in their carboxyterminal domains. A similar homology has been found between those domains and conceptual translations of expressed sequence tags from rice and Arabidopsis thaliana (GenBank D24933 and T23006, respectively). Since a limited but significant sequence homology was observed between the carboxyl terminal domains of the fly, fungal and mammalian enzymes and several bacterial glycosyltransferases that use UDP-Glc or UDP-Gal as donor substrates, it may be speculated that the highly conserved G T carboxyl terminal portions are responsible for UDP-Glc recognition.
7.5 The Role o j Monoglucosyluted Oliqosucchurides in Glycoprotein Folding
123
The amino terminal portions of the G T sequences show a lower degree homology than the carboxyl terminal ones. This is especially noticeable upon comparison of the rat liver and S. pombe sequences. Such variations in the extent of primary sequence similarity in different regions in a family of proteins are reminiscent of those seen in the Hsp70 family of proteins which includes the ER chaperone BiP. Here, their amino termini, containing the ATPase activity have a high degree of homology, whereas the carboxyl terminal portions, containing the hydrophobic peptide binding sites, are much less conserved [ l l ] . As mentioned above, under physiological salt and pH conditions, the GT binds hydrophobic peptides exposed in misfolded glycoproteins. The G T shares this property, therefore, with known chaperones and it may well perform this function in vivo. It may be speculated that the amino-terminal domains of G T are responsible for binding the hydrophobic patches, and since they have to recognize a wide variety of different structures, they may be constituted from quite diverse amino acid sequences in different organisms.
7.5 The Role of Monoglucosylated Oligosaccharides in Glycoprotein Folding Glycoproteins acquire their proper tertiary structure in the ER. This process requires participation of chaperones and other folding-assisting proteins. Glycoproteins that fail to properly fold are retained in the ER and further transported to the cytosol where they are degraded in the proteasomes. On the other hand, properly folded species may continue their transit through the secretory pathway to their final destinations. Two unconventional chaperones (calnexin and calreticulin), that recognize monoglucosylated high mannose-type oligosaccharides, have been described in the ER lumen of mammalian cells [12]. Membrane-bound calnexin and soluble calreticulin display a 35% similarity in their amino acid sequence. It has been shown that interaction of calnexin/calreticulin with monoglucosylated glycoproteins generated either by partial deglucosylation of the transferred oligosaccharide or by reglucosylation of glucose-free compounds by G T facilitates glycoprotein folding in mammalian cells by preventing aggregation and suppressing formation of nonnative disulfide bonds [ 131. Moreover, the above mentioned interaction provides one of the alternative mechanisms by which cells retain misfolded glycoproteins in the ER [14]. In vivo disruption of the calnexin/calreticulin-glycoprotein interaction would occur upon GII-mediated deglucosylation of glycoproteins. Not properly folded species would be reglucosylated by G T and recognized again by calnexin/ calreticulin. On the contrary, no reglucosylation would occur upon proper folding and glycoproteins, finally liberated from their calnexin/calreticulin anchors, would be free to continue their transit through the secretory pathway. The calnexin/ calreticulin interaction with monoglucosylated glycoproteins has been considered to be, therefore, a quality control of glycoprotein folding. Although the bulk of evidence supporting the model of quality control as proposed comes from experi-
124
7 The Role of' UDP-Glcyglycoprotein Glucosyltrunsferuse
/
Glc MxI&IcNAC~-P-P-DOI
, G~~,~~,G~CNAC,@
\ GOLGI
i
DEGRADATION
Figure 2. Schematic representation of the role proposed for monoglucosylated oligosaccharides in glycoprotein folding. Sad and happy faces represent not properly folded and properly folded protein moieties, respectively. CNX: calnexin; CRT: calreticulin; OT: oligosaccharyltransferase; GI: glucosidase 1; GII: glucosidase 11 and GT: UDP-Glcyglycoprotein glucosyltransferase. CNX and CRT interact with monoglucosylated oligosaccharides created either by partial deglucosylation of the transferred oligosaccharide or by GT-mediated reglucosylation of glucose-free oligosaccharides exclusively linked to not properly folded protein moieties. The interaction facilitates proper folding of glycoproteins and prevents their exit to the Golgi when not properly folded. Glycoproteins are liberated from the CNXjCRT anchor upon acquisition of the correct tertiary structure. Permanently misfolded species are bound to degradation in the proteasome.
ments performed with mammalian cell systems, available evidence indicates that the same features of the model occur in the yeast S. pombe, in plants and in protozoa [2, 5 , 10, 15-18]. A schematic representation of the role of monoglucosylated oligosaccharides in glycoprotein folding is depicted in Figure 2. It has been more recently reported that ERp57, an ER lumenal redox enzyme (also called GRP58, ERp61, ER 60, HIP70, Q2 or P58) of the protein disulfide isomerase family specifically interacts with partially deglucosylated glycoproteins, as it catalyzes oxidative refolding of glycoproteins recruited by calnexin or calreticulin [ 19-21]. In addition, it has been reported that these two chaperones mediate the interaction of ERp57 with
7.5 The Role of Monoylucosylated Oligosaccliorides in Glycoprotein Folding
125
soluble and membrane-bound chaperones devoid of cysteine residues. This result suggests that ERp57 might have a chaperone-like role not related to its oxidative catalytic properties. We have obtained two single S. pomhe (a yeast having calnexin but not calreticulin) mutants affected in either one of the two pathways leading to the production of monoglucosylated oligosaccharides [22].Alg6 mutants transferred Man9GlcNAcz to nascent polypeptide chains and y p t l mutants lacked GT. Both single mutants were found to grow normally at 28 " C . On the other hand, gptl/ulg6 double mutant cells grew very slowly and with a rounded morphology at 28 "C and did not grow at 37 " C . The wild type phenotype could be restored upon transfection of double mutants with a GT-encoding expression vector. It was concluded that glycoprotein folding facilitation mediated by the interaction of monoglucosylated oligosaccharides with calnexin is essential for cell viability under conditions of severe ER stress such as underglycosylation of proteins caused by the alg6 mutation and high temperature. A glsZ/alg6 double mutant that transferred Man9 GlcNAc2 and was unable to remove the glucose units added by G T as it lacked GI1 had a wild type phenotype of growth and morphology at 28 "C and was able to grow at 37 "C thus indicating that glycoprotein folding facilitation mediated by the interaction of calnexin and monoglucosylated oligosaccharides does not necessarily require cycles of reglucosylation-deglucosylation catalyzed by G T and GI1 [22]. There is a controversy on whether calnexin/calreticulin behave as lectins that exclusively recognize the above mentioned oligosaccharides or if. alternatively, such recognition is the first and necessary step for an interaction between misfolded glycoprotein protein moieties and calnexin/calreticulin. According to this last interpretation, once the protein-protein interaction is established, recognition of monoglucosylated oligosaccharides would be irrelevant for the stability of the complex. Liberation of glycoproteins from the complex would result from a conformational change in the substrate polypeptides. On the other hand, according to the first interpretation release of glycoproteins from the complex would exclusively occur through the action of GII. In both cases, properly folded species would not be able to be reglucosylated by G T and thus no further interaction with calnexin/ calreticulin would occur [ 121. Evidences supporting the second model (interaction of calnexin/calreticulin first with monoglucosylated oligosaccharides and then with the protein moieties of folding glycoproteins) were provided by experiments in which several transmembrane glycoproteins (murine MHC class I heavy chain Kb and Db, human MHC class I HLA heavy chain, MHC class I1 DRa and DRP and invariant chains, T cell receptor a chain) were found to immunoprecipitate with anticalnexin antibodies even after enzymatic removal of all oligosaccharide chains [23-26]. The possibility exists, however, that as both calnexin and the substrates are transmembrane species, they may have remained trapped in the same detergent micelles after removal of oligosaccharides. On the other hand, a soluble glycoprotein as a1 -antitrypsin was also found to be apparently immunoprecipitated by anticalnexin antiserum after removal of oligosaccharides. In this case the above mentioned possible colocalization of calnexin and the substrate glycoprotein in the same micelles may be ruled out [25]. However, a poor solubility of the yet not properly folded a,-antitrypsin
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7 The Role Gf UDP-Glcyglycoprotein Glucosyltransjierase
(and also of the above mentioned deglycosylated transmembrane glycoproteins) may explain its appearance in the immunoprecipitate. Evidence for the role of calnexin/calreticulin as lectins that exclusively recognize monoglucosylated oligosaccharides comes from experiments in which the interaction of glycosylated bovine pancreas RNase with calnexin/calreticulin was studied either in a dog pancreas microsome-rabbit reticulocyte translation system or with isolated RNase and calnexin [27, 281. It was concluded in both studies that removal of oligosaccharides with endoglycosidases or of the glucose units present in the oligosaccharides with GII, completely abolished calnexin/calreticulin interaction with monoglucosylated RNase. The main drawback of the above mentioned conclusion is that the model glycoprotein chosen (RNase) is practically devoid of hydrophobic domains as revealed by a Kyte and Doolittle hydrophilicity plot. An extremely weak interaction of the protein moiety of RNase with calnexin/calreticulin would be expected if such interaction involved hydrophobic domains in the substrate glycoprotein as has been described for folding proteins and classical chaperones. Trypanosoma cruzi is a protozoan parasite that belongs to an early branch in evolution and lacks several features of the pathway of protein N-glycosylation and oligosaccharide processing present in the ER of higher eukaryotes but that nevertheless displays GT and GI1 activities. We have found recently that this microorganism also expresses a calreticulin-like molecule. No calnexin-encoding gene was detected. Addition of anticalreticulin serum to extracts obtained from cells pulse-chased with [ 35S]Met plus [ 35S]Cys immunoprecipitated two proteins that were identified as calreticulin and the lysosomal proteinase cruzipain (a major soluble glycoprotein). A treatment of the calreticulin-cruzipain complexes with endo H either before or after addition of anticalreticulin serum completely disrupted calreticulin-cruzipain interaction. In addition, mature monoglucosylated but not unglucosylated cruzipain isolated from lysosomes was found to interact with recombinant calreticulin. It was concluded that T. cvuzi calreticulin is a lectin that exclusively recognizes monoglucosylated oligosaccharides in glycoproteins [29]. Results obtained with this protozoan obviate drawbacks mentioned above for conclusions supporting either one of both models of the calnexin/calreticulin-glycoprotein interaction. Both calreticulin and cruzipain are soluble proteins (i.e., they can not be trapped in micelles) and the substrate glycoprotein has several hydrophobic domains interspersed along the entire molecule. Moreover, the conclusion that calreticulin behaves exclusively as a lectin that recognizes monoglucosylated oligosaccharides was derived from experiments performed in intact cells, using a naturally expressed glycoprotein. Glycoprotein folding facilitation mediated by the interaction of protein-linked monoglucosylated oligosaccharides and ER lectins is, therefore, a process that appeared early in evolution. Acknowledgments Work performed by the author has been mainly supported by grants from the United Nations Development Program/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases, the National Institutes of Health (USA) and the Howard Hughes Medical Institute.
References
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References 1. R. Kornfeld, S. Kornfeld. Annu. Rea. Bioc.hcn7. 1985, 55, 631-664. S. E. Trombetta, M. Bosch, A. J. Parodi. Bioc.lzm/~7isfr.y.1989, 28, 8 108-8 1 16. C. G. Parker, L. I. Fessler, R. E. Nelson, J. H. Fessler E M 5 0 .I. 1995, 14 1294--1303. S. E. Trombetta, A. J. Parodi. J . Bid. Clie/n. 1992, 267, 9236-9240. F. Fernindez, S. E. Trombetta, U. Hellman, A. J. Parodi. J. Biol. Chem. 1994, 269. 30701-
2. 3. 4. 5.
30706. 6. M. Sousa, M. A. Ferrero-Garcia. A. J. Parodi. Biochemistry. 1992, 31, 97- 105. 7. M. Sousa, A. J. Parodi. E M 5 0 J. 1995, 14. 4196-4203. 8. A. Dessen, D. Gupta, S. Sabesan, C . F. Brewer. J. C. Sacchettini. Biochemistry. 1995, 34, 4933-4942. 9. R. B. Trimble, F. Maley. A n d . Biocltcwi. 1984, 141, 515-522. 10. F. Fernindez, M. Jannatipour, U . Hellman, L. Rokeach, A. J. Parodi. EMBO J. 1996, 15, 705 713. 11. .IP. . Hendrick, F. U. Hartl. Annu. Reil. Bioclietn. 1993. 62, 349- 384. 12. A. Helenius, E. S. Trombetta, D. N . Hebert, J. F. Simons. Trends Cell Biol. 1997. 7, 193~-200. 13. D. N. Hebert, B. Foellmer, A. Helenius. E M B O J. 1996, 15, 2961-2968. 14. J.-X. Zhang, 1. Braakman, K. E. S. Matlack, A. Helenius. Mol. Bid. Cell. 1997, 8. 1943-1954. 15. M. Jannatipour, L. Rokeach. J. Biol. Clrrn7. 1995, 270, 4845-4853. 16. F. Lupattelli, E. Pedrazzini, R. Bollini, A. Vitale. A. Ceriotti. P l m t Ct4. 1997. 9, 597-609. 17 F. Parlati, D. Dignard, J. J. M. Bergeron, D. Y. Thomas. EMBO J. 1995, 14, 3064-3072. 18. C. Labriola, J. J. Cazzulo: A. J. Parodi. J. Cc4 Biol. 1995, 130, 771-779. 19 J. D. Oliver, F. J. van der Wal. N. J. Bulleid, S. High. Science. 1997, 275. 86-88. 20 J. G. Elliott, J. D. Oliver, S. High. J. Biol. Client. 1997, 272, 13849- 13855. Darby. D. C. Tessier, M . Michalak, J. J. Bergeron, D. Y. Thomas. J. Biol. 21 Chem. 1998, 273, 6009-6012. 22 S. Fanchiotti, F. Fernindez. C. D'Alessio, A. J. Parodi. J . Cdl Bid. 1998. 143. 625-635. 23 B. Arunachalam, P. Cresswell. J. Biol. Chetn. 1995, 270, 2784-2790. 24 M. J. Bennett, J. E. M. Van Leeuwen, K. P. Kearse. J. Biol. Chetn. 1998. 273, 23674423680, 25. F. E. Ware, A. Vassilakos, P. A. Peterson, M. R. Jackson, M. S. Lehrman, D. B. Williams. J. Biol. Chem. 1995. 270, 469774704. 26. Q. Zhang, M. Tector, R. D. Salter. J. Bid. C%e/n.1995, 270, 3944-3948. 27. A. R. Rodan, J. F. Simons. E. S. Trombetta. A. Helenius. E M 5 0 J . 1996, 15. 6921-6930. 28. A. Zapun, S. Petrescu, P. M. Rudd, R. A. Dwek, D. Y. Thomas, J. J. M. Bergeron. Cell. 1997, 88. 29-38. 29. C. Labriola, J. J. Cazzulo, A. J. Parodi. Mol. Biol. CeN 1999, 10, 1381 1394
Carbohydrates in Chemistry and Biology Beat Ernst, Gerald W. Hart, Pierre Sinay copyrightQ WILEY-VCH Verlag GmbH, 2000
8 Mannosyltransferases Peter Orlean
8.1 Introduction Covalently-linked mannose is present in the glycans, glycoproteins, glycolipids, and sugar nucleotides made by eubacteria, archaea, and eukaryotes. Attachment of mannose can be through 0-glycosidic linkages to other sugars, through 0-linkages to serine or threonine in peptides, through a C-C linkage to tryptophan, and through monophosphates to other sugar residues or polyisoprenoids. Mannosyltransferases (Man-T) are so far known to use one of only two classes of mannosyl donor, GDPMan or polyisoprenoid monophosphate mannose. There is much more diversity in the type of acceptor molecule used and in the type and configuration of linkage formed. Biochemical and genetic studies of glycan synthesis have led to the cloning of many glycosyltransferase genes, and genome sequencing projects have been turning up many more genes encoding homologous proteins. Some of the latter have subsequently been shown to participate in glycan synthesis, whereas the roles of others are as yet unclear. The availability of amino acid sequences of increasing numbers of known and putative glycosyltransferases has in turn started to reveal that these proteins can be grouped into families. Some families have representatives in eubacteria, archaea, and eukaryotes, indicating an early evolutionary origin, but other families so far seem restricted to eukaryotes. Amino acid sequence comparisons have permitted some generalizations to be made about the residues likely to be important for catalysis of glycosyl transfer by certain classes of protein. However, it is not yet possible to correlate conserved primary amino acid sequence features of a glycosyltransferase with the nature of the sugar it transfers or the type of linkage it forms. Much progress has been made in identifying Man-T genes and defining the roles of their products various biosynthetic pathways. Man-T will be discussed in the context of the secretory pathway of eukaryotic cells and the biosynthetic pathways that are localized in the ER and Golgi. Proteins related by amino acid sequence
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similarity will be considered as a group. Within this organizational context, reference will be made to related proteins in eubacteria and archaea.
8.2 Occurrence of Covalently-linked Mannose 8.2.1 Eukaryotic Secretory Glycoproteins
Mannose is present in the N - and O-linked glycans and glycosylphosphatidylinositol (GPI) anchors of secretory glycoproteins [ 1-51, In yeast, N-linked saccharides can bear long, branched mannan chains. Other eukaryotic cells also make mannosecontaining polysaccharides, for example, the gluco- and galactomannans of legume cell walls [6,7] and the glucuronoxylomannan capsular polysaccharide of the human pathogenic yeast Cryptococcus fieqforMzuns [S]. At least some of the latter structures may be elaborated on protein-bound glycans, and, consistent with this, the Man-T involved in their synthesis in plants occur in the endomembrane system of the cell. 8.2.2 Glycophospholipids
Man-containing glycophospholipids serve as donors or intermediates in the biosynthesis of various glycans. Particularly widespread are polyisoprenoid-phosphate Man and polyisoprenoid pyrophosphate-linked, Man-containing glycans, which are made by eubacteria, archaea, and eukaryotes. The former are sugar donors in glycan biosynthetic pathways, whereas the latter serve as carriers of larger glycan structures. Free, phosphoinositol-linked, Man-containing glycans such as glycoinositol phospholipids and lipophosphoglycans are anchored in the plasma membrane of protozoa [9], and mannosylated inositol phosphoceramides are made in fungi and plants [lo]. 8.2.3 Eubacterial and Archaeal Mannose-containing Molecules
Some of the earliest studies of Man-Ts were on polyprenol-P-Man made by Micrococcus Zysodeikticus [ 1I]. Other well-studied molecules are the O-antigen repeats of the lipopolysaccharide (LPS) of the outer membrane of Gram-negative bacteria [ 121, exopolysaccharides such as xanthan [ 131, and the lipoarabinomannan of the mycobacterial cell envelope [ 141. At least one archaeon, Thermoplasma acidophilum, makes a protein-linked glycan containing al,2-, a1,3-, and al,6-linked Man [ 151. 8.2.4 C-linked Mannose
Mannose can become directly linked to protein in a C-C linkage between C-1 of Man and the indole moiety of the first tryptophan in the sequence -WXXW- [16,
8.3 Biochemistry of Munnosyl Trunsjijr
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171. This type of protein mannosylation has been found so far in mammalian RNase 2, but may be more widespread.
8.3 Biochemistry of Mannosyl Transfer 8.3. Many Linkages, Two Donors Mannose is commonly linked to other mannoses, to other sugars, or to inositol in ~ 1 . 2 -al,3-, a1,4-, al,6-, P1,2-, and Pl,4-glycosidic linkages, to polyisoprenoid phosphates in P-linkages, and to serine and threonine hydroxyls, as well as to tryptophan, in a-linkage. Most Man-T transfer single Man residues, but in some cases, polymannose chains can be made. An example of polymerizing activity is the formation of the a1,6-linked backbone of yeast mannan. In this case, a complex of potential Man-Ts is involved (Section 8.4.2), but it is not known whether any one protein transfers all the mannoses, or whether multiple catalytic subunits cooperate. The latter can be achieved in a single protein, MtfAp (or WbdAp), which is involved in 0-9 LPS synthesis in Eschericlzia coli. This protein has two catalytic domains that presumably arose in an earlier gene duplication event, and which allow Man to be transferred to two different acceptor structures in succession. One domain first adds cxl,2-Man to an al,3-linked Man, and the second domain then adds two further al,2-Man to the al,2-Man acceptor the first domain just made [ 18, 191. Use of GDPMan or of polyisoprenoid phosphate-linked Man as donor is not correlated with the type of glycosidic linkage a given Man-T forms: for example, some of the a1,2-, a1,3-, and al,6-Man-Ts involved in assembly of the Dol-PPlinked precursor in N-glycosylation use GDPMan as donor, others, Dol-P-Man. No Man-T is known to use both nucleotide and lipid-linked donors, and the glycosyltransferase families identified so far contain proteins that use only one of the two types of substrate. 8.3.2 Donor Specificity
GDPMan is strongly preferred as donor by sugar nucleotide-requiring Man-T. The specificity seems to be for the GDP moiety, for in vitro, some Man-T can use GDPMan analogs in which the mannose has been modified or replaced with another sugar [20]. The ability of Man-T to recognize analogs of GDPMan has been exploited for photoaffinity labeling of Man-T with GDP-he~anolamine-’~~ Iazidosalicylic acid [21]. So far, however, the structural basis for the specificity of glycosyltransferases for the nucleotide moiety of their donor is not known. Indeed, most families of glycosyltransferases related by amino acid sequence similarity include Man-T as well as enzymes that use other sugar nucleotide donors. Synthesis of the inner core of the LPS of E. coli and Rhizohium leguminosarum provides an example of glycosyltransferases whose natural sugar nucleotide donors
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are different, but which can both use the same non-physiological “hybrid” donor. In E. coli, the inner core contains glyceromanno-heptose (Heptose) linked a1,S to ketodeoxy-manno-octulosonic acid (Kdo), whereas in the Rhizohium core, it is Man that is al,5-linked to Kdo. The E. cnli a1,S heptosyltransferase, RfaCp, uses ADPHeptose as donor, and, in vitro, can also use synthetic ADPMan, but not GDPMan. R. leguminosarurn has a GDPMan-utilizing al,5-Man-T activity that recognizes the same acceptor structure as RfaCp does, but which can also use nonphysiological ADPMan as donor [22]. Comparison of the amino acid sequence of the predicted GDPMan-utilizing Rhizohium al,5-Man-T with that of RfaCp may reveal which amino acid residues are important for discrimination between ADP and GDP. In addition to their specificity for the sugar moiety of their donor, the Dol-PMan-utilizing Man-T of the dolichol pathway, as well as the Pmtlp Dol-P-Man: protein O-Man-T, strongly prefer their donor to have a long-chain, lipophilic polyisoprenoid with a p-Man-1 -P attached to a saturated a-isoprene group [23-25]. 8.3.3 Acceptor Specificity Some Man-T, for example, those involved in the ordered assembly pathway for the Dol-PP-linked oligosaccharide [26], can show high specificity for an acceptor glycan of a particular structure, although the protein-glycan interactions involved are as yet unknown. Additional features of some mannosyl acceptors, for example, the nature of their lipid moieties, can have a profound effect on Man-T activity. The influence of isoprenoid chain length and of saturation or unsaturation of the a-isoprenoid has been explored for Man-T that use polyisoprenoid-Ps as substrate. In vitro, Dol-PMan synthase can use shorter chain dolichols, polyprenols, even phenyl phosphate, as mannosyl acceptors [27, 281, but there is a clear preference for a-saturation and for a chain length greater than seven isoprenoid units, the effect of decreasing chain length being to lower the V,, of the reaction. The Man-T of the early part of the dolichol pathway, however, do not require the polyisoprenoid carrier to be long chain dolichol in vitro, for Man5 can be assembled on GlcNAc2 linked to the tetraisoprenoid phytanol [29]. It is not known how these proteins recognize the polyisoprenoid. A photoactivatable dolichol analog has been described that can be phosphorylated, and could thus be used to identify portions of Man-T that are in close proximity to bound Dol-P derivatives [ 301. The Dol-P-Man-utilizing Man-T that adds the first, al,4-Man to GlcN-PI during GPI biosynthesis varies between species in its acceptor specificity. The Trypanosoma enzyme mannosylates GlcN-PI without prior acylation of inositol, whereas the mammalian Man-T requires the 2’-OH of inositol to be acylated [3I , 321. 8.3.4 Structural Features of Man-T
The majority of Man-T are membrane proteins: Man-T activities sediment with membranes, and the available amino acid sequences of Man-T contain one or more predicted transmembrane helices. In a few cases, the transmembrane topography of
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a Man-T has been investigated, and the results are consistent with the accepted notion that the catalytic domains of Dol-P-Man-utilizing Man-T of the endoplasmic reticulum (ER) face the lumen [ S , 331. The GDPMan-utilizing Man-T of the dolichol pathway are believed to catalyze mannosyl transfer at the cytoplasmic face of the ER membrane, whereas mannose transfer from GDPMan in the yeast Golgi takes place in the lumen of that organelle, as expected for these Type I1 membrane proteins. The GDPMan-utilizing Man-T in general have fewer predicted transmembrane segments than the Dol-P-Man-requiring ones, but the greater apparent hydrophobicity of the latter proteins may not necessarily be indicative of multiple transmembrane segments. Thus, experimental determination of the transmembrane organization of Dol-P-Man-utilizing Pig-Bp reveals the protein spans the membrane only once (section 8.4.1). No detailed structure has yet been determined for any Man-T, however, amino acid sequence comparisons have identified motifs conserved in two glycosyltransferase super families that include Man-T. These motifs contain invariant amino acids with acidic side chains that could be important catalytically or for maintenance of structure. A comparison of P-glycosyltransferase sequences by hydrophobic cluster analysis led to the identification of two regions in these proteins in which aspartates are highly conserved [34]. One of these contains what is now recognized as the “DXD” motif, which is found in at least 13 a- and P-glycosyltransferase families that use a sugar nucleotide donor [ 3 S ] , and its presence is an important criterion in the identification of further such enzymes. Site-specific mutagenesis experiments on the two aspartates in Mnnlp, a yeast a1,3 Man-T, showed that both residues are indeed critical for enzyme activity [3S].The second aspartate-containing region [34] includes the sequence -(aliphatic amino acid)4DD-, which is also conserved in members of the phosphoribosyltransferase family, in which the pair of aspartates interacts (via Mg”) with the OH-groups on the ribose moiety of phosphoribosyl pyrophosphate [36]. In glycosyltransferases, these aspartates may likewise interact with the ribose portion of the sugar nucleotide substrate. A motif containing two invariant glutamates, “EXFGXXXXE” (or, more loosely, “E(X7)E”), occurs in a range of a-glycosyltransferases from eubacteria, archaea, and eukaryotes [IS]. Much information is yet needed before we know how a Man-T distinguishes GDPMan from other sugar nucleotides, and what structural features are correlated with the ability of closely-related Man-T to form different a-mannosidic linkages or to transfer Man-P (as can different members of the Ktrlp family (section 8.4.2)).
8.4 Man-T Families and the Pathways They Participate in The Man-Ts that participate in the pathways leading to N-glycosylation, GPI anchoring, and O-mannosylation of protein in the ER will be discussed first, then the Golgi Man-T families involved in the elaboration of yeast mannan. Protein families whose membership includes eubacterial and archaeal proteins will be identified and examples of eubacterial mannosylation pathways will be given.
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Landmark studies that led to the identification of yeast Man-T genes include the analyses of yeast mannan formation in mnn mutants [37], and the use of [3H]mannose suicide enrichment to isolate yeast a1g mutants in the dolichol pathway [38]. The subsequent finding that late-stage, non-conditional alg mutants have a synthetic phenotype in combination with a defect in oligosaccharyltransferase has permitted the cloning of further ALG genes [3]. The isolation of murine Thy1 lymphoma and Chinese hamster ovary mutants were seminal contributions to our understanding of Man-T and the isolation of mammalian Man-T genes [39, 401. Many Man-T genes that are conserved among eukaryotes were originally identified in Saccharomyces cerevisiae, and yeast also has additional Man-T families whose members may be confined to fungi. Accordingly, the yeast nomenclature for Man-Ts will mainly be used, although the discussion of the ER Man-T is intended to apply to all eukaryotes. Man-Ts will be grouped into “families” for discussion, a “family” being referred to if a given protein has obvious sequence homologs in yeast. 8.4.1 Man-Ts of the ER [l-51
It is generally believed that the Man-T involved in the construction of the Dol-PPlinked oligosaccharide and the GPI precursor are localized in the ER membrane. However, dolichol pathway activities have also been detected in mitochondria [41], but it’s not clear whether known genes or new ones encode the enzymes involved. The Man-T discussed here are regarded as ER proteins. AMP The gene for this p-1,4 Man-T, which transfers mannose from GDPMan to Dol-PPGlcNAc;! at the cytoplasmic face of the ER membrane during assembly of the DolPP-linked precursor, was cloned by complementation of the yeast d g l mutant [38]. Alglp is expressed in E. coli [42]. The protein is conserved among eukaryotes, and although there are no close relatives of this protein in any eukaryote’s genome, Alglp shows limited resemblence to the Alg2p family [3], and has homologs in eubacteria and archaea; however, Algl p lacks the “E(X7)E” signature. AlgZp/Algl l p Alg2p adds either the u1,2- or the u1,6-linked mannose to the p-1,4 Man during assembly of the Dol-PP-linked precursor, presumably at the cytoplasmic face of the ER membrane. Alg2p has a homolog in the yeast protein database, Algl lp, which may therefore also be a GDPMan-dependent transferase that acts early in dolichol pathway. Alg2p and Algl l p are members of a large family of eukaryotic, eubacterial, and archaeal proteins with the “E(X7)E” motif [18]. Most of these proteins seem to be retaining a-glycosyltransferases that use a sugar nucleotide as donor and a sugar or inositol as acceptor. Among the bacterial Man-T in this group are cellobiosyldiphosphoprenyl u-Man-Ts involved in acetan and xanthan biosynthesis [43] and Man-T involved in O-antigen synthesis in Gram negative bacteria (eg. Rhizobium LpcCp and Sulmonella RfbUp) [12, 441.
8.4 Mali-T Funiilirs and the Pathways They Purticipute in
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DPmlP Dol-P-Man synthase catalyzes transfer of Man from GDPMan to Dol-P with inversion of configuration to form Dol-P-PMan. This enzyme activity is ubiquitous in eukaryotes, and Dol-P-Man serves as a donor in the dolichol pathway 12, 31, in GPI biosynthesis [ 11, in 0-mannosylation [5], and in C-mannosylation [ 161. Related enzyme activities have been detected in eubacteria, for example Mycobacterium [ 141, and in archaea 1451, polyprenol-P and Dol-P being used, respectively, as acceptors. Eukaryotic Dol-P-Man synthases fall into two classes 146, 471. One, the “S. cereuisiae” class, consists of a single catalytic Dpml protein that is expressed in E. coli and which has a COOH-terminal transmembrane domain [48]. The other, “human”, class is a complex of two proteins, a Dpmlp subunit that lacks a COOH terminal domain and which is not active in E. coli, and the small, hydrophobic Dpm2p protein. Dpmlp is catalytic, and Dpm2p is required both for localization of Dpmlp to the ER membrane, and for optimal binding of Dol-P to the complex [49J. S. cerevisiue D P M l complements mammalian mutant cell lines defective in either Dpmlp or Dpm2p, but “human” class D P M l genes do not rescue a lethal disruption of S. cerevisiae D P M l . The amino terminal half of Dpmlp resembles that of Dol-P-Glc synthases 1501, and also shows a high degree of homology to eubacterial and archaeal members of a large family of P-glycosyltransferases I341 (section 8.3.4). Dpmlp is sensitive to the physical state of its membrane environment, and lipid matrices that form destabilized bilayers promote the association of the enzyme with Dol-P and are necessary for optimal activity [51]. More specific modulation of Dol-P-Man synthase activity may be achieved through phosphorylation by CAMPdependent kinase 1521, and the six Dpmlp sequences indeed all have a potential site for phosphorylation by that enzyme [46]. Ak3P This Dol-P-Man-utilizing u1,3 Man-T adds the 6th Man during assembly of the Dol-PP-linked precursor in N-glycosylation [ 31, presumably on the lumenal side of the ER membrane. Alg3p has no obvious homologs in prokaryotes.
Alg9p/PIG-Bp family Members of this group are Dol-P-Man-utilizing transferases that function in either the dolichol pathway or in the GPI anchor assembly pathway. ALG9 and PIG-B were cloned by complementation, respectively, of a yeast N-glycosylation mutant [3] and the murine Thyl-B mutant, which is defective in mannose addition to the GPI glycan core 1331 and genes encoding related proteins were subsequently identified in the S. cereaisiue genome 12, 53, 541. Yeast Alg9p and Algl2p act in the dolichol pathway to add, respectively, an al,2-linked Man to the wl,3-Man transferred by Alg3p, and an a1,6-Man to the GDPMan-derived al,6-Man [3, 261. Neither is essential for viability. Pig-Bp, whose counterpart in S. cerevisiue is the essential Gpi10 protein [32, 53, 541, adds the third, wl,2-linked mannose during assembly of GPI precursor. Intriguingly. the S. cerevisiae genome encodes a fourth Pig-Bp homolog, Smp3p, which like GpilOp is
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8 Munnosyltrunsj2rases
essential for growth. smp3 mutants are blocked in GPI anchoring and accumulate a Man3-containing GPI [75],suggesting that Smp3p adds the fourth, al,2-Man that has been detected on the complete yeast GPI precursor [55]. Smp3p has a homolog in fission yeast but, unlike the other Alg9p/Pig-Bp family members, Smp3p has no obvious counterpart in Cuenorhabditis elegans or, so far, mammals. If an essential fourth mannosylation step in GPI biosynthesis is confined to eukaryotic microbes and absent from mammalian cells, it could be exploited as a target for antifungal or antiparasitic agents. Hydropathy analyses of Alg9p/Pig-B family members indicate the presence of multiple potential transmembrane segments. However, experimental determination of the transmembrane topology of Pig-Bp reveals the protein has a short cytoplasmic domain, a single membrane-spanning segment, and a large lumenal, catalytic domain [33].Whether the other family members adopt a similar membrane topology is not yet known. Alg9p/Pig-B have no obvious homologs in eubacteria, but weak similarity can be detected to predicted Archaeaglohus and Py~ococcusproteins. Pmtlp family Dol-P-Man-dependent, 0-mannosylation of protein has been intensively studied in yeast [ 5 ] .In this pathway, a first Man is transferred in the ER from Dol-P-Man to serine or threonine, with inversion of configuration to form an a-mannosyl linkage. Up to four further a-Man residues are added in GDPMan-dependent reactions in the Golgi. Transfer of the first Man is catalyzed in the ER lumen by Pmt proteins, seven of which are encoded in the S. cerevisiae genome. The Pmtp’s have multiple transmembrane domains (seven in the case of Pmtlp [56])and a lumenal catalytic domain, and these proteins can form heterodimers. Studies using synthetic peptides indicate that threonine is a better mannosyl acceptor in vitro, but the in vivo acceptor preferences of the individual Pmtps are not yet known. The existence of multiple Pmtps with differing acceptor specificities, coupled with the potential for these proteins to form heterodimers, may permit a wider range of peptide sequences to be recognized as substrates. However, deletion experiments with yeast P M T genes show that in some cases, a given Pmtp may be predominantly responsible for 0-mannosylation of an individual protein, but that in others, several Pmtps have overlapping functions in 0-mannosylation of a protein. 0-mannosylation of protein is essential for viability in S. cereuisicre, and a deficiency in only one Pmt homolog in the fungal pathogen Candidcr ulbicans lowers virulence [57].Were 0-mannosylation confined to fungi, then the Pmtps could be exploited as targets for antifungal agents. However, Pmt-related proteins are encoded in nematode, insect, and mammalian genomes, and 0-linked mannose is found on mammalian proteins, raising the possibility that these organisms also carry out Pmtp-dependent 0-mannosylation. 8.4.2 Golgi Man-Ts and Fungal Mannan Synthesis
In S. cereuisiue, N-linked saccharides can be extended into a “core” structure containing no more than 15 sugars, or into an “outer chain” containing up to 200 mannoses and consisting of an ctl,6-linked polymannose backbone with short al,2-
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linked Man side-branches that end in a1,3-linked Man [58]. These side chains can also bear Man linked through phosphate [59].Assembly of the outer chain is carried out in the lumen of Golgi compartments by GDPMan-utilizing Man-T, and members of five families of Type TI membrane proteins are involved in these mannosylation reactions. With the exception of the Mnnl 0p family, these proteins have no obvious homologs outside the fungi. An increasingly complete understanding of yeast mannan assembly is emerging [4, 37, 60-651. In brief, mannan formation is initiated on the asparagine-linked GlcNAc2Mans core by addition of an u1,6-Man by Ochlp [60]. A short polymer of al,6-Man is then elongated on this mannose by a complex of Mn