Synthesis and Characterization of Glycosides

Synthesis and Characterization of Glycosides Synthesis and Characterization of Glycosides Marco Brito-Arias Biotechn...

Author: Marco Brito-Arias

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Synthesis and Characterization of Glycosides

Synthesis and Characterization of Glycosides

Marco Brito-Arias Biotechnology Unit National Polytechnic Institute of Mexico (UPIBI-IPN)

Marco Brito-Arias Biotechnology Unit National Polytechnic Institute, Mexico la Laguna Ticomˆa Mexico

Library of Congress Control Number: 2006927420 ISBN 10: 0-387-26251-2 ISBN 13: 978-0-387-26251-2 C 2007 Springer Science+Business Media, LLC All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identiﬁed as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.

9 8 7 6 5 4 3 2 1 springer.com

To Carmina Daniela

Contents

Preface ...................................................................................

xii

1. Glycosides, Synthesis, and Characterization ............................... 1.1. Introduction .................................................................. 1.2. Reactions of Monosaccharides ........................................... 1.3. Chemical Modiﬁcations ................................................... 1.3.1. Oxidations ........................................................... 1.3.2. Periodate Oxidation................................................ 1.3.3. Tollens Reaction .................................................... 1.3.4. Benedict and Fehling Test ........................................ 1.3.5. Nucleophilic Addition............................................. 1.3.6. Enediol Rearrangement ........................................... 1.3.7. KilianiFischer Synthesis .......................................... 1.3.8. The Ruff Degradation ............................................. 1.3.9. Conversion of Pentose to Furfural............................... 1.4. Biosynthesis of Sugars..................................................... 1.4.1. Sugars as Energy Sources......................................... 1.5. Synthesis of Carbohydrates ............................................... 1.5.1. Chemical Synthesis ................................................ 1.5.2. C-Glycosyl Amino Acids ......................................... 1.5.3. Enzymatic Synthesis............................................... 1.5.4. Chemoenzymatic Synthesis ...................................... 1.6. Synthesis of Carbohydrates Mimetics................................... 1.6.1. Iminosugars ......................................................... 1.6.2. Aminosugars ........................................................ 1.6.3. Thiopyranoside Monosaccharides............................... 1.6.4. Carbapyranoside-Saccharides.................................... 1.7. Glycoside Reactivity ....................................................... 1.8. The Leaving Groups........................................................ 1.9. Glycosyl Donors ............................................................ 1.10. Protecting Groups...........................................................

1 1 2 2 3 4 5 5 6 9 9 10 10 11 11 12 12 16 21 23 25 25 29 34 34 35 35 39 43

vii

viii

Contents

1.11. Selective Protections ....................................................... 1.12. Selective Deprotections.................................................... References ...........................................................................

52 59 63

2. O-Glycoside Formation .......................................................... 2.1. General Methods ............................................................ 2.1.1. The Michael Reaction .......................................... 2.1.2. The Fischer Reaction ........................................... 2.1.3. The Koenigs-Knorr Reaction ................................. 2.1.4. The Helferich Reaction ........................................ 2.1.5. The Fusion Reaction............................................ 2.1.6. The Imidate Reaction........................................... 2.1.7. The Sulfur Reaction ............................................ 2.1.8. The Armed-Disarmed Method................................ 2.1.9. The Glycal Reaction ............................................ 2.1.10. Miscellaneous Leaving Groups............................... 2.1.10.1 Fluoride Glycosyl Donors ......................... 2.1.10.2 Silyl Glycosyl Donors .............................. 2.1.10.3 Heterogenous Catalysis ............................ 2.1.10.4 The Pool Strategy.................................... 2.1.10.5 Phosphate Glycosyl Donors ....................... 2.1.11. Enzymatic Approach ........................................... 2.1.11.1 Enzymatic Synthesis of Oligosaccharides ...... 2.1.12. The Solid-Phase Methodology................................ 2.2. Cyclic Oligosaccharides ................................................... 2.2.1. Chemoenzymatic and Enzymatic Synthesis................ References ...........................................................................

68 68 68 70 73 76 78 80 88 95 98 104 104 105 110 112 112 113 117 117 124 127 134

3. N-Glycosides........................................................................ 3.1. Nucleoside Formation...................................................... 3.2. Protecting Groups........................................................... 3.2.1. Ribofuranoside Protecting Groups ........................... 3.3. General Methods ............................................................ 3.3.1. The Michael Reaction .......................................... 3.3.1.1 General Figure and Conditions ................... 3.3.2. The Fischer-Helferich Reaction .............................. 3.3.2.1 General Figure and Conditions ................... 3.3.3. The Davol-Lowy Reaction..................................... 3.3.3.1 General Figure and Conditions ................... 3.3.4. Silyl Coupling Reaction........................................ 3.3.4.1 General Figure and Conditions ................... 3.3.5. Sulfur-mediated Reaction...................................... 3.3.5.1 General Figure and Conditions ................... 3.3.6. Mitsunobu Reaction ............................................ 3.3.7. Palladium-mediated Reaction.................................

138 139 140 142 151 153 153 156 156 157 157 159 159 163 163 163 164

Contents

ix

3.3.8. Microbial/Enzymatic Approach .............................. Oligonucleotide Synthesis................................................. 3.4.1. Phosphoramidite Method ...................................... 3.4.2. Phosphonate Method ........................................... 3.4.3. Modiﬁed Oligonucleotides .................................... References ...........................................................................

166 167 170 172 172 176

4. Nucleoside Mimetics.............................................................. 4.1. Modiﬁed Nucleosides ...................................................... 4.1.1. Heterocycle Modiﬁcations..................................... 4.1.1.1 C-5 Substituted Pyrimidines....................... 4.1.1.2 C-6 Substituted Pyrimidines....................... 4.1.1.3 Purine Formation .................................... 4.1.2. Sugar Modiﬁcations ............................................ 4.1.2.1 2 3 -Dideoxysugars.................................. 4.1.2.2 2 Deoxynucleosides................................. 4.1.2.3 3 -Deoxynucleosides................................ 4.1.2.4 4 -Substituted Nucleosides......................... 4.1.3. Complex Nucleosides .......................................... 4.1.3.1 Fused Heterocyclic Nucleosides.................. 4.2. C-Nucleosides ............................................................... 4.3. Carbocyclic Nucleosides .................................................. 4.3.1. Cyclopropane Carbocyclic Nucleosides .................... 4.3.2. Cyclobutane Carbocyclic Nucleosides ...................... 4.3.3. Cyclopentane Carbocyclic Nucleosides..................... 4.3.4. Palladium Mediated............................................. 4.3.5. Enzymatic Synthesis............................................ 4.3.5.1 Base Ring Formation ............................... 4.3.6. Carbocyclic C-Nucleosides.................................... 4.4. Acyclic Nucleosides........................................................ 4.5. Thionucleosides ............................................................. 4.5.1. Preparation of Thioribofuranosyl Intermediates........... 4.5.2. Glycosidic Bond Formation ................................... 4.5.3. Chloromercuration Promoted Coupling Reactions........ 4.5.3.1 Silylmediated Coupling Reactions ............... References ...........................................................................

179 180 181 181 183 184 185 185 190 192 193 195 199 200 209 211 212 212 214 220 220 223 223 228 230 236 237 237 241

5. C-Glycosides........................................................................ 5.1. Synthetic Approaches for the Preparation of C-Glycosides......... 5.1.1. Electrophilic Glycosyl Donors................................ 5.1.1.1 Glycosyl Donors Bearing Good Leaving Groups ...................................... 5.1.1.2 Other Electrophilic Glycosyl Donors ............ 5.1.2. Concerted Reaction and Ring Formation ................... 5.1.3. Palladium-Mediated Reactions ...............................

247 247 250

3.4.

250 251 255 256

x

Contents

5.1.4. Mitsunobu Reaction ............................................ 5.1.5. Nucleophilic Sugars ............................................ 5.1.6. Cross-Metathesis Reaction .................................... 5.1.7. Samarium Promoted Reaction ................................ 5.1.8. The Ramberg-B¨acklund Reaction............................ 5.1.9. Free Radical Approach......................................... 5.1.10. Exoglycals ........................................................ 5.1.11. The Tether Approach ........................................... References..........................................................................

256 257 260 260 262 263 265 267 270

6. Glycoconjugates.................................................................. 6.1. Biological Function and Structural Information ..................... 6.1.1. Classiﬁcation of Glycocoproteins ............................ 6.1.2. Recognition Sites................................................ 6.1.3. Structural Information of Glycoproteins.................... 6.2. Carbohydrate Binding Proteins ......................................... 6.2.1. Combining Sites................................................. 6.3. Glycopeptide Synthesis .................................................. 6.4. Glycoprotein Synthesis................................................... 6.4.1. Indiscriminate Glycosylation ................................. 6.4.2. Chemoselective and Site-Speciﬁc Glycosylation.......... 6.4.3. Site-Selective Glycosylation .................................. 6.4.4. Enzymatic Synthesis............................................ 6.5. Synthesis of Antigenic Glycoconjugates.............................. References..........................................................................

272 272 273 275 276 277 279 281 285 286 289 289 289 292 301

7. Hydrolysis of Glycosides ....................................................... 7.1. Acidic Hydrolysis ......................................................... 7.2. Basic Hydrolysis........................................................... 7.2.1. Phenolic Glycosides ............................................ 7.2.2. Enolic Glycosides ............................................... 7.2.3. β-Susbstituted Alcohol Glycosides.......................... 7.3. Enzymatic Hydrolysis .................................................... 7.3.1. β-Glucosidases .................................................. 7.3.2. β-Glucanasas, β-Quitinases................................... 7.3.3. β-Cellulase ....................................................... 7.3.4. β-Glucuronidase ................................................ 7.3.5. Glycosidase Enzymatic Activity Detection................. 7.3.6. Regarding β-1,4-Glucanases (EG)........................... 7.3.7. Fluorescent O-Glycosides..................................... 7.3.8. O-Glycosides Measured by Absorption .................... 7.3.9. Histochemical O-Glycosides ................................. References..........................................................................

304 304 306 306 306 306 308 308 308 308 308 309 309 310 311 311 313

8. Nuclear Magnetic Resonance of Glycosides .............................. 8.1. NMR of O-Glycosides ...................................................

314 314

Contents

xi

8.2. N-Glycosides ............................................................... 325 References.......................................................................... 328 9. X-Ray Diffraction of Glycosides ............................................. 9.1. X-Ray Diffraction of O-Glycosides.................................... 9.2. X-Ray Diffraction of Nucleosides...................................... References..........................................................................

330 331 337 340

10. Mass Spectrometry of Glycosides ........................................... 342 References.......................................................................... 348 Index...................................................................................... 349

Preface

There is no doubt that glycoside chemistry continues to be a dynamic and exciting ﬁeld related to organic chemistry. Within sugar chemistry, glycosides are of special interest not only because of the challenges represented by their synthesis and structural characterization, but also due to their important biochemical relevance, and hence their applications in a number of essential disciplines, such as pharmaceuticals, food, and biotechnology. Important biomolecules such as DNA and RNA, or cofactors such as ATP and NAD, are some of the natural glycosidic structures playing key rules at a biochemical level. Also, a considerable number and variety of natural and synthetic glycosides are being extensively used as antibiotics, antiviral, and antineoplasic agents. There are also a signiﬁcant number of chromophoric glycosides being used in molecular biology as substrates for detection of enzymatic activity of gene markers. Solid-phase oligosaccharide synthesis, despite the great progress recently reported by different groups, continues to be a challenging task considering the diversity and complexity of glycosides, especially those present in cellular membranes. However, based on the satisfactory evolution of this approach, it is expected that many complex molecules will be prepared in just in the same way that solidphase chemistry is currently used to prepare oligopeptides and oligonucleotides. The aims of this book are to prepare methods and strategies for the formation of glycosides, illustrated by the synthesis of important biologically active glycosides, and also to present an overview of the basic tools needed for the characterization of glycosides through NMR spectroscopy, X-ray diffraction, and mass spectrometry. From the overwhelming number of excellent articles related to glycoside chemistry, it hasn’t been an easy task to select those that are biologically important, and perhaps most importantly serve as didactic models for understanding more about the process of glycoside bond formation. The text should also serve as a helpful guide to those professionals interested in sugar chemistry, especially the design of synthetic routes, by evaluating suitable protecting and leaving groups, and the best reaction conditions needed for the preparation of glycosides. The author would like to thank COFAA and SIP-IPN for ﬁnancial support. MBA June 2004 xii

1 Glycosides, Synthesis, and Characterization

1.1 Introduction Monosaccharides are generally deﬁned as aldoses and ketoses connected to a poly hydroxylated skeleton.1 In an aqueous solution, the monosaccharides are subject to internal nucleophilic addition to form cyclic hemiacetal structures. When the addition occurs between -OH at C(4) or -OH at C(5) with the carbonyl group, a ﬁveor a six-member ring is formed known as a furanose or a pyranose, respectively. It is also known that equilibrium exists between the open and the cyclic form, being displaced to the latter by more than 90%. Therefore, in aqueous solutions, it is more accurate to consider that most of the sugars are present as cyclic molecules and behave chemically as hemiacetals. The Haworth structure is a useful way to represent sugars. However, as it is known that for any 6-membered rings, a nonplanar conformation is assumed. The conformation exclusively preferred is called chair and the two possible conformations are 4 C1 and 4 C1 . The ﬁrst conformation is used for the D enantiomeric form and the second for the L form (Figure 1.1). On a chair conformation type 4 C1 , an α anomeric hydroxyl group is positioned in the axial orientation while a β hydroxyl lies equatorial (Figure 1.2). As a result of this reversible ring formation process, a diastereomer mixture of anomers α and β is produced, as indicated in Table 1.1 for some of the most common monosaccharides.1,2 The pioneering work in 1890 by Fischer3 allowed him to determine the relative conﬁguration and the synthesis of the most known aldohexoses. Based on the assumption that in D-glyceraldehyde, the hydroxyl group was placed to the right, he was able to propose correctly the structure of tetroses, pentoses, and aldohexoses (Figure 1.3). The relative conﬁguration of D-glyceraldehyde was later conﬁrmed by X-ray diffraction by Bijvoet in 1951. Consequently, all the resulting biologically active distereoisomeric aldoses derived from D-glyceraldehyde conserve always the secondary alcohol next to the primary one to the right side in the Fischer projection. Ketoses with three to six carbons are naturally produced from 1,3Dihydroxyacetone, according to the tree shown in Figure 1.4.

1

2

1. Glycosides, Synthesis, and Characterization FIGURE 1.1. α-D-glucopyranose-4 C1 and α-L-glucopyranose-1 C4 .

CH2OH O HO HO OH

OH

mirror plane

OH HOH2C

O

HO

OH

HO

TABLE 1.1. Distribution of αβ of some D-monosaccharides in solution at 31◦ C. Carbohydrate

Glucose Galactose Mannose Rhamnose Fructose Ribose Xylose

% Pyranose

% Furanose

α

β

α

β

38 30 65.5 65.5 2.5 21.5 36.5

62 64 34.5 34.5 65.0 58.5 63.0

0.1 3 0.6 0.6 6.5 6.4 0.3

<0.2 4 0.3 0.3 25 13.5 0.3

1.2 Reactions of Monosaccharides Carbohydrates own their reactivity to the hemiacetalic centre and to the hydroxyl groups, being primarily more reactive than the secondary. Aldoses and ketoses are susceptible to nucleophilic addition and the latter is less reactive due to steric hindrance. The cyclic forms are adopted when the hydroxyl group positioned at C-5 veriﬁes an intramolecular nucleophilic addition to the carbonyl group producing an anomeric mixture of pyranosides (Figure 1.5).

1.3 Chemical Modiﬁcations The classical reactions on monosaccharides were used initially for identiﬁcation or sugars or to distinguish between aldoses and ketoses. They have been also very useful for preparing key intermediates in the construction of glycosides. Some of the common reactions used to identify monosaccharides are

1.3. Chemical Modiﬁcations

3

OH

H

OH

OH

H

OH

H

OH

H

OH

O

HO

CHO

OH

O

OH OH

OH OH

OH OH OH

O

HO

CH2OH

OH

OH

D-Allose

OH OH OH O HO

CHO HO

OH

H

H

OH

H

OH

H

OH

OH

O OH

OH

OH OH

OH

OHO

OH

HO

CH2OH

OH D-Altrose

OH OH O HO HO

CHO H HO H H

OH

OH H OH OH

OH OH

O OH

OH OH

OH OH

CH2OH D-Glucose

O HO HO

OH OH

FIGURE 1.2. Fischer projections, Haworth structures, and 4 C1 chair conformation of Daldohexoses.

1.3.1 Oxidations The oxidation of nonprotected aldoses may undergo carboxylic acids formation depending on the reaction conditions. Thus, with aqueous bromine the monocarboxylic acid (aldonic acid) is formed, whereas with nitric acid the dicarboxylic acid is favored (aldaric acid) (Figure 1.6).

4

1. Glycosides, Synthesis, and Characterization OH OHO HO HO

CHO HO

H

HO

H

H

OH

H

OH

OH

OH

O OH

OH OH

OH

OH

OHO HO HO

CH2OH

OH

D-Mannose

OH OH O CHO H H

OH

OH OH

OH

OH

O

OH OH

OH HO H

H OH

OH OH O

OH OH

CH2OH

OH D-Gulose

OH OH OH OH OHO

CHO HO

OH

H OH

H

OH

O

OH

OH

OH HO H

H OH

OH

OH OH OHO

OH

CH2OH

OH D-Idose

OH

FIGURE 1.2. (continued)

1.3.2 Periodate Oxidation Periodic acid is a strong oxidating agent and is capable of breaking 1,2-cis diols to generate after cleavage of the C-C bond carbonyl fragments (Figure 1.7).

1.3. Chemical Modiﬁcations

5

OH OH O CHO H

OH HO

H

HO

H

OH OH

O OH

OH H

HO

OH

OH

OH OH

OH

O

OH

CH2OH

OH

HO OH

D-Galactose

OH OH OH O CHO HO

H

HO

H

HO

H

H

HO

OH OH

OH

O OH OH

OH OH OH

OH

OHO

CH2OH

HO

OH

D-Talose

FIGURE 1.2. (continued)

1.3.3 Tollens Reaction This classical reaction has been very useful for aldose identiﬁcation and consists of the oxidation of the aldehyde function with a moderate oxidative agent (a silver ammonium salt) to afford the glucuronide ammonium salt and metallic silver, which produce the silver mirror effect (Figure 1.8).

1.3.4 Benedict and Fehling Test The test consists in the use of a cooper citrate (Benedict reagent) or cooper tartrate complex (Fehling reagent), which upon treatment with the sugar under study produces the glucuronide ion along with cooper (I) oxide which is detected as brick-red precipitate (Figure 1.9). Based on the Tollens, Benedict or Fehling test, the sugars are classiﬁed into reducing when positive or non-reducing sugars if negative. Reducing sugars are hemiacetals in equilibrium with small amounts of the open forms. Under basic conditions, the aldoses and ketoses give positive the Tollens test and/or Benedict/Fehling test as result of equilibrium aldose-ketoses via enediol intermediates.

6

1. Glycosides, Synthesis, and Characterization O

H

C H

OH CH2OH

D-Glyceraldehyde

O

O C

O

H

C

C

H

OH

HO

H

OH

H

D-Erythrose

D-Threose

O HO H H

OH OH OH

H H H H

H

O C

OH HO OH H OH H OH H CH2OH

D-Allose

O

H

C

H OH OH

H HO H

O C

H H OH HO OH H OH H CH2OH

D-Altrose

H

O

OH HO H HO OH H OH H CH2OH

D-Glucose

C

C

H H OH CH2OH D-Lyxose

D-Xylose

O

H

C

H H H H OH HO H OH CH2OH D-Mannose

H

O

OH HO OH H H HO OH H CH2OH

D-Gulose

H

HO HO H

OH H OH CH2OH

D-Arabinose

H

O

H

C

CH2OH

D-Ribose

C

OH CH2OH

CH2OH

O

H

CH2OH

H

H H H

H

C

H H H OH HO H HO OH H

CH2OH D-Idose

O C

H

O C

OH HO H HO H HO OH H CH2OH

H H H H OH

CH2OH

D-Galactose D-Talose

FIGURE 1.3. The Fischer projections of D-aldoses.

1.3.5 Nucleophilic Addition Aldose and ketone may react with a variety of nucleophiles, giving as results addition/elimination products such as osazones and oximes, or addition products such as reduced derivatives when reacted with hydrides.

1.3. Chemical Modiﬁcations

7

CH2OH C O CH2OH 1,3-Dihydroxyacetone

CH2OH C O H C OH CH2OH D-Erythrulose

CH2OH C

CH2OH

O

C O

H C OH

HO C H

H C OH

H C OH

CH2OH

CH2OH

D-Ribulose

D-Xylulose

CH2OH

CH2OH

CH2OH

CH2OH

C O

C O

C O

C O

H C OH

HO C H

H C OH

H C OH

H C OH

H C OH

CH2OH D-Psicose

CH2OH D-Fructose

H C OH HO C H H C OH CH2OH D-Sorbose

HO C H HO C H H C OH CH2OH D-Tagatose

FIGURE 1.4. Fischer projections of the 2-ketoses.

The reaction that allowed E. Fischer to determine the structure of the common aldoses was the osazone formation and consisted in the reaction between hydrazine with aldoses (Figure 1.10) to yield crystalline derivatives that can be identiﬁed through their melting points values. The carbonyl group can be reduced by hydrogenation or hydride addition to produce the corresponding alditols (Figure 1.11). These reduced sugars are present

8

1. Glycosides, Synthesis, and Characterization OH

OH O

HO HO

OH OH

HO HO

O

HO HO

O

OH

OH

OH

OH H

OH

intramolecular nucleophilic attack

Anomer α

Anomer β

FIGURE 1.5. The pyranose ring formation. O

O

C H

HNO3

C OH

(CHOH)n

(CHOH)n C

O

H2C

OH

Br2

C OH

H 2O

(CHOH)n

OH

H2C

OH

O

FIGURE 1.6. Oxidative aldose transformation into mono- and dicarboxylic acids.

HC OH HC

H5IO6

HC O

OH

HC

+ H3IO4

O

FIGURE 1.7. Oxidative cleavage of diol by periodic acid. O

O

C H

Ag(NH3)2OH

C O NH4

(CHOH)n H2C

(CHOH)n

OH

H2C

+

OH

o Ag mirror effect

FIGURE 1.8. Tollens reaction.

HO HO

non-reducing end

OH O OH

reducing end

OH O

O HO

OH OH

O C

O H

(CHOH)n H2C

OH

2−

2+

−

HOC(CO2H)(CH2CO2H)2 Cu /4 OH

C OH (CHOH)n H2C

OH

FIGURE 1.9. Benedict and Fehling test.

+ Cu2O red pp

+ 2 H 2O

1.3. Chemical Modiﬁcations HC NNHPh

CHO H

NNHPh

OH

HO

9

H

HO

2 PhNHNH2

H + NH3 + PhNH2

H

OH

H

OH

H

OH

H

OH CH2OH

CH2OH

FIGURE 1.10. Osazone formation. O C

H

H2-Ni or NaBH4

(CHOH)n H2C

H2C OH (CHOH)n

OH

H2C

OH

FIGURE 1.11. Carbonyl reduction for the preparation of sorbitols.

in various fruits such as cherries, pies, apples, etc,, and are used as sugar substitute for diabetics.

1.3.6 Enediol Rearrangement This transformation occurs at basic medium and allows the conversion of epimers, deﬁned as isomeric forms that differ in the position of the hydroxyl group at C-2. In this way it is possible to transform through the enediol intermediate glucose to mannose and vice versa (Figure 1.12). Another important isomerization process through the enediol rearrangement is the interconversion of glucose and fructose. Thus, the enolization proceeds by migration of proton at position 2, to carbon at 1 (Figure 1.13).

1.3.7 KilianiFischer Synthesis This sequence was used to increase the number of carbons in a sugar. The reaction involves cyanohydrin formation by nucleoﬁlic addition of cyanide to the aldehyde.

CHO H

OH

CHOH −

OH OH, H2O

CHO −

OH, H2O

HO

H

(CHOH)n

(CHOH)n

(CHOH)n

CH2OH

CH2OH

CH2OH

FIGURE 1.12. The enediol rearrangement.

10

1. Glycosides, Synthesis, and Characterization

CHOH OH

FIGURE 1.13. The enediol rearrangement.

CH2OH −

OH, H2O

O

(CHOH)3

(CHOH)3

CH2OH

CH2OH

The diastereoisomeric mixture of cyanohydrins obtained was partially reduced to produce the epimeric forms (Figure 1.14).

1.3.8 The Ruff Degradation The process of reducing the monosaccharide skeleton in one carbon is known as Ruff degradation and consists in the oxidation of the aldehyde to the carboxylic acid through the use of calcium salt and subsequent peroxide treatment in the presence of ferric salts to produce the aldose reduced in one carbon (Figure 1.15).

1.3.9 Conversion of Pentose to Furfural Pentoses subjected to high acid concentrations can be transformed to furfural in quantitative yields. The sequence involves a tautomeric keto-enol equilibrium, dehydration and intramolecular nucleophilic addition of the primarily alcohol to the aldehyde to generate furfural (Figure 1.16). CN CHO H

OH

HO H

H OH

−

CN

CN

H

OH

HO

H

H

OH

H

OH

HO H

H OH

HO H

H OH

CH2OH

+

CH2OH

CH2OH

H2, H2O Pd, BaSO4

CHO H HO

H

H H

CHO

OH OH OH CH2OH

D-Glucose

+

HO

H

HO

H

H H

OH OH CH2OH D-Mannose

FIGURE 1.14. The Kiliani-Fischer synthesis.

1.4. Biosynthesis of Sugars CHO H

OH

HO

H Br2, H2O

H

H H

(COO−)2Ca2+

COOH

OH OH

HO H H

CH2OH

H

OH CaCO3

H

HO H H

OH OH

CHO

OH H2O2, Fe3+

H

HO H H

OH OH

H OH + CO32+ OH CH2OH

CH2OH

CH2OH

11

Aldopentose

Aldohexose

FIGURE 1.15. Ruff degradation. CHO H

OH

HO H

H OH

CHO

CHO

O

OH

H2SO4 conc

H

CH2OH

H H

H OH

O

O

H OH

−H 2 O

CH2OH

CH2OH OH

CHO

CH2OH

−H2O

CHO

O

CHO

FIGURE 1.16. Conversion of pentoses to furfural.

1.4 Biosynthesis of Sugars The synthesis of carbohydrates in plants occurs through a mechanism of carbon dioxide ﬁxation, and was understood through the use of long-lived radioactive isotope of carbon 14 C. After considerable investigations it was founded that the initial CO2 acceptor was the ﬁve-carbon compound ribulose 1,5-bis-phosphate (RuBP) which after incorporation of carbon dioxide produce a six-carbon molecule. The resulting molecule is fragmented into two molecules of 3-phosphoglycerate (PGA) that is one of the intermediates of glycolysis. This transformations takes place in the chloroplast by a large multisubunit enzyme, ribulose biphosphate carboxylase “Rubisco”. The following reaction sequence is cyclic and constitutes what is known as the Calvin cycle which consists after formation of PGA in reduction to glyceraldehydes 3-phosphate (GAP), and regeneration of RuBP. The overall process requires 6 CO2 molecules ﬁxed, 12 molecules of GAP produced which rearrange to regenerate 6 molecules of the ﬁve-carbon CO2 acceptor RuBP (Figure 1.17).

1.4.1 Sugars as Energy Sources Metabolically the main monosaccharide useful for the production of energy is glucose. During the glycolysis process glucose is enzymatically transformed and degraded to piruvate and is a preamble which is further introduced into the Krebs cycle.

12

1. Glycosides, Synthesis, and Characterization

CH2 OPO 32− O C OH + C C OH O HC OH

RuBP Carboxylase

O CH 2OPO3 2−

CH2 OPO 3 2− HC OH + − O C O

C C OH O−C O HC OH

−

O C O HC OH

3-PGA

CH 2OPO3 2−

CH2 OPO 32−

CH2OPO3 2−

RuBP Enediol form

ATP

ADP +Pi

O C

NADPH

NADP

+

OPO3 2−

CH OH CH 2 OPO3 2− BPG

CH 2OPO3 2−

O C

ATP

ADP + Pi

H

CH OH CH 2 OPO3 2− GAP

C O HC OH HC OH CH2OPO3 2− RuBP

FIGURE 1.17. Carbohydrate synthesis from CO2 ﬁxation.

Carbohydrates are responsible of several biological events mainly related with the storage and production of energy, as metabolic intermediates and signal molecules. They are also constitutive structural units of essential biomolecules such as polysaccharides (starch, glucogen, and cellulose), glycoproteins, glycolipids, and nucleotides. The process by which glucose is used as energy source, to produce ATP and pyruvate is known as glycolysis and consist in series of events represented in Figure 1.18. The second cycle of glycolysis is divided in four steps.

1.5 Synthesis of Carbohydrates The chemical synthesis of carbohydrates can be accomplished by chemical, enzymatic or the combined approach (chemoenzymatic). Their preparation by either of the mentioned methods has received considerable attention especially because they can be used as starting materials for the synthesis of biologically active carbohydrate derivatives known as mimetics or the synthesis of complex molecules such as oligosaccharides or glycopeptides.

1.5.1 Chemical Synthesis Access to potentially useful sugar or congeners can be obtained from natural sugars such as arabinose and mannose.4 Thus, convenient routes have been implemented for the preparation of KDN from D-mannose,5 3-deoxy-D-manno-

1.5. Synthesis of Carbohydrates OH O

HO HO

OPO32− Hexokinase

+ ATP

OH OH

2−

O3POH2C

+ ADP + H+

OH OH glucose-6-phosphate

D-Glucose

phosphoglucose isomerase

O

HO HO

O OH

2−O POH C 3 2

CH2OH

CH2OPO32−

O OH

phosphofructokinase

OH

OH

fructose-6-biphosphate

fructose-6-phosphate

CH 2OPO 3 2−

aldolase

O

CHO H

+

OH CH2OPO 3 2−

CH 2OH

glyceraldehyde 3-phosphat e

dihydroxy acetone phosphate

glyceraldehyde-3phosphate deshydrogenase

CHO H

O C

+ NAD + Pi+

OH

H

CH2OPO32−

OPO32− OH

+ NADH + H+

CH2OPO32− 1,3-bisphosphoglycerate

glyceraldehyde 3-phosphate

O phosphoglycerate kinase

C

O−

H

O phosphoglycerate mutase

OH CH2OPO3

ADP

+ ADP + H+

OH

OH

C

CH2OH

3-phosphoglycerate

enolase

C

O− OPO32−

H2O

OPO32−

H 2-

ATP

O

O−

CH2

2-phosphoglycerate

O piruvate kinase

C

O− O

ADP + H+

ATP

phosphoenolpyruvate

FIGURE 1.18. The glycolysis pathway.

CH2OH

13

14

1. Glycosides, Synthesis, and Characterization OH

OH

OH

HO

i

CHO

OH

OH ii

HO CO2Me

OH

OH

OH

OH

OH

OH

OH

iii

HO

OH

HO HO

CO2Me OH

OH

HO

O

O

CO2R

HO

OH iv

R = Me R=H

i) methyl 2-(bromomethyl)acrylate, H 2O. ii) O 3, MeOH, −78o C, then Na2SO 3. iii) spontaneous ciclization. iv) KOH, MeOH.

CO2Et

O

CO2Et

CHO HO

O i

ii

O

O

HO OH

HO

O O

iii

O

OH O

CO2− NH4+

HO

O

O

O

O

O

O

OH

i) ethyl α-(bromomethyl)acrylate,10% formic acid, aq. MeCN. ii) O3, MeOH, −78oC, then Me2S, MeOH, −78oC to r.t.. iii) aq. TFA, then NH4OH.

O

EtO

OEt

CHO OAc

HO OH

i

ii

AcO

iii

AcO

AcO

OH

OAc

OAc

OH

OAc

OAc

OAc

OAc

OAc OAc OAc

FIGURE 1.19. Chemical synthesis of sugar congeners from natural sugars.

2-octulosonic acid (KDO) from 2,3:4,5-di-O-isopropylidene-D-arabinose,6 D-glycero-D-galacto-heptose from D-arabinose,7 and KDN from D-mannose8 (Figure 1.19). Different approximations for the preparation of monosaccharides from other sources have been reported. One method consists in the asymmetric synthesis of D-galactose via an iterative syn-glycolate aldol strategy. The general method is shown in Figure 1.20.9

1.5. Synthesis of Carbohydrates

15

OEt

EtO

OAc AcO AcO

OH OH

HO v

iv

O

OAc

HO

OH OH

OAc OAc

i) allyl bromide, ultrasonication, then Ac2O, Py, DMAP. ii) OsO4, KIO4, then TBAF. iii) H3O +, (HC(OEt)3. iv) OsO4, NMO, then Ac2O, Py, DMAP. v) NaOMe, MeOH, then H3O+.

O

OH

OH

OH

i

OH

OH

OH

O2N

OH

ii

H OH

HO HO

OH

OH

OH

OH

OBn

OBn

OH

OBn

iii

iv

OBn

O

OHC OH

HO

OBn

OH NHCbz OBn

OBn

OBn O

OBn

OBn

OBn

OBn

v

OBn OBn

OBn MeO2C

MeO2C

OBn

OBn

HO

OH

OBn

OH

vi

HO HO

O

CO2Me

HO

i) MeNO2, DBU. ii) Nef oxidation iii) EtSH, HCl, then NaH, BnBr, DMF, then MeI, Na2CO3. iv) (Et)2P(O)CH(NHCBz)CO2Me, NaH, CH2Cl2. v) H2, Pd-C. vi) H2, Pd(OH)2, then Dowex H+, MeOH.

FIGURE 1.19. (continued)

A promising and simple concept based on a two-step reaction sequence for preparing monosaccharides via the enantioselective organocatalytic direct aldol reaction of α-oxyaldehydes is recently described. The summarized sequence is illustrated in Figure 1.21.10 An interesting strategy for preparing KDO and 2-deoxy-KDO from 2,3-Oisopropylidene-D-glyceraldehyde was reported, based on a hetero Diels-Alder

16

1. Glycosides, Synthesis, and Characterization O

O

O i

NH

O

OP

O ii

N

O

H OR

R

OP

OR

OR

R O

O

OP

OH

O

iii

OR O

iv

N OR

OH

HO HO

OR

OH

R

OH

i) a) glycolate aldol. b) protect. ii) DIBAL-H cleavage. iii) iterative glycolate aldol. iv) cleavage and deprotection.

FIGURE 1.20. Asymmetric synthesis of D-galactose.

reaction, followed by pyranoside ring formation. Diol formation and double inversion at C-4 and C-5 produced the target molecules (Figure 1.22).11 C-methyl heptoses were suitable prepared from nonracemic butenolide as starting material. Asymmetric conjugate addition provided protected lactone which by methylation afforded α-methyl lactone. Each of them under DIBALH treatment, produced C-methylheptoses (Figure 1.23).12 Naturally occurring sugar amino acids are another class of interesting modiﬁed carbohydrates found as structural components in nucleoside antibiotics. Most of them consist of N- and O-acyl derivatives of neuraminic acids, while other presents a ipso-hidantoin furanosides (Figure 1.24).13 Some of these sugars amino acids have been synthesized via azide furanosides,14,15 as it was the case for β-sugar amino acids shown in Figure 1.25.13

1.5.2 C-Glycosyl Amino Acids It has been mentioned that natural glycopeptides are classiﬁed into O-glycopeptides when the sugar residue establishes an O-glycosyl linkage with L-Serine or L-Threonine and N -glycopeptides if the linkage is with Asparagine. There has been an increasing interest for preparing unnatural C-glycol amino acids O O

O O

OX

OH OY

H

H

O

OH

XO

X

X

aldol 2

aldol 1

OX

OX

OX

XO

OY OH

FIGURE 1.21. Two-step carbohydrate synthesis.

1.5. Synthesis of Carbohydrates

O

BnO

O

BnO

OBn

BnO

i

ii

O

HO

O

OBn

CO2Me

OH

CO2Me

OBn

BnO BzO

+

BnO BzO

O

O CO2Me

BzO

iii

BnO

BnO

CHO

17

BzO CO2Me

i) HCOCO 2Bu, 130oC, then MeOH, TsOH. ii) OsO4, NMO. iii) Tf2O, Py, then PhCO 2NBu4.

FIGURE 1.22. Synthesis of protected KDO and 2-deoxy-KDO.

as a potential building block in the assembling of modiﬁed glycopeptides that may serve in preparing therapeutically useful mimetics, with higher resistance to hydrolytic enzymes and also displaying superior properties than the natural ones. O

O

OTMS

H3 C i

O

OTMS O O

OTMS

H 3C ii

O H3 C

O

O

O

O

O

O iii

iii

OH

OH HO HO

HO HO

O

O H3 C

H3 C OH

CH3

i) Me2CuLi, CH2Cl2. ii) LiHDMS, THF, then MeI. DIBAlH, CH2Cl2, then 3N HCl, THF.

FIGURE 1.23. Synthesis of-methyl heptoses.

OH

18

1. Glycosides, Synthesis, and Characterization

R4

R5 CO2H R2 O OH

R3 R1 R1

R2

R3

R4

R5

glucosaminuronic acid

NH 2

H

OH

OH

H

galactosaminuronic acid

NH 2

H

OH

H

OH

monnosaminuronic acid

H

NH2

OH

OH

H

H

OH

NH 2

H

OH

4-amino-4-deoxy-glucuronic acid

O OH

HO

COOH

OH

HO

O

H 2N

O

HN

OH CO 2H

NH

OH

HO

HO

OH OH

NH NHAc

O

Hydantocidin

Neuraminic acid

Siastatin B

FIGURE 1.24. Naturally occurring sugar amino acids. OH O

O OH

O

O

i

H

O

O

ii

H

O

O

N3

HO O

O

O O

iv

O

O

O

NH

O

N3

O

O

N3

iii

H

O

O

HO

O

HO

Fmoc HO O HO iv

HO

H

H

O NH

Fmoc

O

v

O

HO

O

O NH

O

Fmoc

i) a) Tf2O, Py. b) NaN 3, Bu4NCl (cat.), 69%. ii) 77% AcOH, quant. iii) a) NaIO 4. b) KMnO 4, 50% AcOH, 90%. iv) H2, Pd/C, FmocCl, NaHCO 3. v) NaOCl, TEMPO (cat.), KBr, NaHCO 3, Bu4NCl, 62%.

FIGURE 1.25. Synthesis of protected sugar amino acids.

1.5. Synthesis of Carbohydrates Ph N BnO

CONH2 BnO

O

i

N Ph

OBn OBn

O

BnO

CHO

O

OH OH

OH

OBn OBn

OBn OBn

HO

O

ii

CONH2 iii,iv

19

CONH2

NH2

HO +

O

NH2

OH OH

i) TsOH. ii) a) NaCN, K2CO3, H2O. b) H2O2. 91%. iii) a) MsCl. b) LiN3. iv) a) aq. HCl. b) H2, Pd/C.

FIGURE 1.26. Synthesis of anomeric ribofuranosyl glycines.

A recent review describes methods for the preparation of C-glyosyl glycines, alanines, serines, asparagines tyrosines, and tryptophans.16 For instance the synthesis of ribofuranosyl glycine was described under Strecker conditions, starting from 2-(2,3,5-tri-O-benzyl-β-D-ribofuranosyl)-1,3diphenylimidazolidine, which was after hydrolysis tosylated and reacted with cyanide and peroxide to give the α-hydroxy amide as racemic mixture. The anomers were separated as O-mesyl derivatives which were transformed to the azide and further reduced to the corresponding ribofuranosyl glycines (Figure 1.26).17 A method reported for the preparation of C-glycosyl alanines involves the use of (R)-methyleneoxazolidinone, which was linked to the peracetylated iodosugars under promoted radical additions. The α-linked C-glycoside was subjected to hydrogenolysis to give α-D-galactosyl D-alanine and the α-D-glucosyl isomer (Figure 1.27).18 C-analogues of glycosyl serines have been prepared by a number of methods and among them Strecker, Witting, and Sharpless asymmetric aminohydroxylation reactions19 . One of them describes their synthesis via coupling of anomeric pyridyl sulfone with an electrophiles center under samarium catalysis. The resulting Cglycosylation proceeded with α-selectivity (3.3:1). Final deprotection produced the C-glycosyl serine analogue in good yield (Figure 1.28).20 More recently, the stereoselective synthesis of a C-glycoside analogue N-fmocserine β-N-acetylglucosaminide was described employing the Ramberg-B¨acklung (RB) rearrangement. This procedure involves the coupling reaction between isothiourea and protected iodide to produce thioglycoside in good yield. Oxidation to the sulfone was followed by the RB conditions (KOH/Al2 O3 in tBuOH/(CBrF2 )2

20

1. Glycosides, Synthesis, and Characterization R1 OAc

R1

NCbz

O

R2 AcO

O

OAc

OAc NCbz

t-Bu

O

I

O

R2 AcO

i

+

OAc

O

R1 = OAc, R2 = H

t-Bu

O

R1 = H, R2 = OAc

R1

OAc O

R2 AcO

ii

OAc

CO2H NH2

i) Bu3SnH, NaCNBH3. ii) H2 Pd-C.

FIGURE 1.27. Synthesis of α-D-galactosyl D-alanine and the α-D-glucosyl isomers.

at 50◦ C affording the exoglycal derivative. Final steps which involves hydrogenolysis, deprotection, and oxidation provided the desired C-glycosyl analog (Figure 1.29).21 β-2-Deoxy sugar bearing uridin diphosphate (UDP) is a substrate used by a variety of glycosyltransferases. It is described the chemical synthesis of UDP β-2deoxysugars starting from α-glycosyl chlorides as indicated in Figure 1.30. These

OBn OBn

OHC

O

RN

AcHN

O

i

O

BnO

+

BnO

OBn OBn

O O

AcHN HO

SO2Py R = t-BuMe2Si-

OBn OBn O ii-vi

BnO

NHBoc

AcHN

OH O i) SmI2. 82% ii) deoxygenation. iii) TBA. iv) Boc2O. Cs2CO3, MeOH. vi) Jones.

FIGURE 1.28. Synthesis of C-glycosyl serine analogue.

RN O

1.5. Synthesis of Carbohydrates

21

OAc O

AcO AcO BocN

NH2HCl

S NHAc

O

I

OAc

NH

S

i

NHAc

tBu2

tBu2

Si O S

O

tBu2

iv

BocN

O

O

O S

NHAc

BocN

NHAc

O

Si

O O TBDMSO

iii

O O TBDMSO

ii

O

AcO AcO

O BocN

Si

O O TBDMSO

O

AcO AcO AcO

v

O NHAc

BocN

BocN

O

NHAc

AcO AcO AcO

vi

O

NHFmoc CO2H

O NHAc

i) K2CO3, Na2S2O5/acetone-H2O, 98%. ii) a) Et3N/MeOH-H2O. b) tBu2Si(OTf)2, 2,6-lutidine/DMF, 88%. iii) mCPBA, Na2HPO4/CH2Cl2, 77%. iv) KOH/Al2O3, CBrF2CBrF2/tBuOH, 50oC, 38%. v) a) H2, Pd(OH)2/EtOAc, 78%. b) TBAF/THF. c) Ac2O/Py, 68% (two steps). vi) a) TFA/CHCl3. b) FmocCl/iPrNEt/CH2Cl2-MeOH, 69% (two steps). c) Jones oxidation/acetone, 77%.

FIGURE 1.29. Synthesis of C-glycoside serine analogue by Ramberg-B¨acklung rearrangement.

useful intermediates are essentials for studying glycosyltransferases involved in the synthesis of biologically active natural products.22

1.5.3 Enzymatic Synthesis The enzymatic synthesis of monosaccharides and carbohydrates mimetics by enzyme catalysts is performed mainly by a group of lyases known as aldolases. This O

R1 R2

OH

i,ii

O

R1

NHCbz

R1 = OAc, R2 = H R1 = H , R2 = OAc

R2

OUDP

NHCbz

α/β 1:2

i) TMS-Cl, TMS-I, –78oC. ii) UDP, Bu4N salt, 60%.

FIGURE 1.30. Synthesis of protected UDP epi-vancosamine.

22

1. Glycosides, Synthesis, and Characterization O

O

O X

R

+

H

OH

Aldolase

R

R

R X

Donor

Acceptor

Aldol condensation

X = H, NH 2, OH

FIGURE 1.31. General scheme of enzymatic-mediated aldol condensation.

enzymes effects the conversion of hexoses from their three-carbon components via an aldol condensation.23 There are over 30 aldolases identiﬁed and isolated, being classiﬁed in two types depending on the mechanism involved: aldolase type 1 and type 2, which is Zn-dependent. The general reaction that they catalyze is the stereospeciﬁc addition of a ketone donor to an aldehyde acceptor (Figure 1.31). The aldolases used for synthetic purposes are classiﬁed in ﬁve groups depending on the ketone donor and the products formed. Dihydroxyacetone phosphate (DHAP) aldolase Pyruvate aldolase 2-Deoxyribose 5-phosphate aldolase Glycine aldolase Other aldolases Examples of each of them are indicated in Table 1.2; Aldolases have been also very useful for the preparation of a variety of common and uncommon monosaccharides. Fructose-1,6-diphosphate (FDP) aldolase effects the conversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde3-phosphate (G3P) to D-fructose-1,6-diphosphate (FDP). Table 1.2 summarizes the natural substrates, de donors, and the products obtained through this reaction. Broken lines indicates the bond formed or broken.24 DHAP-aldolases catalyze the reversible asymmetrical aldol condensation of DHAP to L-lactaldehyde or to D-glyceraldehyde 3-phosphate (G3P). There are four types of DHAP aldolase, which are classiﬁed on the basis of the condensation product formed. D-fructose 1,6-diphosphate (D-FDP) aldolase, which condenses DHP with G3P. D-Tagatose 1,6-diphosphate (TDP) which utilizes the same substrates, Fuculose 1-phosphate, catalyzing the condensation reaction between DHAP and L-lactaldehyde to produce L-fucolose 1-phosphate, and Lrhamnulose 1-phosphate aldolase, which recognizes the same substrates to produce l-rhamnulose 1-phosphate (Figure 1.32).24 Likewise, DHAP-dependend aldolases are involved in the incorporation of dihydroxyacetone phosphate (DHAP) on pentose and hexose phosphate introducing consequently three carbons and two quiral centers (Figure 1.33).25 Another enzymatic aldol type reaction takes place on N-acetylneuraminic acid also known as sialic acid, which after a reversible aldol reaction of N-acetyl-D-

1.5. Synthesis of Carbohydrates

23

TABLE 1.2. Natural substrates for aldolases. O

OH

O CH3

PO

OH OH

O

OH OH OH OH

-O2C

OH NHAc

sialic acid synthetase

O

OH

-O2C

OH NHAc

rhamnulose-1-P aldolase

OH OH OH

sialic acid aldolase

OH

-O2C

OP OH

3-deoxy-2-oxo-6-Pgalactonate aldolase

O

O NH3+

-O2C

-O2C

H

Donor

O

Donor

OH CO2–

-O2C

OH

O

O OH

-O2C

Donor

OH

NH3+

-O2C

OP

OH

OH 3-deoxy-2-oxo -L-arabinoate aldolase

4-hydroxy-2-oxoglutarate aldolase

O HO

CH3

4-hydroxy-4-methyl -2-oxoglutarate aldolase

O

D-Thr aldolase

NH3+

OH

O

CO2–

-O2C

2-deoxyribose -5-P-aldolase

OH

-O2C

OH

L-Thr aldolase

NH3+

O

-O2C

CH3

-O2C

OH 3-deoxy-2-oxo -D-pentanoate aldolase

CO2–

CH3

-O2C

-O2C

OH

-O2C

CH3

OH 3-deoxy-2-oxo -D-glucarate aldolase

hydroxybutyrate aldolase

Ser-hydroxymethyl transferase

mannosamine and pyruvate, produces N-acetyl-5-amino-3,5-dideoxy-D-glyceroD-galacto-2-nonulosonic acid (NeuAc) (Figure 1.34).26 Ketoses can be transformed to aldoses through the use of isomerases.27 In this way glucose derivatives can be obtained from fructose as shown in Figure 1.35.28

1.5.4 Chemoenzymatic Synthesis The chemoenzymatic approach is a combination of the chemical and the enzymatic methodologies and intends to explode the versatility and availability of the chemical reagents with the high stereo- and regioselectivity of the enzymes when they act as catalysts. For instance, the enzymatic synthesis of dihydroxyacetone phosphate (DHAP) is too expensive on large scale, and therefore the combined approach becomes

24

1. Glycosides, Synthesis, and Characterization OPO32−

O PO

OH

-O2C

Donor

O

OH

O

PO

OP OH OH

O

O

OH OH

-O2C

OP

-O2C

OP OH

DAHP synthetase

OH

O CH3

OH

3-deoxy-2-oxo-glutamate aldolase

OH OH

-O2C

O OP

OH OH

OP OH OH KDO aldolase

O

O

O D-FDP

OH

+

OH OH

-O2C

KDO synthetase

fuculose-1-P aldolase

PO

OH

OH

FDP aldolase

PO

Donor

H

PO

OP

OP

OH

OH

O

O D-TDP

PO

OH

+

H

OH

PO

OP

OP

OH DHAP

OH

(S,R) D-fructose 1,6-diphosphate

G3P

DHAP

O

OH

G3P

OH

OH

(S,S) D-tagatose 1,6-diphosphate

FIGURE 1.32. DHAP-dependent aldolases.

the best choice. The reported procedure consists of the phosphorylation of dihydroxyacetone dimmer with (PhO)2 POCl followed by hydrolysis of the dimmer to generate dihydroxyacetone phosphate in 61% yield.29 The chemically prepared DHAP is then used as an important material for the synthesis of natural monosaccharides and carbohydrates mimetics (Figure 1.36).

1.6. Synthesis of Carbohydrates Mimetics O

O

O L-FucP

PO

OH

+

OH

PO

H OH

OH

L-Lac

DHAP

+

OH

O L-FucP

OH

OH

(R,R) L-Fuculose 1-phosphate

O

O PO

25

PO

H OH

OH

L-Lac

DHAP

OH

(R,S) L-Rhamnulose 1-phosphate

FIGURE 1.32. (continued) PO HO HO

PO

OH O

OH

OH

i

OP HO HO

OH

O

OH

HO

i) rabbit muscle aldolase (RAMA), DHAP.

OP PO O

OH OH

i

OH

OH

OH OH O OH

OH

OP

i) rabbit muscle aldolase (RAMA), DHAP.

FIGURE 1.33. Enzymatic preparation pentose and hexose phosphate.

1.6 Synthesis of Carbohydrates Mimetics 1.6.1 Iminosugars This class of isosteric sugars also recognized as azasugars have been the subject of intense study because their signiﬁcant activity as α-glycosidase inhibitors, which is a promising strategy in the treatment against diabetes mellitus type II and other glycosidase associated disorders. It is believed that the mechanism for glycosidase inhibition and to some extent for glycosyltransferases involves the binding of the aza sugars to the active site by charge-charge and hydrogen bond interactions.30 A signiﬁcant variety and diversity of either naturally occurring or

26

1. Glycosides, Synthesis, and Characterization O HO HO HO

OH

NHAc O

CO2H i

HO

CO2H

OH

OH

O

AcHN OH

OH

i) NeuAc aldolase

OH HO

OH CO2H

O

AcHN OH

OH

FIGURE 1.34. Enzymatic preparation of sialic acid analogs. O

OH

R3

OH

OH

R3

i

H

HO R1

R2

O

R1

R2

R1 = OH; R2 = H; OH i) glucose isomerase

R 3 = H; OH; OCH3; F; N3

FIGURE 1.35. Enzymatic isomerization of fructose to glucose derivatives.

synthetic aza sugars with glycosidase and glycosyltransferase inhibition activity have been reported.31−35 The common feature of these derivatives is the replacement by chemical or enzymatic methods of the cyclic oxygen by a nitrogen atom. The representative example is known as deoxynojirimycin (Figure 1.37), which has shown strong inhibition against a variety of α-glycosidases. Representative examples of natural and synthetic aminoglucosides implicated in inﬂammation, metastasis, and blocking infection processes are depicted in Figure 1.38. Series of 1-N-iminosugars including D-glucose-type, D-galactose-type, Lfucose-type, D-glucuronic acid-type, and D-xylose-type were synthesized and evaluated as glycosidase inhibitors (Figure 1.39). A general procedure for the preparation of 1-N-iminosugars consisted in the azido substitution of a 5-tosyl-1-O-benzoate, followed by aldol reaction, Perlman hydrogenation and cyclization (Figure 1.40). Another chemical approach described for the preparation of iminosugars consisted in the use of protected L-serinal, which was subjected to Wittig elongation, diol formation, and 2-lithiothiazole treatment, to produce a common thiazole

1.6. Synthesis of Carbohydrates Mimetics

O

EtO

OH

O

OP

O

EtO

i

27

ii

HO HO

O

OEt

PO

OP

OEt

O

DHAP i) a) (PhO)2POCl. b) H2/PtO2. ii) H2O, 65oC.

TPI

O HO

OH

OP

O D-Gd-3P

dihydroxyacetone phosphate

Aldolase

DHAP

fructose 1,6-bisphosphate FDP

OP

OP OH O

HO HO

OP H+ (Dowex 50)

fructose 6-phosphate F-6-P

OP OH O

HO HO

Pase 100%

OH

HO HO

OH OH O OH

PGI

OH

OP O

glucose-6-phosphate HO HO G-6-P

OH

Pase

OH 100%

HO HO

O OH

OH

FIGURE 1.36. Chemoenzymatic preparation of glucose. R NH2 HO

HO

OH

FIGURE 1.37. Iminosugar deoxynojirimycin.

derivative. This intermediate under the appropriate conditions will give access to L-(-)-nojirimycin or L-(-)-mannonojirimycin (Figure 1.41).36 Chemoenzymatic preparation of glycosidase inhibitors Deoxynojirimycin and Deoxymannojirimycin was described by using RAMA-aldolase for the aldol

28

1. Glycosides, Synthesis, and Characterization O

HO HO

OH NH

HO HO

N

HO HO

HO

HO OH

HO

NH

N OH

OH

OH castanospermine

deoxymannonojirimycin

O

OH N

kifunensine

swainsonine

OH

HO

OH OH

CO2H

AcHN

OH OH

OH

OH

siastatin B

HO

OH

OH

HO

H3CS mannostatin

O

N

H N

NH2

OCH3 OH

Synthetic sucrase inhibitor

OH

N

NH

HO HO

HO

OH OH Glucosidase inhibitor

Nectricine

FIGURE 1.38. Representative aminosaccharides. OH

OH

OH

NH

HO

NH

OH

HO HO

OH

L-Fuc-type

D-Gal-type

D-Glc-type

CO2H

NH

H3C

HO HO

HO HO

NH D-GlcUA-type

NH D-Xyl-type

FIGURE 1.39. Series of 1-N-iminosugars. OH

OH O HO HO

TsO

i

OH

O O O

ii

N3

OBz

HO HO

OH OH

N

Boc

OH

HO

vi

v

OH

iii,iv

OH

OBz BzO BzO

O O O

HO HO

+

NH.HCl OH

i) a) Me2CO, H2SO4. b) TsCl/Py, 0oC to r.t. c) BzCl/Py. ii) NaN3/DMSO, 100oC. iii) a) NaOMe/MeOH. b) K2CO3/HCHO-MeOH. iv) a) H2 Pd(OH)2/MeOH. b) 1N HCl. v) a) Boc2O/Et3N/MeOH. b) BzCl/Py. vi) a) MeOCOCOCl/DMAP/CH3CN. b) Bu3SnH VAZO/CH3Ph. c) SiO2-iPrOH/H2O/NH2OH.

FIGURE 1.40. Synthesis of 1-N iminosugars.

OH

NH

NH.HCl

1.6. Synthesis of Carbohydrates Mimetics

NBoc O

i

NBoc O

CHO

O

N

CO2Et

O

O

O

iii

NBoc O

NBoc O N

O

iv

S

O

N O

OTBS

OAc v

NBoc O

NBoc O O

CHO O

O

CHO O

OTBS

OAc

vi

H N

HO HO

S

O

v

HO

S

NBoc O

ii

O

29

vi

OH OH

H N

HO HO HO

OH OH

i) a) Ph 3PCHCO 2Et, b) OsO 4, NMO, c) DMP, TsOH. ii) 2-lithiothiazole, Et2O iii) a) NaBH 4. b) TBSCl, imidazole iv) a) Red-Al, toluene, b) Ac 2O, Py, DMAP. v) a) MeI, MeCN. b) NaBH 4 c) HgCl2, MeCN, H 2O. vi) TFA, H 2O.

FIGURE 1.41. Chemical synthesis of L-(-)-nojirimycin and L-(-)mannonojirimycin.

condensation and hydrogenolysis for azide reduction and ring formation (Figure 1.42).37 Signiﬁcant achievements have been made for the synthesis of aza sugars based on aldolase reactions particulary fructose-1,6-diphosphate,30 2-deoxyribose-5phosphate,38 fuculose-1-phosphate,39 sialic acid aldolase, and Pd/C-mediated reductive amination (Figure 1.43).

1.6.2 Aminosugars Aminosugars are another class of naturally and nonnaturally sugars which might be considered distinct to the previous in that the nitrogen is exocyclic. Their significance is clearly seen in a family of aminoglycoside antibiotics such as neomycin, kanamycin which are widely used against both gram-positive and gram-negative

FIGURE 1.42. Chemoenzymatic synthesis of iminosugar.

O

O PO

OH

+

H

N3 OH

i

OP

OP O OH OH

N3

O OH OH

+

OH

OH

ii

ii

HO

HO NH

HO HO

N3

OH NH

HO HO

OH i) FDP aldolase. ii) a) Pase. b) H2 /Pa

From fructose-1,6-diphosphate aldolase

OH

R NH

HO

HO HO

NH

NH

HO HO

R

OH

OH

R OH NH

HO NHAc

OH

R = OH, H

R = OH, H

R NHAc NH

HO HO

OH

OH

R

CH3

OH

HO

H

NH

R = H, CH3

R

N HO OH

HO

OH

OH

From fuculose-1-phosphate aldolase

From 2-deoxyribose-5-phosphate aldolase

HO

R

H N

NH HO

HO

OH

H N

R HO HO

OH OH NH

OH

R NH

HO OH

R = CH 3, CH2F

FIGURE 1.43. Aza sugars prepared by aldolase reactions.

R = OH, H

1.6. Synthesis of Carbohydrates Mimetics OH From sialic acid aldolase

OH

OH

HO

Me HO

OH

H

N

H N

31

CH3

OH

H N

HO

CO2H

OH

OH HO

OH HO2C

OH

H N

OH OH OH

FIGURE 1.43. (continued)

bacteria. Although there is no uniﬁed protocol for the synthesis of aminosugars, they have been roughly classiﬁed in (a) non-azido (Figure 1.44) and (b) azido approaches (Figure 1.45).40 (a) The non-azido methodologies usually involves the introduction of an amino group at C-2, and glycals are usually the starting materials.

OR OR RO RO

OR

i

O

O

RO RO BnO

RO RO

N N O

i) BnO2C N

ii

OBn

ref.41 OR

OR O

i

OR' NHAc

O

N CO2Bn, hv. ii) a) R'OH, Lewis acid b) Raney Ni. 3) Ac2O, Py

RO RO

O

O

RO RO

SPh NH

F3C O i) (saltmen)Mn(N)(CF3CO)2O. ii) PhSH/BF3-OEt2

ref.42 FIGURE 1.44. Non-azido methods for the preparation of aminosacharides.

32

1. Glycosides, Synthesis, and Characterization OR

OR

i

O

RO RO

O

RO RO

OR

NH

H3C O

i) a) thianthrene -5-oxide, AcNHSiMe3, Tf2O, Et2NPh. b) Amberlyst -15, HOR. 43

ref.

FIGURE 1.44. (continued)

(b) The azido approach is a more common procedure for amino introduction on sugars due its relative stability, good solubility in organic media, and easy conversion to amines through catalytic hydrogenolysis. Some of the methods reported involves the use of glycals, or protected saccharides containing free primary or secondary alcohols. Epimerization of hydroxyl groups can be achieved by following an oxidationreduction sequence in which a secondary alcohol is converted into a keto group, followed by stereoselective hydride reduction and nucleophilic substitution. It has been observed that epimerization by following the Mitsunobu protocol has not been

OAc

OAc O

AcO AcO

i

O

AcO AcO

OAc

N3

i) ClN3, CH2Cl2 (PhCO2)2

ref.44 OBn

OBn

OBn O

BnO

OBn O

i BnO

N3 i) Me3SiN3, Me3SiONO2

ref.45 FIGURE 1.45. Azido methods for the preparation of aminosacharides.

1.6. Synthesis of Carbohydrates Mimetics OBn

OBn

OBn

OBn O

i

O

33

OR

AcO

AcO

N3

R= NO2

ii

R=H i) CAN, NaN3, CH3CN, –15oC, 45%. PhSH, DIEA, 91%.

ref

64

OTs

OH

HO HO

i

O

ii

O

HO HO

O

HO HO OH

OH

N3

OR

OH

OR

i) TsCl, Py, r.t. ii) NaN3, DMF

ref 1 OBn

N3 i, ii

O

HO BnO

OBn O

BnO

OBn OMe

OBn OMe

i) Tf2O, Py/ CH2Cl2. ii) NaN3, DMF

ref 46 OAc AcO AcO AcO

OTf

i

O OAc

AcO AcO

O OAc N3

i) NaN3, DMF

ref 47,48 FIGURE 1.45. (continued)

OR

34

1. Glycosides, Synthesis, and Characterization OEt

O

SH

i

EtO

O ii

OH HO HO HO

S OH OH

iii

OH AcO S

AcO AcO

SH

HO

H

O

OH

iv

OH OAc

OH

AcO AcO AcO

S OAc

i) a) AcSK, AcSH. b) HCl. ii) a) DHAP FDP A. b) Pase. iii) Ac2O, Py. iv) Et3SiH, BF3.OEt2.

FIGURE 1.46. Synthesis of thiomonosaccharides.

probed satisfactorily due to steric hindrance of the secondary hydroxyl groups on the pyranose ring.40

1.6.3 Thiopyranoside Monosaccharides Thiosugars Sugars are another class of interesting carbohydrate mimetics. The synthesis of these derivatives can be achieved by using aldolases RAMA for the aldol condensation reaction.. The following reaction sequence was used successfully for the preparation of deoxygluco, manno, galacto, and altropyranosides (Figure 1.46).49 Another strategy for the synthesis of thiosugars involves the replacement of one of the oxygen atoms at the anomeric carbon of the glycoside by a sulfur atom leading to two distinctly different thiosugars, namely a 5-thio and 1-thioglycosides.50

1.6.4 Carbapyranoside-Saccharides More recently this type of sugar mimics have received increasing attention since some of them present α-glucosidase activity and therefore considered for therapies for non-insulin-dependent diabetes mellitus. Also they have been found to be active as agricultural antibiotics, and because of their recognition by glycosidases and glycosyltransferases as substrates and stability against enzymatic degradation, they have been used also to study oligosaccharide-chain biosynthesis.51,52 The pseudotetrasaccharide Acarbose (Figure 1.47) has been the ﬁrst α-glucosidase inhibitor to be explored in humans as an antidiabetic agent along with the amino sugar 1-desoxynojirimycin Miglitol. The pathway in Figure 1.4853 has described the chemical synthesis of carbamaltose, carbacellobiose and related carbadisaccharides of biological interest. Pseudo-N-acetyllactosaminides were found to be acceptors substrates for human-milk α-(1→ 3/4)-fucosyltransferase. A small scale reaction of the

1.8. The Leaving Groups

35

OH HO HO

OH HN HO

CH3

O OH

OH

O HO

O OH OH

acarbose

O HO

O OH

OH

FIGURE 1.47. Pseudotetrasaccharide Acarbose.

mentioned pseudodisaccharides with GDF-fucose resulted in conversion to pseudotrisaccharides (Figure 1.49).51

1.7 Glycoside Reactivity The reactivity for the anomeric carbon C(1) is the typical for acetals, and therefore the nucleophilic addition may occur. On the other hand, the other hydroxyl groups behave typically for alcohols. For coupling reaction with sugars, the anomeric carbon is involved to produce a glycosidic bond, and usually must be activated with a good leaving group in order to form a new linkage (Figure 1.50). A glycoside is formed when the anomeric carbon of a sugar is connected through a heteroatom (except with C-glycosides) with an aliphatic or aromatic fragment known as aglycon. The glycosidic bond is formed when a nucleophile (alcohol, amine, thiol, or carbanion) substitutes the hydroxyl group at the anomeric position, which has been previously substituted by a good leaving group. Therefore, when the nucleophiles are an alcohol, amine, or carbanion, O-, N -, or C-glycosides are generated as result, as can be observed in Figure 1.51.

1.8 The Leaving Groups As mentioned above, the anomeric hydroxyl group can be replaced under suitable conditions with a good leaving group. Initially, the use of halogens such as ﬂuorine, chlorine, and bromine was the strategy of choice, and particulary the last since it presents the best balance between reactivity and stability and this is why it has been extensively used for preparing glycosides. However, halides are in most cases labile and undergo decomposition. Consequently a number of other leaving groups have been designed for glycoside chemistry, and among them, imidates, sulfur, sulfonates, silyl groups, phoshates, and acetates are equally important alternatives.

36

1. Glycosides, Synthesis, and Characterization OBzl

Ph

O

O

O BzlO

Ph

O

+ HO

OR

O O BzlO

i

OBzl

BzlO BzlO

O BzlO

OMe

O BzlO

OMe

iv

Ph

O O BzlO

ii

iii

OBzl O O BzlO

Ph

BzlO

BzlO O

O BzlO

O

v

BzlO

O

O

OMe

O OBzl

OMe v

v

Ph

O O BzlO

OBzl OR O

Ph

O BzlO

BzlO O

O BzlO

BzlO

OMe

BzlO O

O OR

OR

OMe

Ph

OR

O O BzlO

iv

BzlO O

OMe

O OBzl

iv

AcO AcO AcO AcO

AcO AcO OAc AcO AcO

BzlO

79%

AcO 72%

iv

O OAc

OAc

O

O AcO

AcO O

OAc

OAc

AcO AcO AcO

AcO

OAc

AcO O

OAc

O OAc

60%

i) DMF, NaH, 15-crown-5 ether, 50oC, 60% ii) DMSO, Ac2O, r.t., 72% iii) DBU, PhCH3, 70oC, 56%. iv) a) FeCl3, Ac2O, – 20oC b) H2, Pd/C, EtOH. c) Ac2O, Py. v) NaBH4, CH2Cl2/MeOH, OoC.

FIGURE 1.48. Synthesis of carbapyranoside-disaccharides.

1.8. The Leaving Groups

FIGURE 1.49. Chemoenzymatic synthesis of pseudotrisaccharides.

OH HO

O

X HO

O(CH2)7CH3

OH

OH

37

NHAc i

OH HO

X

O O

O(CH2)7CH3

OH

OH

NHAc O

OH

H3 C X=O X = NH

HO OH

i) fucosyltransferase, GDP-Fuc.

The use of iodide has been restricted due to its low reactivity and ﬂuoride although limitedly has been more used for preparation of some α-glycosides.54,55 It has been found that in the absence of selective conditions, a leaving group can be found as a mixture of anomers, as in the case of the acetates. However, some others such as bromide and imidate can be introduced preferentially at the α-position (Figure 1.52). A well-accepted hypothesis that explains the α-stereoselective preference assumed by the leaving group (halogens and imidate) is based on the anomeric effect, consisting in the electronic effect produced by the ring oxygen, which gives rise to a repulsive effect between one of the oxygen lone pairs and the leaving group, forcing the later to assume such position56 (Figure 1.53).

O

OH

O

X

X C

C

H

H OH

O C

H

X

X

O C

H

X = Halogen, sulfonyl, imidate, sulfur, acetate, etc.

FIGURE 1.50. Anomeric carbon and activation to a good leaving group.

38

1. Glycosides, Synthesis, and Characterization OR

OR

O

OR

O

OR OR

X

O

Nu

OR

OR

OR

X

Nu

OR

OR

+

OR

OR

Nu = R-OH, R-NH2, R-SH, RCH2X = halogen, acetate, imidate, sulfur, etc.

O

O

O

N

O

O

O

S

R

O

N

R

R

C

R

R

R

O

O

S

O

C R

R

FIGURE 1.51. Nucleophile displacement on the anomeric carbon and general types of glycosides.

OAc

OAc

O

OAc

O

X

OAc

OAc

OAc OAc

OAc

+

AcO

X OAc

FIGURE 1.52. Stereoselectivity of halides at the anomeric position.

OAc

OAc O

O

AcO AcO

AcO AcO OAc

X OAc

X

FIGURE 1.53. Anomeric effect on halogens.

1.9. Glycosyl Donors

39

1.9 Glycosyl Donors This term is used to deﬁne a glycosidic moiety that contains a leaving group at the anomeric position. When a glycosyl donor is reacted in the presence of a catalyst (also known as promoter) with a free alcohol called glycosyl acceptor, it will produce an O-glycosidic linkage. The ﬁrst glycosyl donors developed and used speciﬁcally for glycoside formation were the glycoyl halides. As mentioned above, glycosyl bromide and chloride are the most widely used halides, and are the glycosyl donors used for the preparation of O-glycosides according to the methods reported by Michael, Koenigs-Knorr and Helferich (see O-glycoside formation). 2,3,4,6-Tetraacetyl-α-D-glucopyranosyl bromide also known as acetobromoglucose is one of the most extensively used sugar intermediates for preparing glycosides derived from glucose.57 The preparation involves in the initial peracetylation of glucose with acetic anhydride in the presence of a catalyst, commonly pyridine, triethylamine and dimethylaminopyridine, or sodium acetate and zinc chloride, in dichloromethane as solvent. The resulting 1,2,3,4,6-Pentaacetyl-α,β-D-glucopyranoside (as mixture of anomers) is treated with a 33% solution of HBr-acetic acid in dichlorometane at 5◦ C during 12 h. The ﬁnal product is obtained after crystallization from isopropyl ether to yield acetobromoglucose as a white solid. The chloro derivative could be obtained similarly by using a controlled stream of HCl gas until saturation, or SOCl2 in DMF (Figure 1.54). When the sugar contains acid sensitive groups such as azide (Figure 1.55) or acid-sensitive protecting groups such as acetonide, or benzylidine (Figure 1.56), alternative milder conditions have been developed. Such is the case of fosgene in DMF and bromotrimethylsilane (TMS-Br) for chlorination and bromination, respectively.22 OAc O OH

OAc

O

O

ii OH

i OH

OAc

OAc OAc

OAc iii

OH

OH

OAc

Br OAc OAc

OAc

O OAc i) Ac2O, DMAP, Et3N, CH2Cl2. ii) HBr/AcOH 33% iii) HCl (g), CH2Cl2.

OAc

Cl OAc

FIGURE 1.54. Standard conditions for preparation of acetobromo and acetochloroglucose.

40

1. Glycosides, Synthesis, and Characterization Cl O

AcO

OH

i

AcO

N3

O N3

i) (COCl)2, DMF, CH2-CH2

FIGURE 1.55. Preparation of azide glycosyl donors.

Ph

O O AcO

i

O OAc

OAc

Ph

O O AcO

O OAc Br

i) TMS-Br, CH2Cl2.

FIGURE 1.56. Anomeric bromination in acid sensitive sugars.

The 1 H NMR spectrum of acetobromoglucose shows signals for each of the ring protons, as well as for the primarily alcohol and acetates. The well-deﬁned spectrum allows the net identiﬁcation of each proton, starting from the anomeric proton at δ 6.60 shifted downﬁeld due to the presence of the halogen, with coupling constant of 4 Hz indicating an equatorial-axial interaction with H-2. Diaxial interactions are evident as triplets for H-3 and H-4, and axial-equatorial as double of double for H-2 (Figure 1.57). Besides their extensive use in the preparation of glycosides, glycosyl bromide can also be useful for conversion to other suitable glycosyl donors (Figure 1.58),

FIGURE 1.57. 1 H NMR spectrum of acetobromoglucose in CDCl3 .

1.9. Glycosyl Donors OAc

41

OAc O

AcO

Zn/AcOH

AcO

O AcO AcO

OAc Br

OAc

OAc

O

O

i

AcO AcO

AcO AcO

50oC, 12 h, 85%

OAc

O

Br

O

H 3C

OR

i) EtOH, sym-collidine, Tetra-n-butyl-ammonium bromide OAc O

HO

OAc

O

O AcO AcO

+

HO HO

OAc

i

AcO AcO

OH

O

O

H3C

O

OMe

Br

O HO HO

OH

i) AgOTf, Lutidine, DMF, r.t.

OMe

OAc

OAc AcO AcO

O

PhSNa

OAc

EtOH-H2O

O AcO AcO

SPh OAc

Br

OAc

OAc

Bu4NSAc

O AcO AcO

OAc OAc

O AcO AcO

SAc OAc

Br OAc O

Acetone, H2O

AcO AcO

Ag2CO3

OAc Br

75-80%

O AcO AcO OAc OH

FIGURE 1.58. Some glycosyl donors obtained from acetobromoglucose.

42

1. Glycosides, Synthesis, and Characterization OAc

OAc O

NaN3

AcO AcO

O AcO AcO

OAc

N3 OAc

Br

OAc

OAc

HO

O

O

AcO AcO OAc

AcO AcO

i

O OAc

Br i) Hg(CN)2, HgBr2, CH3CN, 4 MS, 12h, 60%

FIGURE 1.58. (continued)

such as glycals,58 othoesthers,59 ad thiols.60 Also, the glycosyl halides can be transformed to glycosyl imidate through the anomeric hydroxyl formation,61 or to amines via a reaction with azide salt and hydrogenolysis.52 More recently a “disarmed” glycosyl iodide derived from glucuronolactone was designed for O-glycosidation. The iodosugar has proved to be a ﬂexible donor, reacting under mild conditions (Figure 1.59).62 Glycosyl acetates are also important glycosyl donors and can be used directly under the fusion strategy for the preparation of O- and N -glycosides. The fusion method consists of the reaction between the glycosyl acetate as glycosyl donor with the glycosyl acceptor in the presence of a Lewis acid as a promoter to generate the corresponding glycoside. Likewise, acetates can also be suitable precursors for the preparation of glycosyl donors such halides, thiols.63 and to imidates, the latter by a two-step process. The ﬁrst step involves the removal of the anomeric acetate with base, among them hydrazine, benzylamine, ammonia, and piperidine, which are the most preferred. The resulting hydroxyl group is obtained as a mixture of anomers and is subsequently used for the preparation of the glycosyl imidate (see Imidate

CO2Me O

Me3CCO2 Me3CCO2

CO2Me R-OH

i

Me3CCO2

Me3CCO2 Me3CCO2 Me3CCO2

I i) NIS/I2, then TMSOTf.

FIGURE 1.59. Glucuronyl iodide as a glycosyl donor.

O OR

1.10. Protecting Groups

43

Method). Another use of glycosyl acetates is the transformation into anomeric amines, through the introduction of the azide group with trimethylsilyl azide under a Lewis acid catalyst, and further hydrogenolysis.64 This reaction is useful for the preparation of some glycopeptides. Likewise 2-thiophenyl glycosides of Neu5Ac are suitably obtained by treatment of 2-O-acetyl, 2-chloro, or 2-chloro Neu5Ac glycosyl donors with PhSH in the presence of NIS/TfOH as promoter system (Figure 1.60). Other activated agents for preparing S-alkyl and S-aryl glycosyl donors are methyl triﬂuoromethanesulfonate (MeOTf), dimethyl(methylthio)sulfonium triﬂuoromethanesulfonate (DMTST), iodo dicollidine perchlorate (IDCP), and phenyl selenyl triﬂuoromethanesulfonate (PhSeOTf)65. Thioglycosides are stable glycosyl donors widely used for the preparation of glycosides. The usual conditions for achieving this goal are the glycosyl acceptor and N-iodosuccinimide (NIS), or NIS-TfOH as promoter. Thioglycosides are also important starting material for the preparation of other glycosyl donors such as acetates, ﬂuorine,63 chlorine,68 sulfoxides,69 or anomeric alcohols (Figure 1.61). Glycals are becoming potentially useful glycosyl donors, and an increasing number of simple and complex glycosides have been reported. For this purpose the glycal is usually transformed to the oxirane. and immediately coupled with the glycosyl acceptor in the presence of a Lewis acid (see The Glycal Method). Moreover, glycals are also suitable intermediates for the preparation of a variety of glycosyl donors (Figure 1.62) such as phosphates and thiophosphates,70 deoxysugars,71 Diels-Alder adducts,72 allyl glycosyl donors,73 and imidates.74

1.10 Protecting Groups An important additional requirement for achieving glycosidic coupling reactions, besides the fact that a good leaving group should be present, is the appropriate use of protecting groups. Their function is to shield those groups (particularly heteroatoms) that are wanted to keep unaltered during the coupling reaction and then release them under mild conditions that do not affect the glycosidic bond (Figure 1.63). A signiﬁcant number of protecting groups75 have been used and combined for pursuing the synthesis of complex natural products including glycosides. Due to its acetal character, the glycosidic bond is hydrolyzed under acidic conditions, and is signiﬁcantly more resistant to base, hydride reduction or hydrogenolysis. The use of ethers such as methyl ether (-O-CH3 ), methoxymethyl ether (-O-CH2 OCH3 , MOM), 2-methoxyethoxymethyl ether (-O-CH2 OCH2 CH2 OCH3 , MEM), and tetrahydropyranyl ether (-O-2-c-C5 H9 O, THP) have been widely used for protection of alcohols. However, in glycoside synthesis attention has to be paid since deprotection is carry out under acidic conditions, which might be hazardous for the glycosidic bond. Silyl derivatives are also another important choice for protection of hydroxyl group.65 Some of the most accepted silyl derivatives for carbohydrate hydroxyl protection are tert-butyl dimethylsilyl (TBDMS),

44

1. Glycosides, Synthesis, and Characterization OAc i

O AcO AcO

OAc

SPh OAc

O AcO AcO

OAc OAc

OAc

O

ii

AcO AcO

SMe OAc

i) PhSH (1.1 eq.), SnCl4 (0.7 eq.), CH2Cl2, 0oC, 4h, 82%. ii) MeSH (excess), SnCl4 (0.7 eq.), CH2Cl2, –20oC, 3h, 85%.

OAc

OAc

R

OAc

AcO

CO2Me

O

i

AcO

AcO

CO2Me

O AcO

OAc OAc

OAc OAc R = Cl, Br

i) TiCl4 or TiBr4

Ref.66 CO2Me

OAc AcO

X

O

AcHN

CO2Me

OAc i

AcO O

AcHN

SPh

OAc OAc

OAc OAc X = OAc, Cl, F.

i) PhSH, NIS/TfOH.

Ref.67 OAc

OAc O

AcO AcO OAc

NH2-NH2

OAc

O AcO AcO OAc

OH

FIGURE 1.60. Preparation of glycosyl donors and precursors from glycosyl acetates.

1.10. Protecting Groups

45

OAc Cl3CCN

O AcO AcO

Cs2CO3, CH2Cl2

OAc

NH

O

CCl3 OAc

OAc

OAc

O

AcO AcO

O

Me3SiN3 AcO

OAc

AcO

OAc

OAc

H2-Ni Raney

O

AcO AcO

N3

OAc

FIGURE 1.60. (continued) OAc

OAc O

AcO AcO

AcOH, NIS

SEt OAc

O AcO AcO

OAc OAc

OR

OR O

RO RO

R2SCl2

SR OR

O RO RO

Cl OR

O OTBDPS

O OTBDPS O

O

DMTST, DAST

SPh

O

F

O

OPiv

OPiv

OR

OR O

RO RO

mCPBA

SPh OR

O RO RO

O S

Ph

OR

FIGURE 1.61. Modiﬁcations of thio glycosyl donors.

triisopropylsilyl (TIPS), tert-butyl diphenylsilyl (TBDPS), and triethylsilyl (TES) ethers. Quantitative cleavage is usually achieved upon treatment with tetrabutylammonium ﬂuoride (TBAF) or HF/pyridine. The most suitable protecting groups for the preparation of glycosides are the affordable acetates, benzoates and benzyl protecting groups since they can be

NH2

46

1. Glycosides, Synthesis, and Characterization OR

OR O

RO RO

OR O

RO RO

DMDO

HSP(O)(OEt)2

O

RO RO O

OH

OR

OR NIS

O

RO RO

CH3CN-H2O

OR O

RO RO

X

Y

O

RO RO

Na2S2O4

O X

BnO PMBO

OBn O

i

OH

OR RO RO

OBn

O

RO RO

OH H2O-DMF

I

OR

O S P OEt OEt

Y

OBn O

BnO PMBO

ii

O

BnO PMBO

O OH

O i) DMDO-acetone. ii) ally alcohol, r.t., 16h, 79%.

OBn

OBn BnO BnO

O

i

BnO BnO

O N3 O

i) a) (NH4)2Ce(NO3)6, 53%. b) Cl3CCN, NaH, 98%.

NH CCl3

FIGURE 1.62. Preparation of glycosyl donors and precursors from glycals.

removed under basic and for the later neutral conditions, being the best conditions for preserving the glycosidic bond. The standard conditions for either installing and removing the most common protecting group described are: Acetate (Ac-): The standard procedure involves the use of acetic anhydride in the presence of pyridine or triethylamine as acid scavenger, and 4-(dimethylamino) pyridine (DMAP) that improves the rate of reaction. The cleavage of acetates proceeds smoothly with NaOMe solution also known as Zemplen conditions. Acetates are stable at pH from 1-8 and can be cleaved with lithium aluminium hydride (Figure 1.64).

1.10. Protecting Groups G1P

E X

E

Y

X

1) PG1

Y

X

PG2

Y

1) PG2

2) E

2) E

PG cleavage

PG cleavage

E X

47

E

Y

X

Y

FIGURE 1.63. Schematic representation of protecting group applicability. OH

OAc O

HO HO

i

OH OH

O AcO AcO

OAc OAc

i) Ac2O, CH2Cl2, Et3N, DMAP, r.t.

FIGURE 1.64. Standard protocol for the preparation of peracetylated sugars.

Benzoyl (Bz-): This protecting group is more stable to hydrolysis than acetates and may resist a pH up to 10. The conditions for protection of alcohols are shown in Figure 1.65 and involves the use of benzoyl chloride in pyridine or triethylamine.76 It is stable to hydrogenolysis and borohydrides but not to lithium aluminium hydride. The cleavage is usually achieved in 1% NaOMe-MeOH solution. Pivaloyl (Pv-): This protecting group, which is also known as Trimethylacetyl chloride, is used for protection of primary and secondary alcohols in yield. An example of the use of this group is the protection of the hydroxyl group at position 2 of fucose derivative.77 The standard conditions for protection are pivaloyl chloride in pyridine or DMAP and the cleavage is performed with Bu4 N+− OH at 20◦ C (Figure 1.66). Trityl (Tr-): This bulky protecting group is selective for primary alcohols (Figure 1.67). The protecting reaction proceeds in pyridine or DMAP-DMF.78 The cleavage can be performed under neutral conditions with 1% iodide in methanol, or weakly acidic in formic acid-ether solution. Benzyl (Bn-): This protecting group when attached with alcohol generates an ether (Figure 1.68). However, unlike common ethers, this can be cleaved under neutral condition by hydrogenolysis. The usual conditions for attachment are NaH, THF, and benzyl bromide or chloride.79 The conditions for removing this group are hydrogen, Pd/C 10% or Pd(OH)2 /C 10% in ethanol or ethyl acetate.

48

1. Glycosides, Synthesis, and Characterization O Cl

R-OH

O +

R

Py, OoC

N

Ph

O

H Cl

OTBDMS

OTBDMS

O

BzCl-Py

O HO HO

BzO BzO

O

O NTCP

NTCP

OTBDPS

OTBDPS O

HO

i

SPh

BzO

SPh

HO

O

BzO

OBz

OH

i) PhCOCl (4.0 eq.), Et3N (8.0 eq.), 4-DMAP (0.2 eq), THF, 50oC, 15h, 92%.

FIGURE 1.65. General procedure for the benzoylation of sugars.

p-Methoxybenzyl (PMB-): This benzyl derivative is installed by reacting the free alcohol with PMB-Cl under NaH, DMF conditions at 0◦ C.80 An example of its applications can be seen in the protection at the second position of acetonide thioglycoside shown in Figure 1.69. Deprotection is carried out under neutral

O O

Cl R

R-OH

+

O

Py, OoC to r.t.

O

N H Cl

O

Me O

Me

i

O

O O

OH OBn

OPv OBn

i) PvCl, Py, DMAP, 70oC, 80%.

FIGURE 1.66. Conditions and reagents for protection of alcohols with pivaloyl group.

1.10. Protecting Groups

49

TrO

HO

O

O Tr–Cl

OH

OH

OH

OH

Py

OH

OH

OH

OH Tr- = (Ph)3C-

FIGURE 1.67. Protection of primary alcohol with trityl protecting group.

Br

R-OH

NaH, THF

+

R O

OH

OBn

HO

Ph

O

i

HO

Ph

O

70%

O H3CO

NaBr + H2O

Ph

O

O

H3CO

O

i) BnCl, NaH, CuCl2, Bu4N+I–, THF, reflux, 25 h.

FIGURE 1.68. General conditions for benzylation of carbohydrates.

PMBO

HO PhS

O O O

i

PhS

O O O

i) NaH, DMF, 0oC, 30 min., then 1.2 equiv. PMB-Cl, 2 h, 93%.

FIGURE 1.69. General conditions for benzylation of carbohydrates with PMB.

conditions with 2,3-dichhloro,5,6-dicyano-1,4-benzoquinone (DDQ), in CH2 Cl2 H2 O (20:1), 1 h, at 25◦ C, in a 91% deprotection yield. Acetonide ((CH3 )2 C(O)2 -): This protecting group is useful for protection of cis diols (Figure 1.70) and the conditions are acetone, 2,2-dimethoxypropane, and ptoluenesulfonic acid or camphorsulfonic acid as catalyst.81 Acetonides are usually stable at a pH between 4 and 12, and the regeneration of the diol can be achieved by treatment with aqueous acid.

50

1. Glycosides, Synthesis, and Characterization OH

O

H3 C

H3C

O

i

O

O

HO

O

O

OH

OH

i) (CH3)2C(OCH3)2, camphorsulfonic acid, DMF, r.t., 3 h

FIGURE 1.70. Acetonide formation for protection of diols.

Benzylidene (PhCH(O)2 -): This classical protecting group is usually selected for protection of position 6 and 4, allowing the remaining positions to be modiﬁed. The benzylidene is attached under mild conditions and are useful for either –OH (4) in axial or equatorial positions (Figure 1.71). Deprotection can be effected under different conditions, such as acid conditions, hydrogenolysis, and hydrides such as BH3 NMe3 , AlCl3 , THF, 60◦ C, 1 h.82 Carbonate (O=C(O)2 -): This group is suitable for protection of cis diols, and it has been used in the synthesis of complex oligosaccharides and also in solidphase oligosaccharide synthesis. The reagents and conditions used for protection are phosgene in pyridine at 0◦ C during 1 h (Figure 1.72), and the yield reported is around 70%.83 Boronate (PhB(O)2 -): This group has been proposed in solid-phase oligosaccharide synthesis84 for simultaneous protection of 4,6-OH groups (Figure 1.73). Deprotection is achieved with IRA-743 resin.85 Tert-butyldimethylsilyl (TBS)More recently introduced for protection of primary and secondary alcohols with reported yield protection around 90% (Figure 1.74). The standard conditions are

HO O HO HO

C6H5CH(OMe)2 SPh TsOH, MeCN, 2h

OH

O

Ph

O O SPh

HO OH BnO

O

Ph

BH3NMe3, AlCl3

O O SPh

AcO OAc

THF

O HO AcO

SPh OAc

FIGURE 1.71. Protection of 4 and 6 hydroxyl groups with benzylidene group and partial removal.

1.10. Protecting Groups

51

SPh SPh O

i

O

HO

HO

O HO

O

OH O o

i) (Cl2)C=O, Py, 0 C, 1h, 69%.

FIGURE 1.72. Standard conditions for protection of diols with carbonate protecting group. Ph B O O

OH

OH

O

i

O

SEt

HO

SEt

HO

OH

OH

i) PhB(OH)2.

FIGURE 1.73. Protection of 4,6-OH groups with boronate. OH

OTBDPS O

OTBS

OTBDPS O

i

HO

TBSO

i) TBSOTf, Py. PMBO PhS

PMBO OH

i

PhS

O

OTBS O

OH

OTBS

i) TBSOTf (4.0 eq.), Et3N (10.0 eq.), CH2Cl2, 0oC, 2h, 97%.

FIGURE 1.74. Protection of secondary alcohols with TBS protecting group.

tert-butyldimethylsilyltriﬂate in pyridine,86 and deprotection is usually achieved with butyl ammonium ﬂuoride (Bu4 NF) in THF. tert-butyldiphenylsilyl (TBDPS-): This protecting group is speciﬁc for primary alcohols and the yields reported are quantitatives (Figure 1.75). This bulky silylated

52

1. Glycosides, Synthesis, and Characterization OH

OTBDPS

HO

HO

O

i

HO

O

SPh

HO

SPh

OH

OH

i) t-BuPh2SiCl, imidazole, DMF, 100%.

FIGURE 1.75. Protection of primary alcohols with TBDPS protecting group.

group has been used for the assembly of oligosaccharide libraries and has been compatible with the use of other highly selective groups.87 The standard protection conditions are TBDPS-Cl, imidazole, DMF or THF. Deprotection is achieved with hydrogen ﬂuoride-pyridine or TBAF, cat. AcOH, THF, and a yield of 87%.

1.11 Selective Protections HO i

O

HO HO

TBDMSO O

BzO BzO

O OH

O NTCP

i) TBDMSCl/Py. ii) BzCl/Py.

Ref.76 HO

HO

O

O

OH

O

O

OAllyl

i

O

O

i) CH2=CHCH2OCO2Et, Pd2(dba)3, THF, 65 oC, 4h, 70%.

Ref.88 FIGURE 1.76. Miscellaneous selective protections

1.11. Selective Protections OBn

OBn ZHN

OH

H3CO

ZHN

i

OH

O

OH

H3CO

OPhOCH3

O

Z = benzyloxycarbonyl i) 4-CH3OC6H5OH, THF, D EAD, Ph3P, 80oC, 82%.

Ref.80 OH

OH HO

H3CO

HO

OH OH

O

OH

i

H3CO

O

OTr

i) Ph3CCl, DMAP, DMF, 25oC, 12h, 88%.

Ref.89 OTMS

OH HO

H3CO

OH

TMSO

i

OH

O

H3CO

OTMS

O

OTMS

i) Me3SiCl, Et3N, THF, 25oC, 8h, 90%.

Ref.90 OH

OH HO

EtS

OH

O

AcO

OH

i

CH3

i) MeC(OCH3) 3, TsOH, DMF, 96%.

Ref.91 FIGURE 1.76. (continued)

EtS

O

CH3

53

54

1. Glycosides, Synthesis, and Characterization Ph

O O HO

Ph

i

O OH

O O AcO

O OH

OCH3

OCH3

i) Lipase AK, vinyl acetate, 92% 92

Ref.

OH

OH

HO

OH

HO

i

OH

OH EtO

OAc

O

EtO

O

i) Pancreatin, vinyl acetate, THF, TEA 95%.

Ref.93 H3C

OH

H3C

i

OH

OH EtO

OBz

O

EtO

O

i) Ph3P, DIAD, PhCO2H, THF, 84%. 94

Ref.

Ph

O O HO

Ph

i

O OH

O O HO

O OBz

OAllyl

OAllyl

i) BzOBt, 92% 95

Ref.

OH

OH HO

O

Ph O

H3CO

O

−

i

O

O3SO

Ph O

H3CO

i) Pyr SO3, Pyr, 69%

Ref.96 FIGURE 1.76. (continued)

O

1.11. Selective Protections

HO HO HO

O

i

O

55

O

O O

OH

O

OCH3

OCH3

O i) DMSO, POCl3, 85%.

Ref.97 OH

OH

HO

OH

HO

i

O

OH HO

O

O

H3CO

O

i) CH2=C(CH3)OCH3, DMF, TsOH. 0oC, 95%

Ref.98 OCH3 i

O

H3 C O

OCH3

OPv

O

H3C

O

OH OBn

Ph

Bn = PhCH2−

OPv

i) NBS, CCl4, 75%.

Ref.99 R O

O HO HO HO

R O

EtO

Z Y

i

OEt

R R

O O HO

O Z Y

i) PPTS, ACN. 90%

Ref.100 FIGURE 1.76. (continued)

56

1. Glycosides, Synthesis, and Characterization OH O

HO HO

O O HO

Ph

i

SPh OH

O SPh OH

i) C6H5CH(OMe)2, TsOH, MeCN, 2h. 82

Ref.

OH O

HO HO

OPiv

OH O

O HO

OH

i

HO HO

SPh

OH

OH

OPiv

O O HO

O SPh OH

i) Pivaloyl chloride, Py, 91%.

Ref.101 O

OH

O

HO

OH

O

i

H3CO

O

CH3

H3CO

OH

O

CH3

i) Cl3CCOCl, Py, rt, 80%

Ref.102

O OTBDPS HO HO

Br

Br

Br

O

OTBDPS

O O

O

O O OH

OH

O

OMe

Br 103

Ref.

FIGURE 1.76. (continued)

OMe

1.11. Selective Protections AcS

NaO3S

OBz

OBz O

i

O

57

BzO

BzO

BzHN

BzHN

OMe

OMe

i) aq. H2O2 (33%), AcOH, NaOAc, 80oC, 8h.

Ref.104 OBn

OBn O

BnO BnO

i

O

BnO BnO O

O

i) DMDO, ZnCl2, acetone.

OH O

HO HO

OTIBS i

OTIBS

O

HO HO

ii

O

HO BnO

i) TIBS-Cl, Imidazole. ii) Bu2SnO, BnBr, TBAI, 67%

Ref.105 OH HO HO

OH

O OH

i

HO HO

NH2.HCl

O OH NHAc

i) a) 1eq. NaOMe/MeOH. b) 1.2 eq. Ac2O, 12h, 91%.

Ref.106 Ph

O O HO

O

i

OMe OAc

Ph

O O ROOC

O OMe OAc

i) TMEDA, ClCO2R, CH2Cl2, 0oC.

Ref.107 FIGURE 1.76. (continued)

58

1. Glycosides, Synthesis, and Characterization HO

OTBDPS

OTBDPS

TBSO i

O

O

HO

TBSO

TBDPS = tert-butyldiphenylsilyl TBS = tert-butyl dimethylsilyl i) TBSOTf, Py.

Ref.

86

OCH3

OH

H3CO OH

HO

OBz

OCH3

C6H4-p-OMe

O

BzO

CH3 H3CO

i

OH O

O

H3CO

O

i) pyridinium p-toluensulfonate, 82-100%.

Ref.108 OH HO HO

OAc

O OH

i,ii

OCH3

HO AcO

O OAc

OCH3

i) (Bn3Sn)2O, PhMe. ii) AcCl, r.t.

Ref.109 FIGURE 1.76. (continued) TABLE 1.3. Summary of common protecting and cleavage conditions. Protecting group

Protection conditions

Acetyl (Ac-) Ac2 O, Et3 N, DMAP, CH2 Cl2 Benzoyl (Bz-) Bz-Cl, Py Pivaloyl (Pv-) Pv-Cl, Py, DMAP Trityl (Tr-) Tr-Cl, DMAP, DMF Benzyl (Bn-) Bn-Br, NaH, THF p-Methoxybenzyl (PMB-) PMB-Cl, NaH, THF Acetonide ((CH3 )2 C(O)2 -) (CH3 )2 CO, 2,2-DMP, p-TSOH PhCH(OCH3 )2 , p-TsOH, CH3 CN Benzylidene (PhCH(O)2 -) tert-butyldimethylsilyl (TBS-) TBS-OTf-Py tert-butyldiphenylsilyl (TBDPS-) TBDPS-Cl-imidazole, DMF

Cleavage conditions NaOMe-MeOH NaOMe-MeOH Bu4 NOH 1% I2 -MeOH H2 -Pd(OH)2 -EtOH DDQ, CH2 Cl2 -H2 O AcOH-H2 O AcOH-H2 O, or H2 -Pd(OH)2 Bu4NF-THF HF-Py

1.12. Selective Deprotections

59

1.12 Selective Deprotections TrO

HO

O OAc

AcO AcO

O

i

AcO AcO

OAc

OAc OAc

i) 1% I2, MeOH.

Ref.110 OAc

OAc ThPhN

OAc

PhThN

i

OAc

OAc

HO

O

MPMO

OAc

O

PhTh = phthalimido. MPM = p-Methoxybenzyl ether p-MeOC6H4CH2OR i) CAN or NBS, CH2Cl2, H 2O.

Ref.111 i

O AcO AcO

O

OCH3

HO AcO

OAc

OCH3

OAc

i) Lipase PS, n-pentyl-OH, 93%

Ref.112 OBn BnO

OH

i

OBn Me BnO

H3CO

O

O

O H3CO

O

Me

O

Ph OBn i

BnO

Ph OBn OH

H3CO

O

i) LiAlH4, AlCl3, Et2O, CH2Cl2, heat

Ref.113 FIGURE 1.77. Miscellaneous selective deprotections.

60

1. Glycosides, Synthesis, and Characterization OAc

OAc O

O

BzO

Me

O

Ph

O

O O

O

Br

O

i) BrCCl3, CCl4, hv, 30 min. 100%

Ref.114 Ph O

OH

O

OH

i

O

O

OMe

OMe

i) H2, Pd(OH)2, EtOH, 92%

Ref.94 Ph O

OBz

O i

TBSO

Me

i

O

TBSO

OMe

i) NBS, BaCO3, CCl4,∆

Ref.115 FIGURE 1.77. (continued)

O OMe

Br

1.12. Selective Deprotections

Si

OH

O

HO

O

Si

O Si

HO

i

O

O

Si RO

OH

O

O

61

RO

O

i) DMF, H+, 82%

Ref.116 t-Bu

Ph O Ph HO

O SiMe3

OH

O

HO HO

i

O

O SiMe3

OH

i) Bu4NF.3 H2O, AcOH, THF.

Ref.117 O

AcO AcO

i

O

AcO AcO

OAc OAc

OH OAc

i) CF3COOH, CH2Cl2.

O O H3C

i

O AcO

OR O

OR OH

OCH3 i) AcOH, 100%. 118

Ref.

OAc AcO AcO

OAc

O OAc

i

OAc

AcO AcO

i) a) BnNH2, THF. b) HCl.

Ref.86 FIGURE 1.77. (continued)

O OAc

OH

62

1. Glycosides, Synthesis, and Characterization OTMS

OTMS OTMS

TMSO

OTMS

H3CO

OTMS

TMSO

i

OH

H3CO

O

O

i) K2CO3 , MeOH, 0oC, 45 min, 100%

Ref.119 Ph

O O BnO

BnO HO BnO

i

O O

(CH2)3

NTCP

O O (CH2)3

NTCP

i) a) NaBH3CN. b) HCl, THF, Et2O

Ref.76 OBz

OBz C6H4-p-OMe

O

BzO

OH

BzO

i

CH3 O

H3CO

OH

H3CO

O

O

i) a) SnCl4, CH2Cl2, −78oC. b) Bu4NOH, 90%.

Ref.108 OBn O

BnO

H3CO

OBn i

OH

BnO

O

BnO +

O O

OBn

H3CO

O O 79%

i) a) NaBH3CN. b) HCl, THF.

Ref.120 FIGURE 1.77. (continued)

H3CO

OH O

References

63

OBn OH

BnO

i

OMPM

H3CO

O

OBn OBn

H3CO

OMPM

BnO ii

C6H4-p-OMe

O

BnO

OH

H3CO

O

O

O OBn OH

BnO

iii

OH

H3CO

O

i) NaBH3CN. b) TFA, DMF. ii) a) NaBH3CN. b) TMSCl, CH3CN. iii) CAN, CH3CN-H2O (9:1), 95%.

Ref.121 OBn

OBn O

BnO BnO

i

BnO

N3 O AllO

OPBB

BnO BnO

OBn OBn

O BnO

N3

OPBB

O HO

PBB = p-bromobenzyl i) Pd(OAc)2, (o-biphenyl)P(tBu)2, PhN(H)Me, NaO tBu, 80oC. ii) SnCl4, 84%.

Ref.122 FIGURE 1.77. (continued)

References 1. 2. 3. 4. 5.

J.F. Robyt, Essentials of Carbohydrate Chemistry, Springer, NY (1998). H.S. Khadem, Carbohydrate Chemistry, Academic Press, NY (1988). E. Fischer, Ber. 23, 2114 (1890). G. Casiraghi, F. Zanardi, G. Rassu, and P. Spanu, Chem. Rev. 95, 1677 (1995) T.-H. Chan, and C-J. Li J. Chem. Soc. Chem. Commun. 747 (1992)

OBn OBn

64

1. Glycosides, Synthesis, and Characterization

6. J. Gao, R. H¨artner, D.M. Gordon, and G.M. Whitesides, J. Org. Chem. 59, 3714 (1994). 7. R.H. Prenner, W.H. Binder, and W. Schmid, Liebigs Ann. Chem. 73 (1994). 8. K.-I. Sato, T. Miyata, I. Tanai., and Y. Yonezawa, Chem. Lett. 129 (1994). 9. S.G. Davies, R.L. Nicholson., and A.D. Smith, Synlett 1637 (2002). 10. A.B. Northrup, I.K. Mangion, F. Hettche, and D.W.C. MacMillan, Angew. Chem. Int. Ed. 43, 2152 (2004). 11. A. Lubineau, J. Aug´e, and N. Lubin, Tetrahedron 49, 4639 (1993). 12. G. Casiraghi, L. Pinna, G. Rassu, P. Spanu., and F. Ulheri, Tetrahedron: Assimetry 3, 681 (1993). 13. S.A.W. Gruner, E. Locardi, E. Lohof, and H. Kessler, Chem Rev. 102, 491 (2002). 14. M.P. Watterson, L. Pickering, M.D. Smith, S.J. Hudson, P.R. Marsh, J.E. Mordaunt, D.J. Watkin, C.J. Newman, and G.W.J. Fleet, Tetrahedron: Asymmetry 10, 1855 (1999). 15. N.L. Hungerford, and G.W.J. Fleet, J. Chem. Soc. Perkin Trans. 1 3680 (2000). 16. A. Dondoni, A. Marra, Chem Rev. 100, 4395 (2000). 17. M.J. Robins, and J.M.R. Parker. Can. J. Chem. 61, 312 (1983). 18. J.R. Axon, and A.L.J. Beckwith, J. Chem. Soc. , Chem. Commun. 549 (1995). 19. G. Li, H.H. Angert, and K.B. Sharpless, Angew. Chem. Int. Ed. Engl. 35, 2813 (1996). 20. O. Jarreton, T. Skrydstrup, J.-F. Espinosa, J. Jim´enez-Barbero, and J.-M. Beau, Chem. Eur. J. 5, 430 (1999). 21. Y. Ohnishi, and Y. Ichikawa, Bioorg. Med. Chem. Lett. 12, 997 (2002). 22. M. Oberthur, C. Leimkuhler, and D. Kahne, Org. Lett. 6, 2873 (2004). 23. O. Meyerhof, and K. Lohmann, Biochem. Z. 271, 89 (1934). 24. H.M. Gijsen, L. Qiao, W. Fitz, and C.-H. Wong, Chem. Rev. 96, 443 (1996). 25. M.D. Bednarski, H.J. Waldmann, and G.M. Whitesides, Tetrahedron Lett. 27, 5807 (1986). 26. W. Baumann, J. Freidenreich, G. Weisshaar, R. Brossmer, and H. Friebolin, Biol. Chem. 370, 141 (1989). 27. G.J. Boguslawski, J. App. Biochem. 5, 186 (1983). 28. J.R. Durrwachter, and C.-H. Wong, J. Org. Chem. 53, 4175 (1988). 29. S.-H. Jung, J.H. Jeong, P. Miller, and C.-H. Wong, J. Org. Chem. 59, 7182 (1994). 30. G.C. Look, Ch.H. Fotsch, and C.-H. Wong, Acc. Chem. Res. 26, 182 (1993). 31. T. Aoyagi, T. Yamamoto, K. Kojiri, H. Morishima, M. Nagai, M. Hamada, T. Takechi, and H. Umezawa, J. Antibiot. 42, 883 (1989). 32. R. Saul, R.J. Moylneux, and A.D. Elbein, Arch. Biochem. Biophys. 230, 668 (1984). 33. H. Kayakiri, K. Nakamura, S. Takase, H. Setoi, I. Uchida, H. Terano, M. Hashimoto, T. Tada, and S. Koda, Chem. Pharm. Bull. 39, 2807 (1991). 34. B.C. Baguley, G. R¨ommele, J. Gruner, and W. Wehrli, Eur. J. Biochem. 97, 345 (1979). 35. a) J. Swenden, C. Borgmann, G. Legler, and E. Bause, Arch. Biochem. Biophys. 248, 335 (1986). b) C. McDonnell, L. Cronin, J.L. O’Brien, P.V. Murphy, J. Org. Chem. 69, 3565 (2004). 36. A. Dondoni, P. Merino, and D. Perrone, Tetrahedron 49, 2939 (1993). 37. T. Ziegler, A. Straub, and F. Effenberger, Angew. Chem. Int. Ed. Engl. 27, 716 (1988). 38. C. Aug´e, and C. Gautheron, Adv. Carbohydr. Chem. 49, 175 (1991). 39. Y.-F. Wang, D.P. Dumas, and C.-H. Wong, Tetrahedron Lett. 34, 403 (1993). 40. R. Rai, I. McAlexander, and Ch.-W.T. Chang, Org. Prep. Proced. Int. (OPPI), 37, 339 (2005).

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41. Y. LeBlanc, B.J. Fitzsimmons, J.P. Springer and J. Rokach, J. Am. Chem. Soc., 111, 2995 (1989). 42. J. Dubois, C.S., Tomooka, and E.M. Carreira, J. Am. Chem. Soc., 119, 3179 (1997). 43. J. Liu and Y.D. Gin, J. Am. Chem. Soc., 124, 9789 (2002). 44. N.V. Bovin, S.E. Zurabyan and A.Y. Khorlii, Carbohydr. Res., 98, 25 (1981). 45. Reddy et al J. Org. Chem. 69, 2630 (2004). 46. B. Elchert, J. Li, J. Wang, Y. Hui, R. Rai, R. Ptak, P. Ward, J.Y. Takemoto, M. Bensaci, and C.-W. Chang, J. Org. Chem., 69, 1513 (2004). 47. V. Pavliak and P. Kovbk, Carbohydr. Res. 210, 333 (1991) 48. F. Dasgupta and P.J. Garegg, Synthesis, 262 (1988) 49. W.-C. Chou, L. Chen, J.-M. Fang, C.-H. Wong, J. Am. Chem. Soc. 116, 6169 (1994). 50. R.V. Stick, K.A. Stubbs, Tetrahedron Asymmetry 16, 321 (2005). 51. S. Ogawa, N. Matsunaga, H. Li, and M.M. Palcic, Eur. J. Org. Chem. 631 (1999). 52. a) T.D. Heingtmann, and A.T. Vassela, Angew. Chem. Int. Ed. 38, 750 (1999). b) A. Bianchi, A. Russo, A. Bernanrdi, Tetrahedron Asymmetry 16, 381 (2005). 53. S. Ogawa, M. Ohmura, S. Hisamatsu, Synthesis 312 (2001). 54. R.M. van Well, K.P. Kartha, and R.A. Field, J. Carbohydr. Chem. 24, 463 (2005). 55. M. Shimizu, H. Togo and M. Yokohama Synthesis 779 (1998). 56. E. Juaristi, and G. Cuevas, The Anomeric Effect, CRC, Boca Raton 4 (1995). 57. W. Koenigs, and E. Knorr, Chem. Ber. 34, 957, (1901). 58. a) W. Roth, and W. Pigman, in Methods in Carbohydrate Chemistry; Whistler R.L. and Wolfrom, M.L., Eds.; Academic Press: New York, 1963, Vol. 2, 405 (1963).; b) B.K. Shull, Z. Wu, and M. Koreeda, J. Carbohydr. Chem. 15(8), 955 (1996).; A. F¨urstner, and H. Weidmann, J. Carbohydr. Chem., 7(4), 773 (1988). 59. R.U. Lemieux, and A.R. Morgan, Can. J. Chem. 43, 2199 (1965).; W. Wang, F. Kong, J. Org. Chem. 63, 5744 (1998). 60. M. Blanc-Muessen, J. Defaye, and H. Driguez, Carbohydr. Res. 67, 305 (1978). 61. Ch. McCloskey, and G.H. Coleman, Org. Synth. 3, 434, (1955). 62. J.A. Perrie, J.H. Harding, C. King, D. Sinnott, and A.V. Stachulski, Org. Lett. 5, 4545 (2003). 63. K.C. Nicolaou, J. Li, and G. Zenke, Helv. Chim. Acta 83, 1977 (2000). 64. Z. Gy¨orgyde´ak, L. Szil´agyi, and H. Paulsen, J. Carbohydr. Chem. 12(2), 139 (1993). 65. G.-J. Boons, and A.V. Demchenko, Chem Rev. 100, 4539 (2000). 66. H. Paulsen, and H. Tietz, Carbohydr. Res. 125, 47 (1984). 67. H. Maeda, K. Ito, H. Ishida, M. Kiso., and A. Hasegawa, J. Carbohydr. Res. 14, 387 (1995). 68. S. Sugiyama, and J.M. Diakur, Organic Lett. 2, 2713 (2000). 69. D. Kahne, S. Walker, Y. Cheng, and D. Van Engen, J. Am. Chem. Soc. 111, 6881 (1989). 70. O.J. Plante, and P. Seeberg, J. Org. Chem. 63, 9150 (1998). 71. V. Constantino, C. Imperatore, E. Fattoruso, and A. Magnoni, Tetrahedron Lett. 41, 9177, (2000). 72. A. Dios, A. Geer, C.H. Marzabadi, and W.R. Franck, J. Org. Chem. 63, 6673 (1998). 73. F. Bosse, L.A. Marcaurelle, and P.H. Seeberger, J. Org. Chem. 67, 6659 (2002). 74. R.R. Schmidt, Angew. Chem. Int. Engl. 25, 213 (1986). 75. P.G.M. Guts, and T.W. Greene, Protecting groups in Organic Synthesis; Wiley, New York, (1991). 76. L. Olsson, Z.J. Jia, and B. Fraser-Reid, J. Org. Chem. 63, 3790 (1998).; K.C. Nicolaou, N. Winssinger, J. Pastor, and F. De Roose, J. Am. Chem. Soc. 119, 449 (1997).

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77. D.P. Larson, and C.H. Heathcock, J. Org. Chem. 62, 8406 (1997). 78. L. Liu, and H. Liu, Tetrahedron Lett. 30, 35 (1989). 79. B. Classon, P.J. Garegg, S. Oscarson, and A.K. Tiden, Carbohydr. Res. 216, 187 (1991). 80. K.C. Nicolaou, T. Ohshima, F.L. van Delft, D. Vourloumis, J.Y. Xu, J.S. Pfefferkorn, and S. Kim, J. Am. Chem. Soc. 120, 8674 (1998). 81. I. Kitagawa, K. Ohashi, N.I. Baek, M. Sakagami, M. Yoshikawa, and H. Shibuya, Chem. Pharm., Bull. 45(5), 786 (1997). 82. V. Ellervik, and G. Magnusson, J. Org. Chem. 63, 9314 (1998). 83. D. Crich, and H. Li, J. Org. Chem. 67, 4640 (2002). 84. J.M. Frech´et, Polymer-supported Reactions in Organic Synthesis; Hodge, P., Sherrington, D.C., Eds.; Wiley. (1980) 85. G.G. Cross, and D.M. Whitﬁeld, Synlett 487 (1999). 86. J.L. Koviak, M.D. Chapell, and R.L. Halcomb, J. Org. Chem. 66, 2318 (2001). 87. C.-H. Wong, X.-S. Ye, and Z. Zhang, J. Am. Chem. Soc. 120, 7173 (1998). 88. R. Lakhmiri, P. Lhoste, and D. Sinou, Tetrahedron Lett. 30, 4669 (1989). 89. S.K. Chaudhary, and O. Hern´andez, Tetrahedron Lett. 95 (1979). 90. T.W. Hart, D.A. Metcalfe, and F. Scheinmann, J. Chem. Soc. Chem. Commun. 156 (1979). 91. M. Oikawa, A. Wada, F. Okazaki, and S. Kusumoto, J. Org. Chem. 61, 4469 (1996). 92. I. Matsuo, M. Isomura, R. Walton, and K. Ajisaka, Tetrahedron Lett. 37, 8795 (1996). 93. F. Theil, and H. Schick, Synthesis 533 (1991). 94. A.B. Smith III, and K.J. Hale, Tetrahedron Lett. 30, 1037 (1989). 95. H. Yamada, T. Harada, and T. Takahashi. J. Am. Chem. Soc. 116, 7919 (1994). 96. L. Leau, N. Goren, Carbohydr. Res. C8, 131, (1984). 97. M. Guiso, C. Procaccio, M.R. Fizzano, and F. Piccioni, Tetrahedron Lett. 38, 4291 (1997). 98. J. Cai, B.E. Davison, C.R. Ganellin, and S. Thaisrivongs, Tetrahedron Lett. 36, 6535 (1995). 99. R.W. Binkley, G.S. Goewey, and J.C. Johnston, J. Org. Chem. 49, 992 (1984). 100. A. Arasappan, and P.L. Fuchs, J. Am. Chem. Soc. 117, 177 (1995). 101. L. Jiang, and T.-H. Chan, J. Org. Chem. 63, 6035 (1998). 102. Tsatsuda et al., Bull. Chem. Soc. Jpn. 58, 1699 (1985). 103. S.V. Ley, and S. Mio, Synlett 789 (1996). 104. J.G. Fernandez-Bola˜nos, J.G. Garc´ıa, J. Fernandez-Bola˜nos, M.J. Di´anez, M.D. Estrada, A. L´opez-Castro, and S. P´erez Garrido, Tetrahedron Assymetry 14, 3761 (2003). 105. J.G.S. Lohman, and P.H. Seeberger, J. Org. Chem. 68, 7541 (2003). 106. H. Liang, and T.B. Grindley J. Carbohydr. Chem. 23, 71 (2004). 107. M. Adinolﬁ, G. Borone, L. Guariniello, and A. Iadonisi, Tetrahedron Lett. 41, 9305 (2000). 108. S. Fors´en, B. Lindberg, and B.-G. Silvander, Acta Chem. Scand. 19, 359, (1965). 109. K. Koch, and R. J. Chambers, Carbohydr. Res 241, 295 (1993). 110. J.L. Wahlstrom, and R.C. Ronald, J. Org. Chem. 63, 6021 (1998). 111. B. Classon, P.J. Garegg, and B. Samuelson Acta Chem Scan. Ser. B, 38, 419 (1984). 112. R. L´opez, E. Montero, F. Sanchez, J. Ca˜nada, and A. Fernandez-Mayoralas, J. Org. Chem. 59, 7029 (1994). 113. R. Miethchen, J. Holz, H. Prade, and A. Liptak, Tetrahedron 3061 (1992).

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114. P.M. Collins, A. Manro, E.C. Opara-Mottah, and M.H. Ali, J. Chem. Soc. Chem. Commun. 272 (1988). 115. S. Hanessian, and N.R. Plessas, J. Org. Chem. 34, 1035 (1969). 116. C.A.A. Van Boeckel, and J.H. van Boom, Tetrahedron 41, 4545 (1985). 117. M. Wilstermann, and G. Magnusson, J. Org. Chem. 62, 7961 (1997). 118. S. Hanessian, and R. Roy, Can. J. Chem. 63, 163 (1985). 119. D.T. Hurst, A.G. McIness, Can. J. Chem. 43, 2004 (1965). 120. P.J. Garegg, Acc. Chem. Res. 25, 575 (1992). 121. R. Johansson, and B. Samuelson, J. Chem. Soc. Perkin Trans 1 2371 (1984). 122. X. Liu, and P.H. Seeberger, Chem. Commun. 1708 (2004).

2 O-Glycoside Formation

2.1 General Methods When a monosaccharide (or sugar fragment of any size) is condensed with either an aliphatic or aromatic alcohol, or another sugar moiety through an oxygen, a glycoside bond is formed. General examples of O-glycosides are shown in Figure 2.1. The most common coupling reaction methodologies used for preparing the vast majority of O-glycosides known thus far are1 The Michael reaction The Fischer reaction The Koenigs-Knorr reaction The Helferich reaction The Fusion method The Imidate reaction The Sulfur reaction The armed-disarmed approach The Glycal reaction The Miscellaneous leaving groups The solid-phase approach

2.1.1 The Michael Reaction O PO

O

promoter

X

+

ArOH

PO

P = protecting group X = Br, Cl

68

Promoter

Conditions

NaH K2 CO3 , NaOH

THF acetone

OR

2.1. General Methods OH

69

OH O

O

HO HO

HO HO

O OH

O OH

OH OH O HO HO

O

O OH

OH

HO OH

FIGURE 2.1. Examples of O-glycosides.

This pioneering methodology for O-glycosylation consists of the condensation reaction between 2,3,4,6-Tetraacetyl-α-D-glucopyranosyl chloride and potassium phenoxide to generate the acetylated derivate that undergoes basic hydrolysis to give phenyl-β-D-glucopyranoside (Figure 2.2). Since its original methodology, some modiﬁcations have been introduced especially for aromatic glycosides. Some of the main features associated with this methodology are Preserves the pyranose or furanose ring Drives the addition of the aromatic aglycon to the anomeric position Uses protecting groups which are easily removed in basic medium Produces exclusively the β-O-glycoside as a result of neighboring group participation OAc

OK

OAc

O OAc OAc

i

+

Cl OAc

OO

NO2

OAc OAc

NO2

OAc OH

ii

OO

NO2

OH OH OH

i) acetone or DMF. ii) MeONa/MeOH.

FIGURE 2.2. Synthesis of paranitrophenyl-β-D-glucopyranosyl tetraacetate.

70

2. O-Glycoside Formation OH

CH3

HO

O

O

NO2

Cl

OH

N

N

OH

Br

N Ac

FIGURE 2.3. O-glycoside chromophores used for enzymatic detection.

This reaction has been employed for the preparation of O-glycosides that are used as substrates for detection and measurement of enzymatic activity of most of the known glycosidases. Using this methodology, several chromophores have been attached to most of the common monosaccharides. After O-glycoside cleavage by the enzyme, the release of the chromophore will indicate the sites and eventually will quantify the enzymatic activity. Some of the chromophores currently used for these purposes are represented in Figure 2.3. The highly ﬂuorescent O-glycoside substrate 7-hydroxy-4-methylcoumarin-βD-glucopyranose is prepared by condensation between acetobromoglucose with 4-methylumbelliferone in the presence of potassium carbonate in acetone. The intermediate is deacetylated under basic conditions to afford umbelliferyl β-Dglucopyranoside (Figure 2.4). Anderson and Leaback2 were able to prepare 5-Bromo indoxyl-β-D- Nacetylglucopyranoside, a hystochemical substrate for enzymatic detection of quitinase by condensing 3,4,6-triacetyl-β-D N-acetylglucopyranoside chloride with 5-bromo-hydroxy-N acetyl indole at 0◦ C under nitrogen atmosphere (Figure 2.5).

2.1.2 The Fischer Reaction O HO

O

promoter

OH

+

ROH

HO

Promoter

Conditions

HCl gas pTsOH

CH2 Cl2, r.t. CH2 Cl2, r.t.

OR

2.1. General Methods

71 O

OAc O

HO

OAc OAc

O O

OAc

O i

+

OO CH3

OAc

Br OAc

CH3

OAc OAc O O OH OO

ii

CH3

OH OH OH

i) K2CO3/acetone. ii) MeONa/MeOH.

FIGURE 2.4. Michael approach for preparation umbellyferyl-O-glycoside.

This straightforward strategy is used specially for the preparation of simple Oglycosides. The advantage of this methodology is that it does not require the use of protecting groups and simply by combining the free sugar with an alcohol under acidic condition we furnish the corresponding O-glycoside. However, contrary to the previous method, this procedure is not stereo selective and therefore it provides a mixture of anomers. Also, it has been found satisfactory only for small aliphatic alcohols (Figure 2.6). The addition of a controlled stream of dry HCl during a period of around 10 min at room temperature generally are the conditions of choice. However, the use Br OAc

HO Br

O OAc OAc

Cl

OH

i,ii

+

N Ac

NHAc

OO OH OH

i) NaOH/MeOH, OoC, N2. ii) MeONa/MeOH.

NHAc

FIGURE 2.5. Synthesis of indole O-glycoside derivative.

NAc

72

2. O-Glycoside Formation OH

FIGURE 2.6. The Fischer glycoside reaction.

OH O

OH

O

OMe

O-

i

HO

OH

HO

OH

OH

OH

i) MeOH-HCl(g).

of Lewis acid, ion exchange resin, and more recently triﬂic acid have been also reported providing good yields.3 It is worth mentioning that besides the main product, a mixture of isomers has been detected, suggesting that a rather complex mechanism is involved. It is also seen that the amount of these isomers depends importantly on the condition reactions employed (Figure 2.7). The Fischer methodology has been applied successfully for the synthesis of benzyl O-glycosides. L-Fucose was converted into benzyl fucopyranoside4 by treatment with benzyl alcohol under saturation with HCl at 0◦ C, to furnish the α and β anomers (ratio 5:1) in 80% yield (Figure 2.8). OH

OH HO

HO OH O

OMe

O OH

O OH

i

OMe

OH

OH

OH ii

OH

OH OH

OH OH

O

O OMe OH

OH OMe

OH

OH OH

OH i) MeOH/ 0.7% HCl, 20oC. ii) MeOH/ 4% HCl reflux.

FIGURE 2.7. The Fischer O-glycoside isomers.

O CH3 OH OH OH

O

i

OH

OBn

CH3 OH

O CH3 OH OH

OH 5:1

OH

OBn

OH

o

i) BnOH/HCl (g), 10 min. r.t, and O/N at 4 C.

FIGURE 2.8. Fischer conditions for preparation of Benzyl L-fucose.

2.1. General Methods

73

2.1.3 The Koenigs-Knorr Reaction O

O

promoter

PO

X

+

ROH

PO

OR

X = Br, Cl

Promoter

Conditions

Ag2 CO3 Ag2 O AgNO3 AgClO4 AgOTf

PhH, drierite (drying agent), I2 s-collidine (acid scavender) HgO (acid scavender) Ag2 ClO3 (acid scavender), THF or toluene, r.t. CH2 Cl2 , r.t.

This reaction reported in 1901 is still one of the most useful reactions for preparing a wide variety of O-glycosides.5 It is useful for coupling reactions with either alkyl or aromatic alcohols as well as for coupling between sugars. The methodology requires silver salts as catalyst and among them the oxide, carbonate, nitrate, and triﬂate silver salts are the most commonly employed (Figure 2.9). Also a drying agent such as calcium sulfate (drierite), calcium chloride, or molecular sieves is recommended. Improved yields are obtained with iodide, vigorous stirring, and protection against light during the course of the reaction. The stereochemistry observed is 1,2 trans type in most of the cases reported, as a consequence of neighboring group participation. When the protecting group is acetate at C (2), there is an intramolecular nucleophilic displacement of the leaving group, generating an orthoester.6 This intermediate is responsible for the incorporation of the alcohol on the β-position (Figure 2.10). Only until recently a method for preparing 1,2-cis glycosides has been developed involving the use of (1S)-phenyl-2-(phenylsulfanyl)ethyl moiety at C-2 of a glycosyl donor to give a quasi-stable anomeric sulfonium ion. The sulfonium ion is formed as a transdecalin ring system. Displacement of the sulfonium ion by a hydroxyl leads to the stereoselective formation of α-glycosides.7 This versatile methodology can be applied for preparation of alky, aryl, and oligosaccharide O-glycosides. A steroidal glycoside cholesterol absorption inhibitor was prepared by condensation between acetobromocellobiose and (3β,5α, OAc

OH

O

HO

OAc OAc

+

O i,ii

Br

OH OH

OAc

OH

i) Ag2O or Ag2CO3/PhH, drierite, I2. ii) MeONa/MeOH.

FIGURE 2.9. The Koenigs-Knorr reaction.

O

74

2. O-Glycoside Formation OAc O OAc OAc

OAc

OAc

O

O

OAc Br

OAc O

OAc

O

OAc

O

O OR +

O

OAc

OR

O CH3

O O

OAc

O

OAc

OAc

R

OAc

H CH3

OAc OAc OAc

ROH/H

FIGURE 2.10. Proposed mechanism for the Koenigs-Knorr glycosidic reaction.

25R)-3-hydroxyspirostan-11-one with anhydrous ZnF2 as catalyst in acetonitrile to provide the steroidal glycoside in 93% yield (Figure 2.11).8 The steroidal glycoside Estrone-β-D-glucuronide was prepared by condensation between Methyl tri-O-glucopyranosylbromide uronate with estrone, employing cadmium instead of silver carbonate (Figure 2.12).9 A comprehensive study about methods for the preparation of diverse O-glucuronides has been described.10 The syntheses of various disaccharides have been reported under Koenigs-Knorr conditions. Gentobiose octaacetate was prepared through condensation of acetobromoglucose with 1,2,3,4-Tetra-O-acetyl-O-Trityl-β-D-glucopyranose in nitromethane using silver perchlorate as catalyst (Figure 2.13).11 B¨achli and Percival12 reported the synthesis of laminaribiose by reacting 1,2,5,6Diisopropylidenglucose with acetobromoglucose in the presence of silver carbonate, iodine, and drierite to produce an acetonide intermediate, which, upon treatment with oxalic acid and sodium methoxide, furnished the 1,3-disaccharide (Figure 2.14). The synthesis of various disaccharides containing N-acetylneuraminic acid (Neu5Ac) was achieved by using acetochloro and acetobromo neuraminic acids as glycosyl donors with active glycosyl acceptors under Ag2 CO3 -promoted reactions conditions (Figure 2.15).13,14 These conditions are also suitable for preparing short oligosaccharides such as the one presented in Figure 2.16. The donor sugar acetobromogentobiose is coupled to the acceptor intermediate using silver triﬂate as glycosidation catalyst.15 Total synthesis of Bleomycin group antibiotic has been achieved by Katano and Hecht.16 Thus, glycoside coupling reaction of protected disaccharide glycosyl donor with histidine derivative using silver triﬂate as glycoside promoter provided Bleomycin key intermediate in 21% (Figure 2.17).

2.1. General Methods

75

OAc O

O

O

Br

OAc O

O

+

O

OAc OAc

HO AcO

OAc OAc

O

O O

OH

i,ii

O

O

OH O

O

OH OH

HO

OH OH i) ZnF2, CH3CN. ii) NaOMe

FIGURE 2.11. Synthesis of steroidal glycoside. CO2Me O

COOH OO

i,ii

+

OAc OAc

HO

CH3

OH Br

OH

OAc

CH3 O

OH

i) Cd2CO3. ii) MeONa/MeOH

FIGURE 2.12. Synthesis of a steroidal O-glycoside. OAc O

+

OAc

OH O O

CH2OTr O OAc i,ii

OAc

O OH

OH OH

OAc

Br OAc

OH

OAc OAc

OH

OH OH

i) AgClO4, CH3NO2. ii) MeONa/MeOH

FIGURE 2.13. Synthesis of gentobiose.

O

76

2. O-Glycoside Formation

O

O

OAc

O OAc

O AcO AcO

O

O OH

+

OAc

i

Br

AcO AcO

O

O

O OAc

O

HO HO

O

O

OH

OH ii

O O

HO

O O OH

OH

OH

i) Ag2CO3, drierite, I2. ii) a)MeONa/MeOH. b) oxalic acid 0.001 N, 100oC.

FIGURE 2.14. Synthesis of laminaribiose. O R

OAc AcO

O CO2Me

O

+

O

AcO

O

O O

OH O

O

Ag2CO3 O

OAc OAc

O

O

OAc

67%

AcO

CO2Me

O AcO OAc OAc

R = Cl, Br

Cl

OAc AcO

O

OH CO2Me

OBn

OBn Ag2CO3 AcO

O

+

AcO

BnO

OAc OAc

CO2Me

OAc

20%

O OBn

O AcO

O

OAc OAc

OBn

BnO

OBn

FIGURE 2.15. Silver carbonate promoted synthesis of Neu5Ac(2→6) disaccharides.

2.1.4 The Helferich Reaction O PO

O

promoter

X

+

ROH

PO

X = Br, Cl

Promoter

Conditions

Hg(CN)2 HgBr2 HgI2

CH3 CN CH3 CN CH3 CN

OR

OBn

2.1. General Methods

77

OBn H3C AcO AcO

O

HO BnO

OAc

OBn

+ OAc

O AcO

O BnO O

O OAc

O OBn Br O O

i,ii,iii

Ph H3C HO HO

O OH

OH O

O HO

OH

OH O

O HO OH

HO O O OH OH OH

i) AgOTf, TMU, CH2Cl2. ii) MeONa/MeOH/C6H12. iii)H2, Pd/C, EtOH-H2O.

FIGURE 2.16. Synthesis of tetrasaccharide.

This methodology is considered a modiﬁcation of the previous one, and the main change being the use of mercury and zinc salts instead of silver. Also, more polar solvents are used such as acetonitrile or nitromethane (Figure 2.18). The yields reported for this reaction are up to 70%, or higher. However, a mixture of anomers is often observed. By following this strategy, Umezawa et al.17 had prepared kanamacin A by condensing 6-O-[2-O-benzyl-3-(benzyloxycarbonylamino)-3-deoxi4,6-O-isopropylidene-α-D-glucopyranosyl]-N,N’-di(benzoyloxycarbonyl)2-deoxyestreptamine, as glycosyl acceptor with 2,3,4-tri-O-benzyl-6-(Nbenzylacetamido)-6-deoxi-α-D-glycopyranosyl chloride, as glycosyl donor. The catalyst employed was mercury (II) cyanide (Figure 2.19). The antitumoral O-glycoside Epirubicine was prepared under Helferich conditions18 using the acetonide form of Adriamicinone and 2,3,6-trideoxi-3triﬂuoroacetamido-4-O-triﬂuoroacetyl-α-L-arabinohexopyranosyl chloride, and a mixture of mercury (II) oxide and bromide as shown in Figure 2.20. Other coupling reactions between sugars under Helferich conditions have been as well described.19 For example, the case of trisaccharide Raﬁnose prepared by

78

2. O-Glycoside Formation O OBn O

AcO

ButOCOHN

Cl O

OBn

OAc O

+

OAc

OBn

HO

O

OAc O

NHAc

N N

i

COOtBu O

ButOCOHN OBn N O OBn O

AcO

N O

OBn

COOtBu OAc O OAc O OAc O NHAc

i) AgOTf, tetramethylurea.

FIGURE 2.17. Glycosilation reaction for preparation of Bleomycin precursor. OAc

OH O OR

O OAc OAc

+

R-OH

i,ii

Br

OH OH

OAc

OH

i) HgCN2, CH3CN. ii) MeONa/MeOH.

FIGURE 2.18. The Helferich general reaction.

condensation between Tetra-O-benzyl-α-D-galactopyranosyl chloride as donor and 2,3,4,1 ,3 ,4 ,6 -hepta-O-acetyl sucrose as acceptor (Figure 2.21). Helferich conditions have been used for preparing disaccharides containing Neu5Ac(2→6)Gal and Glc in good yields, although with low stereocontrol (α:β 3:4) (Figure 2.22).

2.1.5 The Fusion Reaction O PO

O

promoter

X

+

ROH

PO

X = OAc

Promoter

Conditions

ZnCl2 TsOH

120◦ C 120◦ C

OR

2.1. General Methods O

RHN O

OAc

OR

O ZHN

O

O

+

OH

OR

OR

NHR RO Cl ZHN

NHZ i-v

H2N

HO O HO H2N

OH

OH O

OH

O

O OH

NH2 HO H2N

NH2

Z = PhCH2COOR = PhCH2i) Hg(II)CN2, CaSO4/dioxane, PhH. ii) MeONa/MeOH. iii) AcOH. iv) H2, Pd-C.

FIGURE 2.19. Synthesis of a kanamacin A derivative.

O

O

OH MeO

CH3 O

O OH

+

RO RHN R = COF3

Cl OMe

O

O

OH

OH

OH O

MeO O i,ii

OH

OMe

O

OH

O

O

i) HgO-HgBr2. ii) NaOH

FIGURE 2.20. Synthesis of Epirubicine.

CH3 OH NH2

79

80

2. O-Glycoside Formation

BnO

OBn

OH

O

O

OBn

OH OAc O AcO

OAc

+

OO

HO OH

i,ii

OH

O Cl

OAc

OAc

OBn

OAc

OH O HO

O O OH

OAc

OH

OH OH

OH

i) Hg(II)CN2, CaSO4, PhH. ii) MeONa/MeOH. iii) H2, Pd-C.

FIGURE 2.21. Synthesis of Raﬁnose derivative.

Cl

OAc AcO

O

OH CO2Me

OBn

O

OBn

+

AcO

BnO

OAc OAc

CO2Me

OAc i

AcO

O OBn

O AcO

84%

O

OAc OAc

OBn

BnO

OBn

i) Hg(CN)2/HgBr2 (3:1)

FIGURE 2.22. Helferich conditions for the preparation of sialic disaccharide.

This is a process that is mainly used for preparing aromatic glycosides, and generally consists of the reaction between the sugar, having a leaving group either as acetate or bromide with a phenolic aglycon, under Lewis acid conditions, at temperatures above 100◦ C. This methodology has been useful to synthesize 1-naphthyl 2,3,4,6tetra-O-acetyl-α,β-L-idopyranoside by mixing 1,2,3,4,6-penta-O-acety1-α-Lidopyranose, 1-naphthol, zinc chloride and heating up to 120◦ C during 1h. Also aromatic S-glycosides could be effectively prepared under the fusion method. Thiophenol 2,3,4,6-tetraacetyl glucopyranose was prepared as a mixture of anomers (40:60, α:β) when thiophenol was combined with ZnO or ZnCO3 and then reﬂuxed with acetobromoglucose in CH2 Cl2 (Figure 2.23)20 .

2.1.6 The Imidate Reaction O PO

O

promoter

X X = OC(NH)CCl3

+

ROH

PO

OR

OBn

2.1. General Methods OAc O

AcO OAc

OAc HO i

OAc O

AcO

+

OAc

OAc

81

O OAc

i) ZnCl2, 120oC, 1h.

SH

OAc

OAc

O AcO AcO

+

OAc

Br

O

i

AcO AcO

S

OAc

i) ZnO, CH2Cl2 reflux

FIGURE 2.23. Naphtyl O-glycosides and Phenyl S-glycosides. Promoter

Conditions

AgOTf TMSOTf BF3 -OEt2 NaH

CH2 Cl2 , 0◦ C→r.t. CH2 Cl2 or MeCN 0◦ C CH2 Cl2 or MeCN, −20◦ C CH2 Cl2

This is a more recent procedure attributed to Schmidt and coworkers21a−c who introduced trichloroacetimidate as a good leaving group for preparation of Oglycosides. A signiﬁcant number of simple and complex O-glycosides involving the imidate coupling reaction have been described. This strategy involves the use of trichloroacetonitrile that in the presence of a base is incorporated on the anomeric hydroxyl group to generate trichloroacetimidate (Figure 2.24). It should be noted that the resulting imidate derivative is air- sensitive and should be used in coupling reactions immediately following preparation. Imidate formation might be spectroscopically detected by 1 H NMR through a signal appearing down ﬁeld at 6.2 ppm.22 Once the imidate is formed, it can be subjected to nucleophilic attack to provide the corresponding S-, N-, C-, or O-glycoside, depending on the chosen nucleophile. The use of a catalyst such as BF3 .OEt2 , TMSOTf, or AgOTf is necessary to carry out the reaction to completion (Figure 2.25). Although the unquestionable applicability of this approach, an undesirable side reaction has been encountered with glycosyl trichloroacetimidates in the presence of Lewis acid catalysis via the Chapman rearrangement.21b−c Hasegawa et al.23 have prepared the ganglioside shown in Figure 2.26 using 2,3,4,6-tetrabenzylglucopyranosyl-α-acetimidate with the liphophilic alcohol, to generate a ganglioside. The total synthesis of calicheamicin α and dynemicin A has been described by Danishefsky’s group,24 and involves glycosilation of calicheamicinone congener

82

2. O-Glycoside Formation OAc O AcO AcO

OAc O

i

AcO AcO

OAc OAc

OH OAc

OAc O ii

AcO AcO

CCl3

OAc O NH

i) Bn-NH2, HCl, THF. or NH2NH2 ii) Cl3CN, CsCO3/CH2Cl2, r.t.

FIGURE 2.24. Preparation of glycosyl imidate and 1 H NMR of imidate ramnosyl derivative.

with the complex glycosyl imidate using BF3 .OEt2 as Lewis acid catalyst (Figure 2.27). Naturally occurring herbicides known as tricolorin A, F, and G were isolated from the plant Ipomea tricolor and since then synthesized involving glycoside coupling reactions. The ﬁrst total synthesis of tricolorin A was performed by Larson and Heathcock,25 involving three coupling reactions steps with imidate intermediates used as glycosyl donors (Figure 2.28). The lactonization key step for the preparation of the synthesized tricolorins has been achieved either under macrolactonization conditions reported by Yamaguchi26,27 and also under ring clousure methathesis conditions.22

2.1. General Methods OAc O AcO

OAc O

Nu

AcO

AcO AcO

CCl3

OAc O

83

NH Nu

+

Cl3C

OAc

OH

NH

Lewis acid

OAc O AcO

AcO

OAc HN

CCl3 O

Chapman Rearrangement

FIGURE 2.25. Nucleophilic displacement of imidate leaving group. OBn O BnO BnO

N3 CCl3

OBn O

+

i

HO

C13H27 OBz

NH

OBn O BnO BnO

OBn

N3 O

C13H27 OBz

i) NaH, CH2Cl2.

FIGURE 2.26. Coupling reaction for the preparation of ganglioside.

Another hetero-trisaccharide resin glycoside of jalapinolic acid known as tricolorin F has been synthesized involving coupling reactions with imidates as glycosyl donors. In this way disaccharide and trisaccharide were prepared sequentially. The resulting tricoloric acid C derivative was deprotected and subjected to lactonization under Yamaguchi conditions to produce protected macrolactone. Final removal of acetonide and benzyl protecting groups provided Tricolorin F (Figure 2.29).27 A convergent approach for obtaining a tumoral antigen fragment of Lewis X has been developed by Boons et al.28 Condensation of the imidate glycosyl donor and the trisaccharide glycosyl acceptor provided the hexasaccharide, which was

84

2. O-Glycoside Formation

O

O

CCl3 N3

HN

HO O

O

OH

O

O

O

OTBS O

O Me N OH OMe NPhth

Me S

O

TEOC Me

OMe

I

i

OMe O O

Me TBSO MeO O

O

OTBS

N3

HO O

O O

O

O

OTBS

O Me N

O

OH OMe NPhth

Me S

O

TEOC Me

OMe

I

OMe O O

i) BF3.OEt2, CH2Cl2, –78oC (28%).

Me TBSO MeO

OTBS

FIGURE 2.27. Glycosylation of calicheamicinone congener.

further allowed to react with trichloroacetimidate to generate a hexasaccharide glycosyl donor. The ﬁnal coupling reaction with the disaccharide using BF3 .OEt2 , furnished the tumoral fragment Lewis X (Figure 2.30). Selectins (E,P and L) are mammalian C-type lectins involved in the recognition process between blood cells or cancer cells and vascular endothelium. L-selectins play a key role in the initial cell-adhesive phenomena during the inﬂammatory process, whereas E-selectins bind strongly to sialyl Lewis a and x.29,30 It has been found that the tetrasaccharide sialyl Lewis x is the recognition molecule and the preparation of sialyl Lewis x conﬁrmed the hypothesis that sulfation increase the afﬁnity for L-selectins.31 The chemical synthesis of 3e- and 6e-monosulfated and 3e,6e- disulfated Lewis x pentasacharides has been prepared according to Figure 2.31.

CO2Me

O

Me O

CO2Me

NH +

C5H11

O OPiv O

Ph

C5H11

i,ii

Cl3

O

OH

Me O

O OH

O

O O AcO

O

CO2Me OAc O

iii,iv

CCl3

C5H11 O

NH

Me O

v

O

O O

O O AcO

Ph

O

OH NH

C5H11 O

Me O

O O O

O

Ph

O O O

O

CCl3

Me AcO

O Me BnO

OH

O

OAc

O BnO

O

OBn vi

C5H11 O

Me O O

O

Ph

O Me AcO Me BnO

O

O O O

O

O O

O

OAc

O BnO

OBn

i ) AgOTf, CH2Cl2. ii) MeONa/MeOH. iii) AgOTf, CH2Cl2. iv) a)MeONa/MeOH. b) 1 eq. Ac2O, DMAP, CH2Cl2, Et3N. v) a) LiOH, THF, H2O. b) 2,4,6-trichlorobenzoyl chloride, Et3N, MAP, benzene. vi) AgOTf, CH2Cl2.

FIGURE 2.28. Synthesis of tricolorin A precursor.

CO2Me C5H11

CO2Me

RO

O

C5H11 O

CH3

O

O

O

i

RO RO

O

O

O

RO

OH

O

O

OR' OC(NH)CCl3

+

CH3

R = Bn; R' = Ac

O

RO RO

OR'

R = Bn; R' = Ac

ii

R = Bn; R' = H

CO2R'' C5H11 CH3 BnO BnO

CH3

O

O

O

O O

OAc OC(NH)CCl3 RO RO

i

O

O

RO

O

CH3 O RO RO

OR'

R = Bn; R' = Ac; R'' = Me iii R = Bn; R', R'' = H

C5H11

C5H11

O

CH3

O

O iv

O

BnO

OH CH3

O

HO HO

v

O

CH3 BnO BnO

O

CH3

O

O

O O

HO HO

BnO BnO

O

O O

HO HO O

O O -20 oC,

O

1 h; (ii) NaOMe, MeOH , 6h, rt. iii) KOH, MeOH- H2O, 4 h, reflux. iv) 2,4,6trichlorobenzoyl chloride, Et 3N, DMAP, PhH. v) 10% HCl-MeOH, Pd(OH)2 -C 10%, MeOH.

i) BF 3.Et2 O, CH2 Cl2,

FIGURE 2.29. Synthesis of Tricolorin F.

2.1. General Methods OBn

OBn O

OO

O OBn O Me

HO

+

OBn N3

O

CCl3

O

OBn OBn

OBn

OBn

O

87

OO O O OBn O OBn N3 OH Me

OTBDMS

OBn OBn

NH i

OBn

OBn O

O OBn O Me

OO

O

OO O O OBn O OBn N3 OH Me

O

OBn N3

O

OBn

OBn

OBn OBn

OBn OBn

ii

OTBDMS

R = OTBDMS R=H

iii R = Cl3CC(NH)

OBn

OBn HO

O

O BnO

O

O

iv

OBn

N3

O

OBn O BnO OBn

OBn O

O OBn O Me

OO

O

O

OBn N3

O

OBn

OBn OO

O OBn O OH Me

O OBn N3

O O

O

O BnO

O

OBn OBn

OBn OBn

v

Gal-β-(1,4)-GlcNac-β-(1,3)-Gal-β-(1,4)-GlcNac-β-(1,3)-Gal-β-(1,4)-GlcNAc Fuc-α-(1,3)

Fuc-α-(1,3)

i) BF3.OEt2, CH2Cl2. ii) TBAF, AcOH. iii) Cl3CCN, DBU. iv) BF3.OEt2, CH2Cl2. v) a) AcOH. b) H2, Pd-C.

FIGURE 2.30. Covergent synthesis of Lewis X fragment.

N3

OBn

88

2. O-Glycoside Formation OAc

OBn

AcO

O

O OAc OAc

OH

O

+

O AcHN

O

OBn O

HO

OC(=NH)CCl3

OBn

OBn O

O BnO

OBn OBn

OBn

OBn OBn

i

OH OAc AcO

OBn O

O OAc OAc

O O

OBn O

O OBn

OBn OBn OBn

AcHN O

O

O BnO

OBn

OBn OBn i) BF3-Et2 O, CH2 Cl2.

FIGURE 2.31. Coupling reaction for the preparation of Lewis x pentasaccharide intermediate.

Likewise, thioaryl donors can also be suitably converted to acetimidates for performing glycoside coupling reactions. This is the case of arabinosyl thio derivative, which is deprotected under NBS-pyridine conditions affording the lactol in 80% yield as a mixture of anomers (2:1). Treatment with NaH, followed by addition of Cl3 CCN, provided the desired trichloroacetimidate intermediate. This strategy has been succesfully applied in the syntheses of cytotoxic marine natural products Eleutherobin (Figure 2.32).32

2.1.7 The Sulfur Reaction O PO

O

promoter

SR

+

R'OH

PO

R = Me, Et

Promoter

Conditions

NIS-TfOH HgCl2 CuBr2 -Bu4 NBr-AgOTf MeOTf MeSOTf AgOTf-Br2 DMTST NBS-TfOH

0◦ C→r.t. CH2 Cl2 or MeCN 0◦ C CH2 Cl2 or MeCN, −20◦ C Et2 O, r.t. Et2 O, r.t. CH2 Cl2 MeCN, −15◦ C EtCN, −78◦ C

OR'

2.1. General Methods O PO

O

promoter

SR

+

89

R'OH

PO

OR'

R = Ph

Promoter

Conditions

NBS BSP DMTST MeOTf MeSOTf

CH2 Cl2, r.t. CH2 Cl2, MS, r.t

Thioglycosides are useful glycosyl donors widely used in the preparation of O-glycosides. An example of their applicability for the preparation of saccharide synthesis is represented in Figure 2.33. Thus, the synthesis of trisaccharide intermediate was obtained by combining the thioglycoside donor with a monosaccharide acceptor in the presence of methyltriﬂate, to provide the target trisaccharide in 72% yield.33 A convergent synthesis of the trisaccharide unit belonging to an antigen polysaccharide from streptococcus has been performed by Ley and Priepke.34 In this approach ramnosylalkylsulfur was used as the glycosyl donor, and cyclohexane1,2-diacetal as the protecting group (Figure 2.34). Thioalkyl donor are also useful derivatives for the preparation of biologically important natural sugars known as Sialic acids.23 An efﬁcient procedure for introducing thioalkyl groups as leaving groups involves the conversion of acetate into thiomethyl by treatment with methylthiotrimethylsilane in the presence of TMStriﬂate. O-glycosilation reaction proceeds between the thioglycosylsialic donor with a glycosyl acceptor (bearing an -OH group available), using a catalyst such as N-iodosuccinimide-TfOH as promotor (Figure 2.35). 2-Thiophenyl glycosides were used as glycosyl donor for preparing complex oligosaccharides containing sialyl moieties. A remarkable convergent approach was described for preparing a sialyl octasaccharide consisting in the O Me Me

N N

O OMe O OAc Me

FIGURE 2.32. Citotoxic marine glycoside Eleutherobin.

Me O

OH OH

Me

90

2. O-Glycoside Formation O

Ph

H3C

O

O

SEt

O

NPhth

O OBz OBz

BzO BzO

+

OBz

OBz OH O OBz

i

Phth = phtalimido

OBz BzO O

Ph

O O

H3 C

O

O

O OBz

OBz

O

OBz

NH2

OBz OBz i) CF3SO3CH3, Et2O, MS, rt. ii) a) NH2-NH2 .H2O, EtOH reflux. b) Ac2O, Py

FIGURE 2.33. Thioglicoside coupling reaction for preparation of a trisaccharide intermediate.

initial glycosidic reaction between 2-thiophenyl Neu5Ac donor with trisaccharide intermediate to produce the expected tetrasaccharide in 45% having an α(2→6)linkage. The resulting tetrasaccharide was coupled with dimeric sialyl donor to yield hexasaccharide in 42%. Acetal hydrolysis was followed by coupling reaction SEt

O HO HO

OH

ii

i

SEt SEt O MeO

O

O O

BnO BnO

OH

OBn OMe

FIGURE 2.34. Synthesis of an antigen polysaccharide fragment.

2.1. General Methods

91

iii SEt

O MeO O O

O O OMe BnO BnO

OBn

OMe

O MeO O

iv

O

OH

OMe OMe

OMe

O

O

MeO O

HO O

OMe

O

HO

O

v

O

O

MeO O O

O

HO O

HO O O

OMe BnO BnO

OBn

HO HO

i) BnBr, NaH, DMF. ii) 1,1,2,2-tetramethoxycyclohexane. iii) IDCP, 4 Å MS. iv) NIS. v) AcOH-H2O. vi) H2, Pd/C, EtOH.

FIGURE 2.34. (Continued)

OH

CO2Me

OAc

OH

AcO

OBz O

SMe

O

+

Ac2N

OTE

HO OAc

OAc

OH i

CO2Me

OAc

OH OTE

AcO

O

O

O

OBz

Ac2N OAc

OAc

OH

i) NIS/TfOH, MeCN, –40oC.

FIGURE 2.35. Thioalkyl donor for the preparation of sialic acids. OBn OBn CO2Me

OAc AcO

HO NHAc

+

SPh

O

AcHN

O O

O

OAc OAc

O

O BnO

OTE OBn

OBn

O OH

O i

OBn OBn HO NHAc

O O

O

AcO AcHN

OTE OBn

OBn

O

O O CO2Me

OAc

OAc AcO i

O

O

AcO

AcO

O BnO

O O

AcHN OAc

OAc

CO2Me

O O

O AcHN OAc

SPh

OAc

FIGURE 2.36. Convergent synthesis of sialyl oligosaccharide.

2.1. General Methods

AcO AcHN OAc

O

O AcHN

O

R 1,R 2 = H

NHAc O

O O CO2Me

OBz

CO2Me

OAc

SMe

AcO

O

O

O

AcHN OAc

OAc AcO

O AcHN

AcO

O

O

OBn OBn

OAc

OAc

O

OBz NHAc

O

AcHN

O

AcHN AcO OAc

O

O

O BnO

O OTE OBn

OBn

O

OBz OBz AcO AcO

OAc

OBz

CO2Me

O AcHN

OAc CO2Me

O OAc

OBz

OAc

O

OAc OAc

OTE OBn

OR

AcO

OAc

O

O

OBn

O RO

AcO

ii

O BnO

O

OAc

OAc

AcO AcHN

OBn O

OAc

R1,R2 = acetonide

OBn

CO2Me

O

O

OAc

93

OH O O CO2Me

i) NIS/TfOH, 45%. ii) DMTST, 85%.

FIGURE 2.36. (continued)

with Neu5Acα(2→3)GalSMe donor to give the octasaccharide in 85% yield (Figure 2.36).35 Crich and Li36 introduced the use of 1-(Benezenesulﬁnyl)piperidine/triﬂic anhydride as promoter conditions for preparing O-glycosides from thioglycoside donors. These conditions were applied for preparing Salmonella-type E1 core trisaccharide (Figure 2.37).

94

2. O-Glycoside Formation Ph O

OBn O

O

Ph

O BnO

O +

O

O

O SPh O

HO O O

Ph-pNO2

OPiv O

O

S N

BSP =

i

Ph O O O O Ph

OBn O

O O BnO

O

Ph-pNO2

OPiv

O

O

O

O

O o

i) a) BSP, m.s., CH2Cl2, r.t. b) Tf2O, –60 C to 0oC 1h.

FIGURE 2.37. Preparation of Salmonella-type E1 core trisaccharide under BSP-Tf2 O conditions.

Highly ﬂuorinated thiols have been developed and used as donors in the preparation of disaccharides. The reactivity of these novel ﬂuorinated thiols were examined using different acceptors. Thus, disaccharide formation under glycosidic conditions provided the disaccharides in high yields (Figure 2.38).37 OBz OBz BzO BzO

OH

O

+

S OBz

O

BzO BzO

OBz

N H3C

O

i

OCH3

BzO BzO

O O OBz BzO BzO

C8H17

i) NIS (2 eq.), AgOTf (0.2 eq.), CH2Cl2.

FIGURE 2.38. Highly ﬂuorinated thiols glycosyl donor for glycosidation.

O OBz

OCH3

2.1. General Methods O

O Opent

HO OR

Opent

Opent

+

RO

O

RO OR

R = Bn

O

O

promoter

95

OR

OR

glycosyl acceptor

glycosyl donor "armed"

"disarmed"

O HO

O

O

O

OR' OR' promoter

O

RO

O OR'

OR

OR

OR'

R' = Acetonide

FIGURE 2.39. General scheme for the armed-disarmed approach.

2.1.8 The Armed-Disarmed Method This versatile approach has been attributed to Mootoo and Fraiser-Reid,38 and considers the use of a glycosyl donor in the classical sense coined with the term “armed saccharide” (because the reducing end is armed for further coupling reaction), and an acceptor, in this case “disarmed saccharide,” which contains both a free alcohol and a leaving group sufﬁciently resistant for the ongoing coupling reaction. The resulting disaccharide now becomes an armed disaccharide, which in turn is reacted with another glycosyl acceptor or disarmed sugar to produce the oligosaccharide chain elongation (Figure 2.39). This method was ﬁrst implemented in the preparation of 1-6 linked trisaccharide shown in Figure 2.40. As one can see, the disarmed sugar intermediates function as glycosyl acceptor bearing the hydroxyl group at position 6 available for establishing a glycosidic linkage with the armed unit. Despite the usefulness of pentenyl as protecting group, clear preference in the use of thioglycoside donors as armed and disarmed donors is often observed (Figure 2.41).39 This concept was applied successfully in the stereocontrolled synthesis of Lex oligosaccharide derivatives by using two glycosylation steps as described by Yoshida et al.40 The ﬁrst coupling between “armed” thiophenyl fucopyranosyl derivative with “disarmed” thiophenyl lactose derivative under NIS-TfOH conditions provided trisacccharide, which was subjected without puriﬁcation to second condensation with different acceptors, one of which is indicated in Figure 2.42. The construction of α-linked mannoside disaccharide was achieved under the armed-disarmed approach by using armed thiogalactoside donor activated

96

2. O-Glycoside Formation OBn OBn O

BnO BnO

O

OH i

O

OPent + AcO OBn AcO

OPent

BnO BnO

OBn O O

OAc RO RO

Pent =

OPent

OR

R = Ac R = CH2Ph

ii,iii

OBn O

BnO BnO

BnO BnO

O

OBn O

O

OH

OBn O

O

O

O

O

BnO BnO

OBn O O RO RO

OPent

O

OBn

O

i

OR

O

O O

i) I(collidine)2ClO4, 2 eq. of CH2Cl2. ii) NaOMe, MeOH. iii) BnBr, NaH, DMF, (n-Bu)4NI.

FIGURE 2.40. The armed-disarmed approach.

by BSP/Tf2 O and condensed with disarmed thiomannoazide intermediate bearing a free hydroxyl group. Addition of triethyl phosphate prior to the aqueous work up led to the generation of the expected α-linked disaccharide in 74% (Figure 2.43).39 PO O HO

SPh

PO O

"disarmed"

O PO

PO

SPh

O O

SPh

promoter "armed"

"armed"

PO O HO

PO

SPh "disarmed"

promoter

O PO

PO O

O

O O

SPh

P = protecting group

FIGURE 2.41. The general scheme of the armed-disarmed approach with thioglycosyl sugars.

O

2.1. General Methods Me OBz O O

O

OBn OBn

SPh

O HO

O

SPh OBn

O

OBz

O

OBz

OBz

O O

O

i

OBz

OBz

O

97

O OBz

OBz Me

O

OBn

OBn OBn

OBn OBn O

OEt OBz

HO OBn

O O

O

O O

O ii

OBn OBn

OBz

O

OBn

OBz

OBz Me

O

OEt

O

OBn

OBn OBn i,ii) CHCl3, 4 Å, NIS-TfOH, –20oC, 1h.

FIGURE 2.42. Preparation of Lewis X tetrasaccharide using armed-disarmed coupling method.

Recently S-benzoxazol thio glycoside (SBox) was synthesized and introduced as alternative glycosyl donor for preparing disaccharides under the armed-disarmed approach. Thus, the SBox glycosyl donor was used as armed donor and condensed with disarmed thioglycoside to provide the target disaccharide (Figure 2.44).41

Ph

BnO

N3 O

O O HO

O

SPh

O BnO

OBn

BnO

OBn

BnO

SPh

SPh

OBn

OBn

BSP

Ph

OTf Ph

S

O

O

O

O N3

SPh

N

FIGURE 2.43. Synthesis of α-linked mannosyl disaccharide following an armed-disarmed strategy.

SPh

98 R2

2. O-Glycoside Formation

N

O

R1 BnO BnO

OAc

R2

OAc +

i

N O

R1 BnO

HS O

S O

OBn

Br

SBox

R1 = OAc; R2 = H R1 = H; R2 = OAc

OH

OBz

O BzO

R2 SEt

R1 BnO

OBz

OAc O OBn O

OBz ii

O BzO

SEt

OBz i) K2CO3, acetone, 90%. AgOTf, CH2Cl2.

FIGURE 2.44. Armed-disarmed synthesis using S-benzoxazol (SBox) as disarmed glycosyl donor.

2.1.9 The Glycal Reaction O

O

DMDO

PO Acetone

O

promoter +

PO

ROH

PO OH

O

Promoter

Conditions

ZnCl2

THF

The glycals are unsaturated sugars with a double bond located between C1 and C2. These useful intermediates were discovered by Fischer and Zach in 191342 and their utility in the preparation of building blocks for oligosaccharide synthesis is increasingly important. Different routes for the preparation of triacetyl glycals have been examined by Fraser-Reid et al.,43 involving the Ferrier rearrangement. Moreover, a suitable one-pot preparation of glycals has been more recently described, starting from reducing sugars by Shull et al.44 The general procedure for preparing these valuable intermediates is based on the reductive removal of a halogen and neighboring acetate group through the use of zinc in acetic acid (Figure 2.45). The completion of this reaction can be followed by 1 H NMR, where the presence of a signal around 6.3 ppm as double of double with J1,2 = 6.2 Hz, J1,3 = 0.3 Hz is expected for H-1, and a multiple shifted upﬁeld for H-2.

OR

2.1. General Methods

99

FIGURE 2.45. The Fischer-Sachs glucal and 1 H NMR of benzylfucopyranosyl glycal.

More recently the use of alternative catalysts such as titanium complex, Li/NH3 , Sodium, Cr (II), and vitamin B-12 as catalysts has been described as improved method, for preparing especially acid sensitive glycals. As for any double bond, these unsaturated sugars may undergo electrophilic addition, which takes place at the C2 position leaving a positive charge at C1, which instantly reacts with the conjugate base. This reaction is particularly useful for the preparation of 2-deoxypyranosides (Figure 2.46). OAc OAc O

O EX AcO

AcO AcO

AcO

FIGURE 2.46. Electrophilic addition.

E

X

100

2. O-Glycoside Formation O

O OR

OR O RO RO

H3C

CH3

O RO

acetone

RO O

FIGURE 2.47. The Brigl epoxide formation.

A more extended application for glycoside bond formation has been developed recently. Such strategies consist of the conversion of glycals into Brigl’s epoxide, and then further treatment with nucleophiles to effect ring opening. The oxidation of the double bond has been successfully achieved with dimethyl dioxirane (DMDO) in acetone (Figure 2.47). The standard procedure for generation of DMDO was developed by Murray and Jeyaraman45 and optimized by Adam et al.46 Such procedure involves the use of potassium monoperoxysulfate as oxidative acetone agent, and the reaction conditions require temperatures below 15◦ C, an efﬁcient stirring. The DMDO/acetone solution generated must be immediately distilled under moderate vacuum. The concentrations of DMDO are in the order of 0.09–0.11 M (5%), and it is used as acetone solution. The transformation of the glycal to the epoxide can be veriﬁed by 1 HNMR, where the disappearance of the signal at 6.3 ppm for H-1 double bond is observed, and it is expected the presence of a signal at 5.0, as double for H-1 and at 3.1 as double of double for H-2 (Figure 2.48). The stereo selectivity of epoxide formation is protecting group-dependent, observing in the case of acetate protecting group a mixture of epoxide anomers, and preferentially the α-anomers if the protecting groups are benzyl, or methyl groups (α:β ratio 20:1). As expected, the epoxide ring opening by nucleophiles occurs with inversion of conﬁguration, providing β-glycosides exclusively (Figure 2.49). Likewise, alternative epoxide conditions from glycals have been assayed besides DMDO treatment. Among them, cyclization of a bromohydrin,47 mchloroperoxybenzoic acid-potassium ﬂuoride complex oxidation of the glycal,48 and potassium tertbutoxide oxidation of ﬂuoride glycosyl donor49 has been described (Figure 2.50). The potential of 1,2-anhydro sugars as glycosyl donor for the preparation of β-linked saccharides was established by Halcomb and Danishefsky50 and such strategy consist in the treatment of the glucal having available a hydroxyl group at position 6, with the sugar epoxide under Lewis acid conditions (ZnCl2 ) at low temperature. The resulting glucal disaccharide generated as a single coupling product was further converted to the epoxide, which eventually led to the next coupling reaction with another glucal acceptor (Figure 2.51). The tetrasaccharide Cap domain of the antigenic lipophosphoglycan of Leishmania donovani has been prepared under the glycal approach by Upreti and Vishwakarma.51 Thus, the preparation of the hexa-O-benzyl-lactal under stardard

2.1. General Methods

101

FIGURE 2.48. 1 H NMR spectra of 1,2-anhydro-3,4-di-O-benzyl-α-D-fucopyranose (and traces of acetone).

procedures was followed by oxirane formation with dimethyl dioxirane to generate the corresponding oxirane. Methanolysis ring opening and gluco→manno conversion generated the disaccharide intermediate. This was coupled to the mannobiose donor to produce the tetrasaccharide, which after deprotecction led to the tetrasaccharide Cap domain (Figure 2.52). Brigl’s epoxide has been exploited successfully for the preparation of glycosylated peptides such as collagen type II derived glycosides carrying β-Gal and αGlc-1,2-βGal side chains.52 Galactosyl glycal is reacted with DMDO-acetone solution and the resulting epoxide reacted with hydroxylysine and ZnCl2 as promoter (Figure 2.53). General procedures for preparation of glycosidic bond of glycopeptides can be reviewed in the comprehensive study reported by Kunz.53 OAc

OAc

O

O

HO-R AcO

AcO

AcO O

R

AcO

FIGURE 2.49. Ring opening for β-glycoside formation.

O

OH

102

2. O-Glycoside Formation O

OH

RO

i

RO

Br OR

R = Me, Bn

O

O RO

RO

ii

O RO

RO

OR

OR R = Ac, Bn

O

F

RO

iii

RO

OAc OR

R = Bn i) KH or, KHMDS, 18-crown-6, –70oC. ii) MCPBA-KF, CH2Cl2, r.t. iii) t-BuOK, THF.

FIGURE 2.50. Alternative glycal-epoxidations. OBn O BnO BnO

OH O

+

BnO BnO

O

OBn O

ii

i

BnO BnO

BnO BnO

O

O

OH

BnO BnO

OBn O

iii

O

OBn O

BnO BnO

BnO OBn BnO

O

O

O

BnO OBn BnO

O

OH O

OBn O

BnO BnO i

BnO BnO

O

BnO OBn BnO

O

O

BnO OH BnO

i) ZnCl2/THF, –78oC to r.t. ii) NaH, BnBr. iii) DMDO-acetone.

FIGURE 2.51. Epoxide glycal as glycosil donors.

O

OBn

OBn OBn

O

BnO

OBn

BnO

O

OBn OAc

AcO

O BnO

OBn

OBn

iii

OH

BnO

O BnO

O OBn

O

ii

O BnO

OBn OMe

OH O

OMe

O BnO

OR

RO

AcO AcO

O

RO

O O

AcO

O

O

OBn

O

BnO

OBn

i

O BnO

OBn OBn

OBn

OBn

O

RO

AcO AcO

O O

RO CCl3

O

R = Ac R2 = Bn

RO

NH OR2

iv

OR2

RO R2O

O

RO2

R3 = Me

O OR2

O O

OR3

RO R, R2, R3 v H

o

o

i) DMDO, CH2Cl2, 0 C, 2h. ii) MeOH, rt, 4h. iii) a)(COCl)2, DMSO, CH2Cl2, –78 C. b) NaBH4, rt, 4h. iv) TMSOTf, CH2Cl2, –30oC, 45 min. v)a) Pd(OH)2, H2, 4h. b) NaOMe, MeOH. c) Ac2O/AcOH/H2SO4.

FIGURE 2.52. Synthesis of a tetrasacharide using an epoxide disaccacharide as glycosyl donor. NHFmoc HO O

OTBDPS O

OTBDPS O

O i

O

COOR2 R

O

ii

O O

NHFmoc

OTBDPS O O

COOR2

O OH

R

αGlc-1,2-βGal

H2NH2C

H-Gly-Glu-Hyp-Gly-Ile-Ala-Gly-Phe

Gly-Glu-Gln-Gly-Pro-Lys-OH O

1) DMDO-acetone. ii) ZnCl2, THF

FIGURE 2.53. Amino acids glycosidation.

104

2. O-Glycoside Formation

This methodology has been extended for the preparation of E-selectin ligand tetrasaccharide sialyl Lewisx (SLex ), which is located at the terminus of glycolipids present on the surface of neutrophils. The chemoenzymatic sequence consisted in the reaction of the 6-acetylated glucal with β-galactosidase transferase to produce disaccharide, which was subjected to further transformations according to the pathway presented in Figure 2.54.54

2.1.10 Miscellaneous Leaving Groups 2.1.10.1

Fluoride Glycosyl Donors O

PO

O

promoter

X

+

ROH

PO

OR

X=F

Ppromoter

Conditions

SnCl2 -AgClO4 Cp2 HfCl2 -AgOTf SnCl2 -AgOTf

Et2 O, −15 → r.t. CH2 Cl2 , −25◦ C CH2 Cl2 , 0◦ C

Fluorine is considered a poor leaving group, and its use for glycoside bond formation has been more restricted than chlorine and bromine, although display higher thermal and chemical stability. Nonetheless several Oglycoside synthesis involving glycosyl donors with ﬂuorine as leaving group has been described, specially for the preparation of α-O-glycosides with high stereoselectivity.55 Based in the use of ﬂuorine glycosyl donors, the synthesis of the marine algae α-agelaspines, was carried out through the condensation of perbenzylated galactopyranosyl ﬂuorine as anomeric mixture with the long chain alcohol in the presence of a mixture of SnCl2 -AgClO4 as catalyst (Figure 2.55).56 A general procedure for the preparation of ribofuranosyl ﬂuorides and their use as glycosyl donors for O-glycosylation with α-stereocontrol was developed by Mukaiyama et al.,57 and consists of the conversion of 2,3,5-tri-O-benzyl-Dribofuranoside that react under mild conditions with 2-ﬂuoro-1-methylpyridinium tosylate at room temperature to give an anomeric mixture (α:β 58:42) in 84% yield. These two ﬂuorines could be either separate or interconverted by treating the α-anomer with boron triﬂuoride etherate in ether at room temperature (Figure 2.56). It has been observed that the glycosylation reaction between the glycosyl ﬂuorine with different alcohols under Lewis acid conditions provides mainly αriboglucosides in high yield as it is shown in Figure 2.57. Sulfated Lex and Lea -type oligosaccharide selectin ligands were synthetically prepared as described below. Thus, glycosyl donor and acceptor were condensed under Mukaiyama conditions (AgClO4 -SnCl2 ) to form the

2.1. General Methods

105

OH OH O O

HO

AcO AcO

OAc O

OAc i

OH OH iii-vii

HO

O HO O

OH

OAc O

HO HO

ii

OH O

HO

viii

O

HO2C

NHAc

OAc O

O HO O

OH

OH OH OH

OH

OH OH

NO2

OH O

O HO O

OH NHAc

OH

O HO HO

OH

AcHN

OH

OH OH OH

O

H3C OH OH ix

O O

O O

HO2C

AcHN

OH NHAc

OH

O HO HO

OH O

sialyl Lea

OH OH

i) subtilisin, DMF. ii) β-galactosidase. iii) Ac2O. iv) NaN3, CAN. v) H2 cat. vi) Ac2O. vii) saponification. viii) α2-3SiaT, CMP-NeuAc. ix) α1-3/4 FucT, GDP-Fuc.

FIGURE 2.54. Chemoenzymatic synthesis of tetrasaccharide sialyl Lea .

β-glycoside in 90% yield. The sulphated tetrasaccharide was formed by reaction of tetrasaccharide acceptor with SO3 .NM3 complex in anhydrous pyridine (Figure 2.58).58 2.1.10.2

Silyl Glycosyl Donors O

PO

promoter

OR

+

R'OH

O PO

R

Promoter

Conditions

Me3 Si t BuMe Si 2

TMSOTf or BF3 .Et2 O TMSOTf

CH2 Cl2, −5◦ C CH2 Cl2 -acetone, −35◦ C

OR'

106

2. O-Glycoside Formation O OBn

OBn O

(CH2)12CH3

HN

i

+ BnO

F

HO

OBn

(CH2)13CH3 OH O

OBn

OBn O

(CH2)12 CH3

HN

BnO OBn O (CH2)13CH3 OH

i) SnCl2, AgClO4/THF. ii) H2, Pd-BaSO4/THF.

FIGURE 2.55. Fluorine monosaccharide as glycosyl donor.

BnO

BnO

O OH

+

F

OTs

+

F

N H

OBn OBn

O

i

OBn OBn α/β 58/42 i) NEt3, CH 2Cl2

BnO

O

BnO i

F OBn OBn

O

F

OBn OBn

i) BF3.OEt2, Et2O, r.t. 10min, 72%

FIGURE 2.56. The Mukaiyama protocol for preparation of ribofuranosyl ﬂuoride.

2.1. General Methods BnO

BnO

O

F

O

107

i + R-OH

OR OBn OBn

OBn OBn

α/β 72/28 i) SnCl2, Ph3CClO 4, Et2O, MS 4A, 93%

FIGURE 2.57. N-glycosylation reaction using ribofuranosyl ﬂuorine.

t

OBn

OAc OBn

O

O

BuMe2SiO AllylO

+

F NPhTh

HO

OR1 OBn

OBn i

R4O AllylO

OBn OBn

O

O O NR2R3

OBn

OBn

R1 = Ac, R2, R3 = Phth, R4 = SitBuMe2

ii

R1 = H, R2, R3 = H2, R4 = SitBuMe2 iii

R1 = Ac, R2, R3 = H, Ac, R4 = SitBuMe2

iv

R1 = Ac, R2, R3 = H, Ac R4 = H

F OAc OAc

O

OAc OAc

O MCAO

O

RO

OAc

OBn OBn

O

O

O

MCAO

OAc

OAc OBn

OBn

O

F

OBn

NHAc

OBn

v

vii

OBn

R = Allyl

vi

R=H

OH

OAc OAc

OAc OBn

OBn

O

O

O

OAc O

NHAc

OBn vii

OBn OBn

O

O

O

RO

OH

O ix

HO3SO

OBn

O

OH O

O

O

OH OBn

OH

O

O

NHAc OH

R = MCA R=H

viii

OH

OH OH

R = SO3H

FIGURE 2.58. Total synthesis of sulphated Lex .

OH

OH

108

2. O-Glycoside Formation AcO OAc O

OSiMe3

AcO

O

i + COOMe

Me

AcO

OAc

AcO

OAc O

O

O

AcO AcO

OAc COOMe

Me

AcO i) TfOSiMe 3, –40oC.

FIGURE 2.59. Silyl derivatives as glycosyl donors.

Silyl groups are best known as versatile protecting groups, and their use as leaving groups for glycoside bond formation has been more limited. An example of glycoside formation involving a silyl group as leaving group is reported for the preparation of luganol O-glycoside.59 In this work, the glycosyl donor is combined with luganine in the presence of trimethylsilyltriﬂate at low temperature (Figure 2.59). It is worth mentioning that stereoselectivity is dependent on C-2 neighboring group participation. When acetate is the C-2 protecting group, the β-anomer is obtained, while if the protecting group is benzyl, the α-anomer is preferred. The use of selenoglycosides as glycosyl donors and acceptor in glycosilation reactions has also been described by Metha and Pinto.60 A typical glycosidation procedure with phenylselenoglycoside donors involves the glycosyl acceptor, ˚ molecular sieves, silver triﬂate, and potassium carbonate in dichloromethane 4-A (Figure 2.60). Tetrazol has also been tested as a leaving group for the preparation of an antibiotic fragment.61 A coupling reaction with the methoxyphenyl glycosyl acceptor was catalyzed with (Me3 )3 OBF4 as shown in Figure 2.61. 2-Aminodisaccharides were obtained by an elegant [3,3] sigmatropic rearrangement, by Takeda et al.62 The addition of thiophenol to an unsaturated C-1 in the presence of Lewis acid was followed by a sigmatropic rearrangement with an imidate group that migrates from C-4 to C-2. Disaccharide formation was catalyzed with Pd(CH3 CN)2 -AgOTf complex in dichloromethane (Figure 2.62).

2.1. General Methods SePh Ph

O

O

HO

Bn

O

O

i

O

O

+

AcO AcO

O

Ph

O

109

Bn

OMe O

OAc AcO AcO

i) K2CO3 , AgOTf, MS, CH2Cl2.

OAc

FIGURE 2.60. Phenylselenosugars as glycosyl donors.

The total synthesis of the cyclic glycolipid Arthrobacilin A, a cell growth inhibitor was achieved by Garcia and Nizhikawa,63 under zinc p-tert-butylbenzoate salt as glycoside catalyst, obtaining the β-galactoside glycoside in 73% along with α-isomer in 27% (Figure 2.63). X O

OMP X

O AcO

O

+

O

iii O AcO

OH

X = α-OMP

i

X = OH ii

X = α-OMP

i

X = OH

X = Tet OMP

ii

O

O

iii

O O O AcO

i) CAN. ii) 1H-tetrazole. iii) (CH3)3OBF4, MS.

FIGURE 2.61. The use of tetrazol as a leaving group.

X = Tet

OMe

110

2. O-Glycoside Formation

OAc

OAc

OAc

O

OAc O

i

AcO

OH

OH O

ii

SPh

iii

SPh OBn

CCl3

O HN

OTBS O

HO BnO

OTBS O

iv

O

OBn

NHCOCCl3

OMe

v SPh SPh OTBS

OTBS O O

NHCOCCl3

vi

OBn

O

NHCOCCl3

O

O O

O BnO

OAc

OBn

BnO

OMe

OAc NHCOCCl3

O

OBn O

AcO

OBn

OMe

OAc O

vii

OBn

+

O BnO

OBn

AcO AcO

NHCOCCl3

O O BnO

OMe

OBn

OBn

i) PhSH, SnCl4/CH2Cl2. ii) MeONa/MeOH. iii) a) TBSCl-imidazol/CH2Cl2. b) Cl3CCN-NaH/CH2Cl2. iv) xylene, reflux. v) Pd(CH3CN)2-AgOTf, MS 4A/CH2Cl2. vi) m-CPBA/CH2Cl2. viii) Ac2O-AcOH/ BF3.OEt2.

FIGURE 2.62. Sigmatropic rearrangement.

2.1.10.3

Heterogenous Catalysis

Stereocontrolled α- and β-glycosylations by using environmentally benign heterogenous catalyst has been developed as a novel approach for stereoselective formation of β-O-glycosidic linkages. Polymeric materials such as montmorillonite K-10,64 heteropoly acid (H4 SiW12 O40 ),65 sulphated zirconia (SO4 /ZrO2 ),66 and perﬂuorinated solid-supported sulfonic acids (Naﬁon resins)67 have been assayed successfully providing series of stereocontrolled O-glycosides in high yield (Figure 2.64).

OMe

2.1. General Methods OBn

OAc O

i

O +

BnO OBn

OBn

OH

OAc C9H19

O

BnO OBn

Cl

OMe

O

111

OMe C9H19 O

OR OR O RO

O

C9H19 O

O

OR O

O C9H19

O O RO

OR

RO O

O O

RO

C9H19

O

OR i) zinc p-tert-butylbenzoate, 2-methyl-2-butene, MS, CH2Cl2, r.t., 2.5h

FIGURE 2.63. Glycosylation reaction for preparation of Arthrobacilin A. OBn

OBn O

BnO BnO

+

montmorillonite K-10

R-OH

10:1 CH 2Cl2-CH 3CN

OBn OP(OEt)2

heteropoly acid (H4SiW12O40)

O

+

OBn OR

–20oC, 30 min 90%

OBn BnO BnO

O

BnO BnO

R-OH MeCN 90%

OBn OH

β >> α

OBn O

BnO BnO

+

OBn OR α/β 92:8

OBn

OBn BnO BnO

O

OR

SO4/ZrO2

BnO BnO

Et2O

OBn

OBn

O

+

R-OH

SO4/ZrO2 CH3CN

OBn

BnO BnO

OBn

F

BnO BnO BnO

OR

BnO

OBn O Y

+

R-OH

Nafion CH3CN

X

O

BnO BnO

OBn O

OR

X = S(O)Ph, Y= H Y = S(O)Ph, X= H

FIGURE 2.64. Stereocontrolled O-glycosidations using heterogeneous polymeric materials.

112

2. O-Glycoside Formation OBn

OBn

OBz

O

OBz O

+

STol

BnO

STol

HO

OLev

O(NBz)

(BzN)O

O

+

STol O(ClBn)

HO

NHTroc i

OBn

OBz

OBn

OBz (BzN)O

O(NBz) O

O

O

STol O(ClBn)

O

O

BnO

NHTroc

OLev

i) NIS, TfOH.

FIGURE 2.65. One-pot reaction for two β-linkages formation.

2.1.10.4

The Pool Strategy

This term applies to deﬁne a one-step reaction used to build up two β-linkages simultaneously from 3 sugar intermediates.68 This approach has been described for the preparation of the glycosyl ceramide Globo H hexasaccharide identiﬁed as an antigen on prostate and breast cancer cells. The synthesis consisted in the initial synthesis of the trisaccharide building block from the one-pot reaction of the 3 suitable sugar intermediates under N-iodosuccinimide and triﬂic acid conditions in 67% yield (Figure 2.65). 2.1.10.5

Phosphate Glycosyl Donors O

PO

O

promoter

OR

+

R'OH

R P(=O)(OPh)2 P(=S)(Me)2 P(=O)(NMe2 )2 P(=NTs)(NMe2 )2

PO

Promoter

Conditions

TMSOTf TrClO4 TMSOTf BF3 -Et2 O

CH2 Cl2, −5◦ C

OR'

CH3 CN, −40◦ C CH2 Cl2

Phosphorous glycosyl donors are another option for preparing oligosaccharides. These donors have been used for the preparation of sialyl oligosaccharides; however, the yield reported were moderate. This is the case of the preparation of sialyl tetrasaccharide derivative, which was carried out by condensation

2.1. General Methods

AcO

+

AcHN

OMe

O

HO

CO 2Me

O

OBn O

OBn

OP(OR)2

OAc

OH

O

OAc OAc OBn

OBn

O

O OBn

113

OBn

OBn

R = Et : 36% R = Bn : 20%

i CO2Me

OAc AcO

O

O

O

AcHN OAc OAc

OBn O

OBn

OH

O

O OBn O OBn

OMe OBn

OBn

OBn

i) TMSOTf, MeCN, –40oC, 1h.

FIGURE 2.66. Phosphorous glycosyl donors for oligosaccharide synthesis.

between sialyl phosphite with trisaccharide acceptor under TMSOTf as catalyst (Figure 2.66).69

2.1.11 Enzymatic Approach Enzymes in organic chemistry has become an essential tool for the synthesis of important target molecules, and in many cases they are considered the ﬁrst choice, especially for those key steps involving stereospeciﬁcally controlled reaction conditions. In general, enzymes are considered efﬁcient catalysts that perform the desired transformation under mild conditions with high selectivity and speciﬁcity, usually avoiding epimerization, racemization, and rearrangements processes. Besides there is a current need of developing economical and environmentally friendly processes for synthesis. However, some aspects still need close attention in order to fulﬁll thoroughly the requirements especially for high-scale production. Thus, many enzymes are unstable, high cost, difﬁcult to handle, and require expensive cofactors. Glycosyltransferases are important enzymes involved in essential processes related to oligosaccharide biosynthesis, and they have been found also very useful as biocatalyst for the chemoenzymatic synthesis of interesting oligosaccharides

114

2. O-Glycoside Formation OH

HO HO

OH O

OH

i

OH

OPO32–

OH

OH

ii

O

HO HO

iii

HO OH

OUDP

OH

R-OH

O

OH O

HO OH

OUDP

i) UTP, UDP-Glc pyrophosphorylase. ii) UDP-Glc 4-epimerase. iii) Gal transferase.

FIGURE 2.67. Glycosylation with galactosyltransferases.

and nucleotides.70,71 They have been classiﬁed as Leloir if they are involved in the biosynthesis of most of N- and O-linked glycoproteins in mammalians, and requires mono- and diphosphates as glycosyl donors, and non-Leloir enzymes which utilize sugar phosphates as substrates. Glycosylations with galactosyltransferases can be performed through the use of glucose-1-phosphate as donor. A general sequence consists in the conversion by using UDP-Glc pyrophosphorylase to give UDP-glucose. Epimerization with UDP-glucose epimerase affords UDP-galactose, which is used for glycosylation with galactosyltransferase (Figure 2.67).72 Several chemoenzymatic synthesis of α(2→6) and α(2→3)-oligosaccharides have been reported through the use of sialyltransferases for glycosidic coupling reactions. One described approach involves the in situ regeneration of CMP-Neu5Ac, requiring catalytic amount of CMP-Neu5Ac (Figure 2.68).73

OH OH HO

NHAc

HO

O

O O OH

OH

OH

CMP-Neu5Ac i CMP

OH

HO AcHN HO HO

COOH O

O OH

HO

O OH

HO

NHAc

O O OH

OH

i) α-(2-6)-sialyltransferase

FIGURE 2.68. Synthesis of silayl trisaccharide mediated by silayl glycosiltransferase.

OR

2.1. General Methods OH OH HO

NHAc

HO O

O

O

O OH

OH

OH O HO

O OH

OH

NHR

O

OH

115

C13 H27

O

OH OH

CMPNeu5Ac

CMP

OH

COOH

HO

O

AcHN HO HO

O OH

HO O

O

HO

O OH

OH

OH

NHAc

O

OH

O OH

O OH

O HO

OCer

OH

i) α-(2-6)-sialyltransferase

FIGURE 2.69. Enzymatic synthesis of ganglioside.

Sialyltransferases also proved to be efﬁcient biocatalysts in the preparation of gangliosides, being involved in (2→6) linkage formation between the tetrasaccharide ceramide with CMP-Neu5Ac (Figure 2.69).74 Glucosamine may be enzymatically transformed to glucosamine 6-phosphate by treatment with hexokinase from yeast, and ultimately to glucosamine 1-phosphate by the action of phosphoglucomutase (Figure 2.70).75 UDP-glucuronic acid was prepared from UDP glucose by the action of UDPGlc dehydrogenase along with NAD. This cofactor was regenerated with lactate dehydrogenase in the presence of piruvate (Figure 2.71).76

OH HO HO

OPO32– O

NH2

i

OH

HO HO UDP

UTP

OH

O NH2

iii

HO HO

OH

ii

Pyr

PEP

i) Hexokinase from yeast. ii) pyruvate kinase. iii) phosphoglucomutase.

FIGURE 2.70. Enzymatic preparation of glucosamine 6- and 1-phosphate.

O NH2

OPO32–

116

2. O-Glycoside Formation OH O

HO HO

-O2C

i

OH

OH

2 NAD 2 NADH

OUDP

O

HO HO

OUDP

L-LDH

O

OH 2

2

CO2–

CO2–

i) UDP-Glc dehydrogenase

FIGURE 2.71. Enzymatic preparation of UDP-glucuronide.

CMP-N-acetylneuraminic acid has been prepared from CTP and NeuAc under catalysis by CMP-NeuAc synthetase. In a cascade representation, it is observed that CTP is synthesized from CMP with adenylate kinase and pyruvate kinase (Figure 2.72).77

OH O

HO HO

OH NHAc HO–

HO HO HO

O CO2–

NHAc O OH

OH

OH

HO

i

CO2H

O

AcHN OH HO iii

CMP CDP

CTP

iv

ii PEP CDP

CTP iii

Pyr

Pyr

Pyr = pyruvate

OH

OCMP

HO PEP

O

AcHN

CO2H

OH HO i) UDP-NeuAc aldolase. ii) CMP-NeuAc synthetase. iii) pyruvate kinase. iv) adenylate kinase.

FIGURE 2.72. Synthesis of CMP-N-acetylneuraminic acid.

2.1. General Methods

O OH O HO HO

OH

OHO O F

OH

O

F

OH

H

117

OH O

OPhNO2 HO HO

H O

OH HO

OH

O

OPhNO2

OH

FIGURE 2.73. Glycosynthase-catalyzed oligosaccharide synthesis.

2.1.11.1

Enzymatic Synthesis of Oligosaccharides

Mutated glycosidase also known as glycosynthase AbgGlu358Ala in combination with activated glycosyl donors and suitable acceptors can generate synthetic oligosaccharides. Thus, for this transformation the conditions selected were α-glycosyl ﬂuoride as glycosyl donor and p-nitrophenyl as glycosyl acceptor in the presence of ammonium bicarbonate buffer. The proposed mechanism of glycosynthase-catalyzed reaction is illustrated in Figure 2.73.78 The Regioselective preparation of α-1,3 and α-1,6 disaccharides by using αglycosidase as biocatalyst has been described. Thus, by combining p-nitrophenylα-galactose functioning as glycosyl donor, with the glycosyl acceptor methoxygalactose, the expected 1,3- and 1,6-disaccharides were obtained in the form of αand β- anomers (Figure 2.74).79 A transglycosylation reaction mediated by α-L-fucosidase from Alcaligenes sp. was performed by combination of p-nitrophenylglycosides donors, with different acceptors such as N-acetylglucosamine, lactose, D-GlcNAc, and D-Glc, providing the corresponding p-nitrophenyl glycosides of di- and trisaccharides containing a (1→2)-, (1→3)-, (1→4)-, or (1→6)-linked to the α-L-fucosyl group. In the general procedure illustrated in Figure 2.75 the p-nitrophenyl fucoside donor was combined with p-nitrophenyl lactosamine acceptor, being incubated with α-Lfucosidase at 50◦ C to produce the 2- and 3-linked trisaccharides.80 Sulfotransferases provides a versatile method for the preparation of glycoside sulfates. A recent report describes the use of 3 -phosphoadenosine -5’phosphosulfate (PAPS), and GlcNAc-6-sulfotransferase as catalyst (Figure 2.76).81 A chemoenzymatic synthesis of rhodiooctanoside isolated from Chinese medicines was described. The synthesis was carried out by direct β-glucosidation between 1,8-octanediol and D-glucose using immobilized β-glucosidase from almonds with the synthetic propolymer ENTP-4000 to generate the glycoside in 58% yield (Figure 2.77).82 Lactosamine was prepared using an enzymatic approach consisting in the preparation of UDP glucose and condensation with N-acetyl glucosamine (GlcNAc) in the presence of galactosyl transferase (Figure 2.78).83

2.1.12 The Solid-Phase Methodology Perhaps what remains the most challenging task for sugar chemistry is the synthesis of complex oligosaccharides such as those found in bacterial membranes or

OH

OH

O HO HO R = C6H4-p-NO2

OR

OH

OH

OH

OH

O

O

HO

OMe

HO HO

HO OMe

OH

OH

OH

OH

O

O

OH

OH

HO

HO O

HO

HO OH

OMe

O

O

HO

O OMe

HO HO

FIGURE 2.74. Example of microbial catalyzed coupling reaction. OH

OH OH

OPhNO2-p

O

O O H3 C

OH

+

OPhNO2-p

O HO

HO

NHAc

OH

HO OH a-L-fucosidase

OH

OH OH O HO

HO

O HO OH

OPhNO2-p

O HO

O OH O

OH

H 3C

O

O

+

NHAc

O

OH

OH OH

O

O

H 3C

-HOPhNO2-p

OPhNO2-p NHAc

OH

HO OH

FIGURE 2.75. Transglycosylation reaction for the preparation of 2- and 3-linked trisaccharides.

2.1. General Methods OH

OSO32-

GlcNAc-6-sulfotransferase

O

HO HO

119

O

HO HO

OR

OR NHAc

NHAc NH2 N O

N

N

O

N

O

-O S O P O O

NH2

2–

N

O

O–

O3PO

–

O P O

N

OH

2–

N

O

O–

N

OH

O3PO

3',5'-ADP

PAPS

FIGURE 2.76. Transfer of the sulfuryl group from PAPS to the glycoside.

OH

OH HO HO

i

O

+

O

HO HO

Me(CH2)6CH2OH

OH

O(CH2)7Me NHAc

OH

OH HO HO

O O O

HO HO

O(CH2)7Me NHAc

i) β-glucosidase (250u), 50oC, H2O.

FIGURE 2.77. Chemoenzymatic synthesis of rhodiooctanoside.

OH

OH

OH

OH O

O +

HO

HO NHAc OH

OH OUDP

Gal-transferase

OH

OH O

HO OH

FIGURE 2.78. Enzymatic synthesis of lactosamine.

β-D-Gal(1

NHAc HO O

O OH

4)-D-GlcNAc

OH

120

2. O-Glycoside Formation

wall cells and are usually in the form of glycopeptides. Different types of monosaccharides can be present as constitutive parts such as glucose, galactose, mannose, N-acetylglucosamine, silaic acid, and L-fucose. Also, the order of linkage and stereoselectivity between them is rarely conserved. The different nature, stereoselectivity, and linkage sequence have been a formidable obstacle for the development of general procedures of the type used for peptides and oligonucleotides which can be prepared on machine synthesizers with high efﬁciency. The main advantage of the solid-phase methodology is the coupling of sugar units to the resin, which allows easy washing away of the nonreacted reagents, avoiding tedious puriﬁcations steps. Nonetheless, despite the difﬁculties, interesting progress has been made for preparing oligosaccharides84a and glycopeptides,84b suggesting that in the solid phase technology for complex sugars will be affordable. The solid-phase approach involves three elements, namely the glycosyl donor, glycosyl acceptor, and the resin, which is properly activated with a group susceptible for attachment either with the glycosyl donor or acceptor depending on the strategy of choice. Although it appears obvious, it is important to remain that the linkage between the resin and the sugar should be easily cleaved under compatible conditions for the glycoside bond. According to a comprehensive review,85 the synthetic strategies are classiﬁed by (a) Donor-bound, (b) Acceptor-bound, and (c) Bidirectional Strategies. One general approach involves the initial attachment of a glycosyl donor (halides, trichcloroacetimidate, sulfoxides, phosphates, phosphates, thio and pentenyl and glycols) to the resin (polystyrene-base). The attached sugar is selectively deprotected depending on the required position (1,2- 1,3- 1,4- 1,6), transforming the resin-sugar complex in a sugar acceptor which will be coupled to the next glycosyl donor to produce a second linkage. By repeating this sequence an elongated chain is obtained. The ﬁnal release and full deprotection will produce the free oligosaccharide (Figure 2.79).86 An example of the donor-bound strategy is the bounding of sulfur glycoside to polistyrene resin to form a sulfur linkage between the donor and the resin (Figure 2.80). Suitable hydroxyl group from the donor will serve as linkage site with de next sugar unit for chain elongation. It should be noted that the glycosyl donor also contains a position available for the linkage with the next sugar. In other words, the glycosyl donor once attached to the resin becomes a glycosyl acceptor, as can be seen for the next coupling sequence (Figure 2.81).85 The synthesis of β-(1→6) gentotetraose was accomplished by using a benzoyl propionate as resin linker. The glycosyl donor chosen was acetobromoglucose functionalized with trichloroacetate group as a temporary protecting group at position 5. Glycosylation reactions were effected under Helferich conditions and cleavage from resin was performed with hydrazinium acetate (Figure 2.82). Polymer solid phase has been also exploited successfully by Crich et al,87 for the synthesis of sensitive β-mannosides, using a variation of sulfhoxide method, consisting in the transformation of sulfoxide to triﬂic group as leaving group. The

2.1. General Methods O

O

O

O

OR4 i

OR4

ii

OR3

R1O

O

O

OR4

121

OR8

OR2

OR2

OR2

O

O

R1O

OH

R1O

OR7

R5O OR6 O

O

O

O

OR4

i

OR4

ii

O

O

R 1O

O

R 1O

OR8

O

OR8

OR2

OR2

O

R5 O

OH

R5 O

O

OR11

OR6

OR6

OR10

R 9O OR10

O

i

O

OR4 O

R1 O

O

OR8

O oligosaccharide

OR2 O

R5 O

O

OR11

iii

OR6 oligosaccharide

OH

R9 O OR10

i) deprotection. ii) glycosyl donor. iii) cleavage.

FIGURE 2.79. General scheme for solid-phase oligosaccharide synthesis 1,4-linkage case.

subsequent addition of alcohol acceptor to the donor attached to the Wang resin will result in the glycoside β-mannoside formation (Figure 2.83). The enzymatic solid-phase oligosaccharide synthesis relies mainly by the use of glycosyltransferases, glycosidases and glycosynthases. By taking advantage on their high stereo- and regioselectivity, various oligosaccharides and glycopeptides have been prepared usually under mild conditions without the need of using protecting groups. Unfortunately, the enzymatic approach is still in some cases OH O BnO BnO

OBn

SH

+

Cl polystyrene resin

BnO BnO

OH O

S

OBn

FIGURE 2.80. Example of donor-bound strategy for solid-phase glycosilation reactions.

122

2. O-Glycoside Formation OAc O BnO BnO

OH O BnO BnO

OAc O BnO BnO

OBn Br

S

OBn O

OBn

O BnO BnO

OAc O

OH O BnO BnO

BnO BnO

OBn

OBn

OAc O BnO BnO

OBn Br

OBn

O

O O

BnO BnO

S

O

S BnO BnO

OBn

OBn O O

BnO BnO

S

OBn

OAc O BnO BnO

OBn O O

MeI, H2O

BnO BnO

OBn O O

BnO BnO

OH OBn

FIGURE 2.81. Sulfur mediated solid-phase coupling reaction.

unaffordable due its high cost for large-scale processes, lower yields provided, and their limited capability for recognizing a broad range of sugars, especially those not common. Two general approaches have been proposed for the preparation of oligosaccharides through the solid-phase approach (Figure 2.84).88 A solid-phase enzymatic approach for extending the oligosaccharide chain was described by Gijsen et al.88 in which a disaccharide-linker fragment attached to a resin was coupled with the glycosyltransferases UDP-galactose and CMP-NeuAc in the presence of galacosyltransferases and Sialyltransferase as enzymatic catalyst. Final treatment with hydrazine was used to release the tetrasaccharide from the solid support (Figure 2.85).

O O

O

CCl3 HO

O AcO AcO

O

OAc Br i,ii

OH

O

O AcO AcO

O

OAc

O O O

CCl3

O iii

AcO AcO

O

O

OAc AcO AcO

OAc Br

O O

CCl3

O AcO AcO

O

O

OAc AcO AcO

O O

OAc AcO AcO

O

O

OAc

O

AcO

O ii,iii,iv

AcO AcO

OAc Br

AcO

O AcO AcO

O

OAc AcO AcO

O

O

OAc AcO AcO

O

O

OAc AcO AcO

O OAc

i) TBABr, 35oC. ii) MeOH, Py. iii) Hg(CN)2, 30oC. iv) hydrazinium acetate 50oC.

FIGURE 2.82. Solid-phase coupling promoted by Helferich conditions.

OH

124

2. O-Glycoside Formation B

O O BnO

B

OBn O

i,ii

O O BnO

OBn O

OR

SPh iii

HO HO BnO

OBn O

OR

+

B(OH)2

i) BSP, TTBP, Tf2O, –60oC. ii) ROH. iii) Me2CO/H2O.

FIGURE 2.83. Solid-phase synthesis of β-mannoside glycoside.

2.2 Cyclic Oligosaccharides The synthesis of cyclic oligosaccharides involves the preparation of linear saccharides, which ultimately are joined together to form a cyclic macromolecule. There are two main approaches proposed based on the cycloglycosylation step. The ﬁrst involves the preparation of a long chain having at each end the donor and acceptor functionalities that will be interconnected through a glycosidic bond at a ﬁnal step, and the second involving the polycondensation of smallest repeating unit called “saccharide monomers.” It has been observed that the later strategy is considered less laborious however produce cyclic oligomers of different size since under these conditions the ring formation step is not controllable. The chemical synthesis of cyclic oligosaccharides has been mainly driven to obtain cyclic (1→4)-linked oligopyranosides, however (1→3), and (1→6) linked cycloforms are also described. In the case of (1→2)-linked oligosaccharides, the ring closure requires about 17 or more glucopyranoside residues because (1→2)linkage composed of pyranoside connected by one equatorial and one axial bond assumes rigid conformations and cannot cyclize.89 The pioneering total synthesis of cyclic oligosaccharide α-Cyclodextrin was carried out by Ogawa’s group in 1985,89 and since then alternative chemical or enzymatic methodologies appeared for preparing cyclic oligosaccharides. Nowadays the industrial production of cyclodextrins relies on the enzymatic conversion of prehydrolyzed starch into a mixture of cyclic and acyclic oligomers. A full report about cyclic oligosaccharides90 proposes four approaches to the synthesis of cyclic oligosaccharides developed during the last 10 years: 1. the stepwise preparation of a linear precursor that is subjected to cycloglycosylation; 2. the one-pot polycondensation /cycloglycosylation of a small “oligosaccharide monomer” typically, a di-, or trisaccharide that can yield a range of macrocycles of different sizes; 3. the enzyme-assisted synthesis of natural or unnatural cyclic oligosaccharides;

2.2. Cyclic Oligosaccharides

125

A) Immobilized substrate Transferases

O O

OH i

O O

O

O OH

Enzyme recovery

i

O O

O

O

O O OH

i) nucleotide sugar transferase

B) Immobilized enzymes

O O

OH

Transferase 1

Transferase 2

Transferase 3

O O

O O

O O OH

FIGURE 2.84. Two general approaches for immobilized solid-phase oligosaccharide synthesis.

126

2. O-Glycoside Formation OH

OH

OH

O

O

HO HO

O O

H N

O

OH

(CH2 )5

OH

O O

UDO-Gal Gal-T Linker

CMP-NeuAc Sialyl-T

CO2H OH

OH O

OH

OH

AcHN

O HO

OH HO

OH

O

O

OH

O

OH O

O

HO

O

OH

linker

OH

FIGURE 2.85. Enzymatic-solid phase glycosylation reaction.

4. the ring opening of cyclodextrins followed by oligosaccharide chain elongation and cycloglycosylation (Figure 2.86). Despite the signiﬁcant advances observed in cyclic oligosaccharide synthesis, their preparation is time-consuming, producing the target compounds with low regio- and stereoselective in low yields. The total synthesis of α-CD and γ-CD was described according to Figure 2.87.91,92

AG

GD

GD

AG

4 AG = acceptor linear oligosaccharide

disaccharide

O

O

O

GD = donor

O

O

cyclo-oligomerization

O

O

O O

cyclic oligosaccharide

O

O

O enzymatic degradation or

O O

O

O

cyclo-oligomerization

HO

OH n

smaller cyclic oligosaccharide

polysaccharide or disaccharide (n = 0)

FIGURE 2.86. The four suggested approaches to the synthesis of cyclic oligosaccharides.

2.2. Cyclic Oligosaccharides

127

In 1990 it was reported the chemical synthesis of β-(1→3) linked hexasaccharide. The chemical approach involved the glycosidic reaction between benzylidene acceptor and protected glucosyl bromide as glycosyl donor, under silver triﬂatepromoter conditions. As can be seen in Figure 2.88, the construction of the linear oligosaccharide and its ﬁnal cycloglycosylation was performed by using glycosyl bromides, which were prepared by photolytic brominolysis of 1,2-O-benzylidene glucose with BrCCl3 (Figure 2.88).93 The formation of (1→6)-glycopyranosidic linkages might produce cyclic ditri- and tetrasaccharides. An early synthesis of β-(1→6)-glucopyranan under Helferich conditions, generated along with the linear oligomer, a cyclic di- and tetrasaccharide in 12 and 6%, respectively (Figure 2.89).94 An improved synthesis of cyclotetraoside was described by the same group 10 years later, consisting in the preparation from the peracetylated tetrasaccharide into the tetrasaccharide derivative having both the acceptor and the donor components. The ﬁnal cyclization was performed under Helferich conditions providing a mixture of tri- and tetrasaccharide in 22% and 25% yield, respectively (Figure 2.90).95

2.2.1 Chemoenzymatic and Enzymatic Synthesis The use of enzyme is as mentioned for many O- or N-glycosides the parallel possibility for preparing cyclic oligosaccharides. The limitation continues to be

OBn

OBn

O

AcO BnO BnO

OBn O

O BzO

O

HO BnO

+

BnO

F

OBn

OBn O

O BnO

OAll

BnO

OBn HO BnO

i

O OBn O

OBn O BnO

OBnO

OBn O 4BnO

OBn

OBn AcO BnO

O OBn O

OBn O BnO

OBn O

OBn O 6 BnO

OAll

OBn

FIGURE 2.87. Chemical synthesis of cyclic α(1→4)-oligosaccharide γ-CD.

OAll

128

2. O-Glycoside Formation

ii,iii,iv

OBn O

RO BnO

OBn O

OBnO v

BnO

OBn O

OBnO

R = Ac R=H

6

BnO

F

OBn

i

RO OR RO OR

OR O

O

O

RO

O

OR

O OR

O

O

RO

RO

O O

RO

vi

R = Bn R=H

OR

RO

OR

OR O

O

OR

RO

O RO

RO

O

OR O OR

O

O

OR

OR

RO

i) SnCl2/AgOTf. ii) PdCl2/AcOH. iii) SO2Cl2/DMF. iv) AgF/MeCN. v) NaOMe/MeOH/THF. vi) H2, Pd-C, THF-MeOH/H 2O.

FIGURE 2.87. (continued)

the availability and affordability, however. Some enzymes such as glycosidases and cycloglycosyltransferases (CGTases) that are involved in the preparation of cyclodextrins from starch and other α-(1→4)-glucans are accessible and more versatile.95 The feasibility of the chemoenzymatic approach was established in the preparation of cyclic β(1→4) hexa-, hepta- and octasaccharides, from 6-Omethylmaltosyl ﬂuoride when incubated with CGTase. Thus, a mixture of 6I , 6III , 6V -tri-O-methyl-α-CD (42%), 6I , 6III , 6V -tetra-O-methyl-γ-CD (16 %) and in less proportion 6I , 6III , 6V -tri-O-methyl-β-CD was obtained (Figure 2.91).96

2.2. Cyclic Oligosaccharides O O RO

Me

O O HO

Me

O

i-iii

129

O O

OBz Br

Ph

O

R = ClCH2CO-

iv

Me O

O O RO

Me ii,iii

O O O

O v

O

OBz

Ph

R = ClCH2CO-

Me

O O RO

O O O

Me O

BzO

OBz

Me

O

O O O O

Me O

O O HO

O

OBz

Br

O O

Ph

iv,ii,iii

Me

O O RO

O

Me

O

O

O

OBz

Me

O O O

Me O

O O HO

O O HO

BzO 2

O

Ph

O

Me O

O

OBz

Br

O O

OBz

Me

O

O

OBz

R = ClCH2CO-

iv

O

Me

O

O

O

O

O

Me O

O

O

OBz

BzO 4

iv

FIGURE 2.88. Synthesis of cyclic β-(1→3)-linked oligosaccharide.

Br

130

2. O-Glycoside Formation Me O

O

Me O

O O

O O

RO

O

OR O

RO

O

O O

Me

Me

O O O

O

O

OR

O

RO O

OR

O

O O

vi

O

Me

O

R = Bz R=H

Me

FIGURE 2.88. (continued)

HO

HO O

AcO AcO

i

O AcO

O

AcO AcO

O

AcO AcO AcO

O

OAc Br AcO OAc

AcO +

O AcO AcO

OAc OAc O

OAc

AcO O

O

O

OAc AcO AcO

+

AcO

O

O

O

O AcO AcO

OAc OAc

O

O O OAc

OAc

O OAc OAc

i) Hg(CN)2, HgBr2, MeCN.

FIGURE 2.89. Preparation of linear and cyclic β(1→6) di- and tetrasaccharides.

n

2.2. Cyclic Oligosaccharides

131

Cl3CCOO O

AcO AcO

O O

OAc AcO AcO

n=1 n=2

O OAc

n

O

AcO AcO

OAc AcO

i,ii

HO O

AcO AcO

O O

OAc AcO AcO

O OAc

n

O

AcO AcO

Cl AcO

iii

OAc

OAc

AcO AcO

AcO O

AcO O

O

OAc

+

AcO

O

O

O

O

O AcO AcO

O

O O OAc

OAc OAc

OAc OAc

O

O

AcO AcO

OAc

OAc

O OAc OAc

i) Cl2CHOMe, BF3.Et2O/DCE. ii) HgBr2/DCE, MS.

FIGURE 2.90. Improved synthesis of cyclic β(1→6) tri- and tetrasacharides.

Furthermore, under the same conditions it was possible to prepare from the maltotriosyl ﬂuoride the cyclic α(1→4) hexasaccharide (6I , 6II -dideoxy-6I ,6II diiodo-α-CD) in 38% (Figure 2.92).97 An alternative option for the enzymatic preparation of cyclic oligosaccharides besides CGTases are glycosidases, which exert their action on polysaccharides. This possibility was exploited in the preparation of cyclic fructins by conversion of β-(1→2)-fructofuranan by bacterial fructotransferases isolated from Bacillus circulans (Figure 2.93).98

132

2. O-Glycoside Formation MeO HO HO

O HO OH

O

O

HO

OH F

i

O O

OH

HO

OH

HO

O MeO O

OH

HO O

O OMe

HO

OH

HO O

O

OH HO

O OH O HO

OMe O O HO OH OH HO

OH OH O

HO O HO

O

OH HO

O MeO

O HO

O OH

HO O HO

O OH

HO

OH

O OH O

O

O HO O

O OH

O

O OH

OH

OMe

HO

HO O

O HO OH

OH HO

OH OH

O

OMe

O HO

O HO

O

O OMe

HO

O

OH

O MeO

HO

HO HO

O

O

O

MeO O O

HO HO

HO OH

i) CGTase phosphate buffer pH 6.5

FIGURE 2.91. Synthesis of of 6I , 6III , 6V -tri-O-methyl-α-CD, 6I , 6III , 6V -tetra-O-methyl-γCD and 6I , 6III , 6V -tri-O-methyl-β-CD.

OAc

AcO

OAc

O

O O AcO

I O

OAc

AcO O

O OAc

AcO

OAc

AcO O

O

OAc AcO

O

I

OAc O O OAc AcO

O AcO

i) CGTase phosphate buffer pH 6.5

I O

HO HO

OH OH

O

O HO

OH OH

O

O HO

OH F

i

FIGURE 2.92. Enzymatic synthesis of 6I , 6II -dideoxy-6I ,6II -diiodo-α-CD. OH HO HO

HO

OH

O

OH

O OH O

HO

HO

O OH

HO

O OH

O

OH

O O OH O OH HO

i

HO

O OH

O O

n

OH HO

O OH

O O OH

HO O OH

O OH

O OH

HO O OH

OH

OH OH n = ca. 35 i) CFTase phosphate buffer pH 7.0

FIGURE 2.93. Enzymatic synthesis of cycloinulooligosaccharides.

OH

134

2. O-Glycoside Formation

Summary of Preparation of the Main Glycosyl Donors OAc

OAc AcO AcO

O

HBr/AcOH 33%

OAc

OAc CH2Cl2, r.t., 12 h

OAc

OAc AcO AcO

O

AcO AcO

Br

OAc

O

Cl3CCN/Cs2CO3

OAc

OH CH2Cl2, r.t., 12 h

AcO AcO

O OAc

CCl3

O NH

OAc AcO AcO

OAc PhSNa

O

Et2O/H2O

OAc

AcO AcO

Br

OAc

OBn BnO BnO

EtSH/BF3-Et2O

O OAc

SPh

OAc

OAc AcO AcO

O

OAc

CH3Cl

O

AcO AcO

CH2Cl2, 0oC, 1 h

SEt

OAc

OBn

O

O

O

BnO BnO

O

O

References 1. 2. 3. 4. 5. 6. 7. 8.

K. Toshima, and K. Tatsuta, Chem. Rev. 93, 1503 (1993). F.B. Anderson, and D.H. Leaback, Tetrahedron 12, 236 (1961). H.P. Wessel, Carbohydr. Chem. 7, 263 (1988). Y.F. Shearly, C.A. O’Dell, and G. Amett, J. Med. Chem. 30,1090 (1987). W. Koenigs, and E. Knorr, Chem. Ber., 34, 957 (1901). K. Igarashi, Adv. Carbohydr. Chem.Biochem., 34, 243 (1977). J.-H. Kim, H. Yang, J. Park, and G-J. Boons, J. Am. Chem. Soc., 127, 12090 (2005). M.P. De Ninno, P.A. McCarthy, K.C. Duplatiel, C. Eller, J.B. Etienne, M.P. Zawistowski, F.W. Bangerter, C.E. Chandler, L.A. Morehouse, E.D. Sugarman, R.W. Wilkins, H.A. Woody, and L.M. Zaccaro, J. Med. Chem. 40, 2547 (1997). 9. R.B. Conrow, and S. Bernstein, J. Org. Chem. 36, 836 (1971). 10. A.V. Stachulski, and G.N. Jenkins, Natural Products Reports 173 (1998). 11. A. Bredereck, A. Wagner, H. Kuhn, and H. Ott, Chem. Ber. 93, 1201 (1960).

References 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. 40. 41. 42. 43.

135

P. B¨achli, E.G. Percival, J. Chem. Soc. 1243 (1952). A.Y. Khorlin, I. M. Privalova, and I.B. Bystrova, Carbohydr. Res. 19, 272 (1971). H. Paulsen and H.Tietz, Angew. Chem. Int. Ed. Engl. 21, 927 (1982). H.P. Wessel, N. Iberg, M. Trumtel, and M.-C. Viaud, BioMed. Chem. Lett. 6, 27 (1996). K. Katano, H. An, Y. Aoyagi, M. Overhand, S.J. Sucheck, W.C. Stevens Jr., C.D. Hess, X. Zhou, and S.M. Hecht, J. Am. Chem. Soc. 120, 11285 (1998). (a) S. Umezawa, S. Koto, K. Tatsuta, H. Hineno, Y. Nishimura, and T. Tsumura, Bull. Chem. Soc. Jpn., 42, 529 (1969). (b) S. Hanessian, M. Tremblay, and E.E. Swayze, Tetrahedron, 59, 983 (2003). (c) H. Tanaka, Y. Nishida, Y. Furuta, and K. Kobayashi, Bioorg. Med. Chem. Lett., 12, 1723 (2002). H.J. Roth and A. Kleeman, Pharmaceut.Chem. 7, 263 (1988). T. Suami, T. Otake, T. Nishimura, and Y. Ikeda, Bull. Chem. Soc. Jpn. 46, 1014 (1973). N. Bagget, A.K. Samra, and A. Smithson, Carbohydr. Res. 124. 63 (1983). (a) R.R. Schmidt, Angew. Chem. Int. Engl., 25, 213 (1986). (b) R.R. Schmidt, and K.-H. Jung, Carbohydr. Eur., 27, 12 (1999). (c) R.R. Schmidt and W. Kinzy, Adv. Carbohydr. Chem. Biochem., 50, 21 (1994). A. F¨urstner and T. M¨uller, J. Am. Chem. Soc. 121, 7814 (1999). A. Hasegawa, K. Fushimi, H. Ishida, and M. Kiso, J. Carbohydr. Chem. 12, 1203 (1993). S. Danishefsky and M.D. Shair, J. Org. Chem. 16, 61 (1996). D.P. Larson, C.H. Heathcock, J. Org. Chem. 62, 8406 (1997). S.F. Lu, Q.O. O’Yang, Z.W. Guo, B. Yu, and Y.Z. Hui, J. Org.Chem. 62, 8400 (1997). M. Brito-Arias, R. Pereda-Miranda, and C.H. Heathcock, J. Org. Chem. 69, 4567 (2004). (a) G.J. Boons, S. Isles, J. Org. Chem. 61, 4262 (1996). (b) G.-J. Boons, Contemporary Organic Synthesis 3, 173 (1996). (a) S. Komba, H. Galustian, H. Ishida, T. Feizi, R. Kannagi, and M. Kiso, Angew. Chem. Int. Ed. 38, 1131. (1999). (b) Y. Zhang, A. Brodsky, P. Sinay, Tetrahedron: Asymmetry 9, 2451 (1998). (c) A. Hasegawa, K. Ito, and H. Ishida, Kiso J. Carbohydr. Chem. 266, 279 (1995). (a) A. Koenig, R. Jain, R. Vig, K.E. Norgard,-Sumnicht, K.L. Matta, and A. Varki, Glycobiology, 7, 79 (1997). W.J. Sanders, E. J. Gordon, O. Dwir, P. J. Beck, R. Alon, and L.L. Kiessling, J. Biol. Chem. 274, 5271 (1999). A. Lubineau, J. Alais, and R. Lemoine J. Carbohydr. Chem. 19, 151 (2000). K.C. Nicolaou, T. Ohshima, F.L. van Delft, D. Vourloumis, J.Y. Xu, J. Pfefferkorn, and S. Kim, J. Am. Chem. Soc. 120, 8674 (1998). H. Lonn, Carbohydr. Res. 115, 139 (1985). S.V. Ley, and H.W. Priepke, Angew. Chem. Int. Engl. 33, 2292 (1994). K. Hotta, H. Ishida, M. Kiso, and A. Hasegawa, J. Carbohydr. Chem. 14, 491 (1995). D. Crich, and H. Li, J. Org. Chem. 67, 4640 (2002). Y. Jing, ad X. Huang, Tetrahedron Lett. 45, 4615 (2004). D.R. Mootoo, P. Konradsson, U. Udodong, and B. Fraser-Reid, J. Am. Chem. Soc. 110, 5583 (1988). J.D.C. Codee, R.E.J.N. Litjens, R. den Heeten, H.S. Overkleeft, J.N. van Boom, and G.A. van der Marel, Org. Lett. 5, 1519 (2003). M. Yoshida, T. Kiyoi, T. Tsukida, and H. Kondo, J. Carbohydr. Chem. 17, 673 (1998). A.V. Demchenko, N.N. Malysheva, and C. De Meo, Org. Lett. 5, 455 (2003). E. Fischer, and K. Zach, Sitz. ber. kgl. preuss. Akad. Wiss., 16, 311 (1913). B. Freiser-Reid, D.R. Kelly, D.B. Tulshian, and P.S. Ravi, J. Carbohydrate Chem. 2, 105 (1983).

136 44. 45. 46. 47. 48. 49. 50. 51. 52.

53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78.

2. O-Glycoside Formation B.K. Shull, Z. Wu, and M. Koreeda, J. Carbohydr. Chem. 15(8), 955 (1996). R.W. Murray and R. Jeyaraman, J. Org. Chem. 50, 2847 (1985). W. Adam, J. Bialas, and L. Hadjiarapoglou, Chem. Ber. 124, 2377 (1991). C.H. Marzabadi, and C.D. Spilling, J. Org. Chem. 58, 3761 (1993). G. Belluci, G Catelani, G., Chiappe, C., D’Andrea, F., Tetrahedron Lett. 53, 10471 (1997). Y. Du and F. Kong, J. Carbohydr. Chem. 14(3), 341 (1995). R.L. Halcomb and S.J. Danishefsky, J. Am. Chem. Soc. 111, 6661 (1989). M. Upreti, D. Ruhela, and R.A. Vishwakarma, Tetrahedron 56, 6577 (2000). J. Broddefalk, K.-E. Bergquist, and J. Kihlberg, J. Tetrahedron Lett. 1996, 37, 3011. Broddefalk, J.; B¨acklund, J.; Almqvist, F.; Johansson, M.; Holmdahl, R.; Kilhberg, J. J. Am Chem. Soc. 120, 7676 (1998). H. Kunz, Angew. Chem. Int. Ed. Engl. 26, 294 (1987). C-H. Wong, Y. Ichikawa, T. Krach, C. Gautheron,-Le Narvor, D.P. Dumas, and G.C Look, J. Am. Chem. Soc. 113, 8137 (1991). M. Shimizu, H. Togo, and M. Yokohama, Synthesis 6, 779 (1998). M. Morita, T. Natori, K. Akimoto, T. Osawa, H. Fukushima, and Y. Koezuka, BioMed. Chem Lett 5, 699 (1995). T. Mukaiyama, Y. Hashimoto, S. Shoda, Chem. Lett. 1983, 935. K.C. Nicolaou, and N.J. Bockovich,, and D.R. Carcanague, J. Am. Chem. Soc. 115, 8843 (1993). L.F. Tietze and R. Fischer, Angew. Chem. 5, 902 (1983). S. Metha, and B.M. Pinto, J. Org. Chem. 58, 3269 (1993). A. Sobti, K. Kim, and G.A. Solikowski, J. Org. Chem. 6, 61 (1996). K. Takeda, E. Kaji, H. Nakamura, A. Akiyama, A. Konda, and Y. Mizuno, and H. Takayanagi, and Y. Harigaya, Synthesis 341 (1996). D.M. Garcia, H. Yamada, S. Hatakeyama, and M. Nishikawa, Tetrahedron Lett. 35, 3325 (1994). H. Nagai, S. Matsumura, and K. Toshima, Tetrahedron Lett. 43, 847 (2002). K. Toshima, H. Nakai, and S. Matsumura, Synlett 9, 1420 (1999). K. Toshima, K. Kasumi, and S. Matsumura, Synlett 6, 813 (1999). M. Oikawa, T. Tanaka, N. Fukuda, S. Kusumoto, Tetrahedron Lett. 45, 4039 (2004). (a) F. Burkhart, Z. Zhang, S. Wacowich-Sgarbi, and C-H Wong, Angew. Chem. Int. Ed. 40, 1274 (2001). (b) Lahmann, M., Oscarson, S. Org Lett. 2, 3881 (2000). J.M. Coteron, K. Singh, J.L. Asensio, M. Domingues-Dalda, A. Fernandez-Mayoralis, J. Jimenez-Barbero, and M. Martin-Lomas, J. Org. Chem. 60, 1502 (1995). K.G. Nilsson, Carbohydr. Res. 95, 167 (1987). G.A. Freeman, S.R. Shauer, J.L. Rideout, and S.A. Short, Bioorg. Med. Chem. 3, 447 (1995). E.S. Simon, S. Grabowski, G.M. Whitesides, J. Org. Chem. 55, 1834 (1990). Y. Ichikawa, G.J. Shen, C.-H. Wong, J. Am. Chem. Soc. 113, 4698 (1991). J.J. Gaudino, J.C. Paulson, J. Am. Chem. Soc. 116, 1149 (1994). J.E. Heidlas, W.J. Lees, P. Pale,and G.M. Whitesides, J. Org. Chem. 57, 146 (1992). E.J. Toone, E.S. Simon, and G.M. Whitesides, J. Org. Chem. 56, 5603 (1991). J.L.C. Liu, G.-J. Shen, Y. Ichikawa, J.F. Rutan, G. Zapata, W.F. Vann, and C.-H. Wong, J. Am. Chem. Soc. 114, 3901 (1992). L.F. Mackenzie, Q. Wang, Q., R.A.J. Warren, and S.G. Whiters, J. Am. Chem. Soc. 120, 5583 (1998).

References

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79. D.G. Drueckhammer. W.J. Hennen, R.L. Pederson, C.F. Barbas III, C.M. Gautheron, T. Krach, and C.-H. Wong, Synthesis, 499 (1991). 80. X. Zeng, T. Murata, and T. Usui, J. Carbohydr. Chem. 22, 309 (2003). 81. B.N. Cook, S. Bhakta, T. Biegel, K.G. Bowman, J.I. Armstrong, S. Hemmerich, and C.R. Bertozzi, J. Am. Chem. Soc. 122, 8612 (2000). 82. H. Akita, E. Kawahara, and K. Kato, Tetrahedron Asymmetry 15, 1623 (2004). 83. C.-H. Wong, S.L. Haynie, G.M. Whitesides, J. Org. Chem. 47, 5416 (1982). 84. (a) K.C. Nicolaou, N. Watanabe, J. Li, J. Pastor, and N. Winssinger, Angew. Chem. Int. Ed. 37, 1559 (1998) K.C. Nicolaou, N. Winssinger, J. Pastor, and F. De Roose, J. Am. Chem. Soc. 119, 449 (1997) Wong, C.-H., Ye, X.-S., Zhang, Z. J. Am. Chem. Soc. 120, 7137 (1998). (b) S.A. Mitchell, M.R. Pratt, U.J. Hruby, and R. Polt, J. Org. Chem. 66, 2327 (2001). 85. P.H. Seeberger and W.C. Haase, Chem Rev. 100, 4349 (2000). 86. P. Sears. and C.-H. Wong, Science 291, 2344 (2001). 87. D. Crich and M. Smith, J. Am. Chem. Soc. 124, 8867 (2002). 88. H.J.M. Gijsen, L. Qiao, W. Fitz, and C.-H. Wong, Chem Rev. 96, 443 (1996). 89. G. Gattuso, S.A. Nepogodiev, and J.F. Stoddart, Chem. Rev. 98, 1919 (1998). 90. T. Ogawa and Y. Takahashi, Carbohydr. Res. 138, C5 (1985). 91. Y. Takahashi and T. Ogawa, Carbohydr. Res. 169, 277 (1987). 92. P.M. Collins and M.H. Ali, Tetrahedron Lett. 31, 4517 (1990). 93. D. Bassieux, D. Gagnaire, and M. Vignon, Carbohydr. Res. 56, 19 (1977). 94. G. Excofﬁer, M. Paillet, and M. Vignon, Carbohydr. Res. 135, C10 (1985). 95. N. Nakamura, Methods Carbohydr. Chem. 10, 269 (1994). 96. S. Cottaz, C. Apparu, and H. Driguez, J. Chem. Soc., Perkin Trans. 1 2235 (1991). 97. C. Apparu, S. Cottaz, C. Bosso, and H. Driguez, Carbohydr. Lett. 1, 349 (1994). 98. M. Kamakura and T. Uchiyama, Biosc. Biotechnol. Biochem. 57, 343 (1993).

3 N-Glycosides

These types of glycosides are generated when a sugar component is attached to an aglycon, through a nitrogen atom, establishing as a result a C-N-C linkage. Nucleosides are among the most relevant N -glycosides since they are essential components of DNA, RNA, cofactors, and a variety of antiviral and anti-neoplasic drugs. Usually for nucleosides, a pyrimidine or purine base is linked to the anomeric carbon of a furanoside ring. The nucleosides responsible for the formation of the genetic material DNA and RNA are adenine, guanine, cytidine, and thymine, the latter exchanged by uracil in the case of RNA (Figure 3.1). Nucleosides can be classiﬁed in natural nucleosides such as those involved in the genetic storage of information, naturally modiﬁed nucleosides, and synthetic nucleosides. Naturally modiﬁed nucleosides include a signiﬁcant and diverse number of compounds, some of them with slight changes mostly at the base, or major structural modiﬁcations done by enzymes. So far most of them have unknown biochemical function;1 nonetheless they have been strongly associated with antiviral, antitumoral, and growth regulation processes (Figure 3.2). Representative examples of natural modiﬁed nucleosides includes queuosine (Q) and Wye base (W), which have been found in the tRNA of some plants and bacteria, and it plays an important rule in the inhibition of tumor processes. Derived from this relevant biological function the total synthesis of these unique nucleosides have been reported for Q2–4 and W.5 Moreover, the synthesis of complex nucleoside antibiotics has been reviewed.6 The analysis was focused on the challenging synthetic methods for carbohydrate and nucleoside chain elaboration, glycosidation, and methods for controlling stereochemistry and for joining subunits. As a result, the total synthesis of Polyoxin J,7 sinefungin,8 thuringiensin,9 tunicamycin V,10 nikkomycin B,11 octosyl acid A,12 hikizimycin,13 and capuramycin14 was completed (Figure 3.3). Important cofactors playing a key rule as biological catalysts required by the enzymes for the optimal performance of biochemical transformations are nucleotides. Such is the case of adenosine triphosphate ATP and nicotinic acid adenine dinucleotide NAD that are constituted by an adenosine nucleoside combined with phosphate for the former, and phosphate and nicotinamide for the latter (Figure 3.4). 138

3.1. Nucleoside Formation O

NH2 5 4

3N

6 1

2

N

O NH

N

O

NH O

N

HO

HO

O

O 3'

O

1'

2'

OH

OH

R

R = OH R= H

R

OH

thymine

Heterocyclic base = cytosine

uracil

thymidine

DNA nucleoside = 2' deoxycytidine

uridine

RNA nucleoside = cytidine

NH2 N 7 8

9

N

O N

5 6 1N 4 3 2

NH

N

N

N

HO

5'

O 4'

R

R= OH

R= H

HO

O

HO

5' 4'

139

3'

OH

NH2

O 2'

1'

R

OH

R

R=H R = OH Heterocyclic base = adenine DNA nucleoside = 2' deoxyadenosine RNA nucleoside = adenosine

guanine 2' deoxyguanosine guanosine

FIGURE 3.1. DNA and RNA nucleosides.

3.1 Nucleoside Formation Considering a disconnection analysis, there are two major general routes for nucleoside syntheses.15 The ﬁrst is based on the attachment between the aglycon base and the protected sugar activated with a good leaving group at the anomeric position. Under these conditions, the stereoselectivity is conditioned by the protecting group attached at position 2. The second general procedure considers the coupling reaction between a base precursor and the sugar derivative, which contains the free amine linked to the anomeric carbon. The ring closure generally takes place after the glycosidation reaction and the conﬁguration is predetermined by the nitrogen attached to the anomeric carbon. The latter approach has been most efﬁciently used for preparing carbocyclic nucleosides (Figure 3.5).

140

3. N-Glycosides O

O H3CHNH2C

NH N R

O NH

O

N R

dihydrouridine

NH S

N R ribothymidine (T)

5-methylaminomethyl-2-tiouridine (mmm5s2U)

O

O

O

CO2H

MeO NH N R

O

5-methoxyuridine

N R

CH2CO2CH3

N R

uridine-5-oxyacetic acid

CH3

H3C

NHCOCH3 CH3

N R

5-methylthiouridine

O

N 4-cetylcytidine

O

thiouridine

O

NH2

N R thiocytidine

CH2CO2CH3

HN

N O

N R

(m5C)

N N R

NH

5-methylcytidine

(m3C)

(mcm5U)

S

N N R

O

3-methylcytidine

5-(methoxycarbonylmethyl)uridine

S

O

NH2 N

N R

HN

N R

O

NH

O

NH

(acp3U)

O HN

HO2CH2CO

3-(3-amio-3-carboxypropyl)uridine

(mo5U)

O

NH2

N

O

S

S

N R

5-(methoxycarbonylmethyl)-2-thiouridine

(m5S2U)

(mcm5S2U)

FIGURE 3.2. Naturally modiﬁed nucleosides.

3.2 Protecting Groups It has been mentioned in the previous chapter that protecting groups are important components for most of the general methodologies designed for establishing glycosidic bonds. Usually the methods for glycoside formation require prior protection of those elements (usually heteroatoms) within the molecule that are needed

O

O N

N

NH

N R

N

N R

N inosine (I)

N R

CH3

N

NH

N R

N

N

1-methylinosine

N6-methyladenosine

(m1I)

(m6A)

O

NH N

NHCH3

N

CH3

O

N

N

N R

N

CH3

N N R

NH2

N

1-methylguanosine

1-methyladenosine

CH3

(m2G)

NHCO2CH3

O O

N N R

N

2-methylguanosine

(m1G)

(m1A)

NH

NH N

CO2CH3

N

N CH3

NH2

N R

N H

N H

7-methylguanosine

base W

(m7G)

HO

R2O HN

O N

NH

N R

N

NH2

queuosine (Q) R2 = mannose or galactose R, R2 = H queuine

FIGURE 3.2. (continued)

NHCH3

142

3. N-Glycosides

to remain unaltered. Also important is the fact that the cleavage of the protecting group should be carried out under preferentially mild conditions, and in the case of complex nucleosides the installation and removal of the protecting groups for nitrogen, oxygen, and sulfur should be accomplished under compatible conditions. The protection and deprotection of nucleosides can be done by chemical or enzymatic means. Some of the most commonly used protecting groups used in the preparation of O-glycosides are also useful in the synthesis of nucleosides (Figure 3.6).

3.2.1 Ribofuranoside Protecting Groups Enzymes have been found to be interesting alternatives for installing protecting groups on nucleosides. Some of the enzymes used for this purpose are subtilisin mutant (8350)16 and lipases mainly from Pseudomona and Candida strains.17 Representative protections of purine and pyrimidine nucleosides are indicated in Figure 3.7. By using the appropriate lipase it is possible to achieve regioselective acyl protections on nucleosides. For instance, the enzymatic transesteriﬁcation reaction of 5 -ﬂuorouridine with n-octanoic anhydride catalyzed with Candida Antarctica (CAL), Pseudomona sp. (PS), (KIWI-56), and Mucor javanicus (M) lipase was performed, producing 5 -, 3 - and 2 -acylnucleosides, respectively (Figure 3.8).18 Regioselective removal of certain protecting groups such as acetates attached to the ribosyl moiety of nucleosides might be carried out by enzymes. For instance, Subtilisin strain selectively hydrolyzes the 5 -position of purine and pyrimidine tri-O-acylated esters to produce 2 ,3 -di-O-Acylribonucleosides in 40–92% (Figure 3.9).19 On the other hand, diastereoselective deacetylation of peracetylated 2 deoxyribofuranosyl thymine was carried out using wheat germ lipase (WGL) and

CH3 O O

OH

O

CO2H O

H2N

N

NH

N H

O OH

NH2

O HO

OH

polyoxin J

FIGURE 3.3. Complex nucleoside antibiotics.

3.2. Protecting Groups N

NH2 O

143

NH2

N

HO2C

N N

NH2 HO

OH

sinefungin

OH AcHN

OH OH

O

O

H N

(CH3)2CH(CH2)9

O O

OH O

O

N

NH

HO OH

O HO

OH

tunicamycin V

NH O HN

O

O

O

NH2 O

O N

NH

O

HO OH

O H3CO

OH

capuramycin

FIGURE 3.3. (continued)

ATP

NH2 O N

N

H2N O N

O

P

O O OH

O O

P

−

N

O O

−

O

P O

O

N

O

−

OH

OH

OH

NAD+

FIGURE 3.4. Structure of nucleoside cofactors ATP and NAD. R2

R2 R2 N

N

N H

R1O

N

RO Cl

R1O

N

OR1

C Y

N N

R3

X

N N

N

X

RO

X

R3

N

NH2

X

X = O, S, CH2

RO

R1 = Ac, Bz, Tol, Bn R2= NH2, OH R3 = H, NH2

OR OR

FIGURE 3.5. General procedures for N-glycoside formation. Acetate (CH3CO-)

HO

AcO

O OH

O OAc

i

OH OH

i) Ac2O, CH 2Cl2, DMAP, r.t.

cleavage: (1) NaOMe, MeOH. (2) Aqueous NH3, dioxane.

FIGURE 3.6. Common ribose protecting groups.

OAc OAc

OR

3.2. Protecting Groups

145

Benzoyl (PhCO-).

O

O CH 3

HN O HO

CH 3

HN i

N

O

N

BzO

O

OH

O

OBz

i) Bz-Cl, pyridine.

cleavage: (1) R-NH2, EtOH, 100oC. (2) EtOH, KOH, reflux, 3 h. (3) NH3, MeOH

Toluyl (p-MePhCO-)

Cl

Cl N N HO

N

N

N

N

i

TolO

O

O

OTol

OH

i) Tol-Cl, pyridine. cleavage: NH3, MeOH, 100oC, 78%.

Pivaloyl (Me3CCOCl )

HO

T O

PivO i

i) Piv-Cl, pyridine. cleavage: NaOMe, MeOH.

FIGURE 3.6. (continued)

T O

N N

Trityl (Ph3C-)

HO

TrO

O OH

O OH

i

OH OH

OH OH

i) Tr-Cl, pyridine, r.t.

cleavage: (1) 80%, AcOH, 60oC. (2) HCO2H, Et2O.

Benzyl (PhCH2-)

T

HO

T

BnO

O

O

i

OBn OBn

OH OH i) BnBr, NaH, DMF.

cleavage: H2/Pd(OH)2 , EtOH.

Tertbutyldimethylsilyl (tBuMe2Si-)

U HO

O

U i

OH

TBDMSO

O

OH

i) TBDMS-Cl, pyridine, r.t.

cleavage: (1) tetrabutylammonium fluoride (TBAF). (2) pTsOH, MeOH, H 2O, 7h.

FIGURE 3.6. (continued)

3.2. Protecting Groups

147

Tetraisopropylsilyl ([( iPr)2Si]2O-)

HO

O

O

O

O

iPr2Si

i

O

O iPr2Si

HO

O

i) Pr2iSi(Cl)OSi(Cl)Pr2i, imidazole, THF, rt, 90 min.

cleavage: Bu4NF, THF.

FIGURE 3.6. (continued)

porcine liver esterase (PLE), affording pure β-anomer thymidine in 29 and 31%, respectively (Figure 3.10).20 When porcine pancreas lipase (PPL) in phosphate buffer is used for deacetylation of 3 ,5 -di-O-acetylthymine, the removal of the acetyl group at the 5 -position is achieved, leading to the 3’-O-acetylthymidine (Figure 3.11).20 Other suitable selective protections and deprotections useful for chemical manipulations that might occur at the ribosyl moiety are illustrated in Figure 3.12.

HO

B

AcO

B

AcO

O

O i

OH

X

OH

X

B = T, U, C, A X = H, OH i) Subtilisin 8350, DMF. 65-100%

O F

O CO2H

F

CO2H

i

N HO O

N O

R

O O

i) Pseudomona cepacea lipase, RCO2Et, AcOEt, rt, 72h.

FIGURE 3.7. Enzymatic regioselective acylation by oximeacetates and lipases.

148

3. N-Glycosides HO

B O

i

O

R HO

O

O

B O

N +

O

R

OH O

R

B = T, A

B O

ii

O OH i) Pseudomona cepacea llipase (PSL), Pyridine. ii) Candida antartica lipase (CAL), THF.

FIGURE 3.7. (continued) O F NH HO

N

B ii

O

O

R

O R

O

O

B

i

HO

O O

OH OH OH

O R = CH3(CH2)6−

OH

iii

HO

B O

OH

R

O O

i) Candida antarctica lipase (CAL), 90%. ii) Pseudomona sp. lipase (PS), 92%. iii) Mucor javanicus lipase (M), 42%.

FIGURE 3.8. Regioselective acyl protection by lipase.

OH

3.2. Protecting Groups AcO

OAc

HO

B

O

i

B

O

OAc

OAc

149

OAc

B = U, C, A, G, N-2AcG, H i = Subtillisin or PPL, organic solvent, phosphate buffer, pH 7.

FIGURE 3.9. Selective enzymatic 5 -acetyl deprotection. O

O

NH

NH i

N

AcO

N

HO

O

O

O

O OH

OAc

i) WGL, phosphate buffer, 29%. or PLE, phosphate buffer, 31%.

FIGURE 3.10. Lipase-catalyzed deacetylation of anomeric nucleoside.

Regioselective protections and deprotections is often a critical step especially for the preparation of complex nucleosides. Some suitable deprotections of complex nucleosides that do not alter the original composition of the structure have been described (Figure 3.13).6

O

O NH

NH i

N

AcO

O

O

OAc

N

HO

O

O

OAc

i) PPL, phosphate buffer, 98%.

FIGURE 3.11. Selective enzymatic.5 -deacetylation of 3 ,5 -di-O-acetyl thymidine.

150

3. N-Glycosides NBn2

NBn2 N

N

N

N

BnO

O

BnO

N

N i

BnO

N

N

O

BnO

OBn

OH

i) t-BuMgBr, PhH, 80oC, 69%

Ref.21 Cl

Cl N

N

N

N TrO

N

N

N

N

i,ii

HO

O

O

OH OBn

OBz OH i) BnOH, Me2NCON=NCONMe2. ii) NH 3/MeOH

Ref.22 B HO

O

OH OH

B DMTO i-iv

O

OH OTBDMS

B = G, A, C, U i) t-Bu2Si(OTf) 2, Im, DMF, 0oC. ii) t-BuMe2SiCl, Im, DMF, 60oC, 80-87%. iii) HF-Py, CH2Cl2, 0oC, 90% iv) DMT-Cl, Py, 0oC, 90%.

Ref.23 FIGURE 3.12. Miscellaneous chemical protection and deprotection.

3.3. General Methods BnO

BnO

O

O

O

O

OH

i

O

O

151

O

i) DIBAL, NiCl2, Et2O, 0oC, 55%

Ref.24 NH2

NH2 N N ButMe2SiO

N

N

N

i

N

HO

O

O

N

O

OSiMe2But

N

O

OSiMe2But

i) CF3COOH-H 2O (9:1), 0oC, 95 %.

Ref.25 FIGURE 3.12. (continued)

3.3 General Methods The following are general methods that we will explain in this section: r Michael reaction r Fischer-Helferich reaction

NH O O HN O

O

O

AcO

O

NH2 N

OAc

O H3CO

FIGURE 3.13. Suitable deprotection of complex nucleosides.

NH

O

OPiv

1. NaOH, aq MeOH, 2.5 h (cleaves O-Ac and O-Piv)

152

3. N-Glycosides O

Boc-O CH3

O

H N

CO2H

CHO

O

N

N H O

N Boc

HO

OH

1. TFA, 0oC, 15 min. (cleaves O- and N-BOC) 2. H2O, then lyophilize (cleaves acetal)

N3

OPiv OPiv

PivO AcHN

N

O

O OAc

O N

OAc

O OBn

AcO

OAc

OAc

N3 OAc

1. DDQ, CH2Cl2, 58oC, 43h (cleaves O-Bn) 2. n-Bu4NOH, MeOH, reflux, 2 h (cleaves acyls) 3. H2, Lindlar, H2O (reduces azide groups)

TBSO CH3

O

CO2Bn

O O

N

N H TBDPS O

N3 HO

OH

NH O

1. n-Bu4NF, THF, 30 min. (cleaves 2 O-SiR3) 2. H2, 10 % Pd-BaSO4, aq. MeOH, 30 min. (cleaves benzyl ester and reduces -N3)

FIGURE 3.13. (continued)

r r r r r r

Davol-Lowy reaction Silyl-mediated reaction Sulfur-mediated reaction Mitsunobu reaction Palladium-mediated reaction Microbial/enzymatic approach

3.3. General Methods

153

OTBS AcHN

O O

Cbz

H N

O

O O

O OH O

N

BOMO

BOC OH

O OH

HO

1. 10% HCO2H, Pd, 1.5 h (cleaves O-BOM, N-Cbz) 2. 13% HCO2H, MeOH, 40oC, 5 h (cleaves N-BOC, acetonide) 3. HF, MeOH, CH3CN (cleaves O-TBS)

FIGURE 3.13. (continued)

3.3.1 The Michael Reaction 3.3.1.1

General Figure and Conditions

O

N

promoter +

PO

X

O N H

PO

X = Cl, Br

Promoter

Conditions

NaH K2 CO3 KOH-TBA

DMF DMF CH2 Cl2

It is a classical procedure for preparing nucleosides, and it can be considered a modiﬁed O-glycoside approach. In this way, the sugar derivative is an R-Ofuranosyl halide, where R can be acyl-, benzoyl, benzyl, tosyl, or silyl, and the halogen commonly chlorine instead of bromine, since it has probed to be more stable for furanose derivatives than its counterpart. The nitrogen base (purine or pyrimidine) is reacted under basic conditions, usually NaH or K2 CO3 in DMF (Figure 3.14). A variety of antibiotics have been prepared according to this method, as in the case of the nucleoside known as methyltubercidine. For achieving this goal, the 7-deazaguanine was used as the nitrogen base, which was condensed to

154

3. N-Glycosides O O

NH

RO

O i,ii

NH

+

N H

O

N

RO

Cl

O

O

OR OR

OR OR

i) NaH/DMF. ii) MeONa/MeOH

FIGURE 3.14. The Michael modiﬁcation.

2,3,5-tri-O-benzylribofuranosyl bromide under NaH/DMF conditions to afford a 1:1 anomeric mixture of the N-glycoside (Figure 3.15).26 More recently, Battaharya27 reported the synthesis of ﬂuoroarabinotubercidine, toyocamicine, and sangivamicine, under the current N-glycoside formation OCH3 OCH3

CH3

BnO

N

O i

N

+

N H

N

N

N

H3CS

Br H3CS

CH3

BnO

OBnOBn

O

OBnOBn OCH3

CH3

OCH3

N

N N

N ii-vi

CH3

HO

O

O

O

N

N

vii-ix

AcO

O

OH OH

i) NaH/DMF. ii) Ni/EtOH-PhH. iii) HCl/dioxane. iv) H2, Pd-C. v) a) acetone/p-TsOH. b) Ac2O/Py. vii) POCl3. viii) NH3/MeOH. ix) F3CCOOH/H2O.

FIGURE 3.15. Synthesis of methyltubercidine.

3.3. General Methods

155

procedure. Other deazapurines have been described by Seela et al.28 involving the condensation between the purine base, with protected ribosyl halides under basic conditions. According to Seela29 and Kazimierczuk,30 the stereoselective glycosylation of the sodium salts of halopurines, with 2-deoxy3,5-di-O- p-tolouyl-α-D-erytro-pentofuranosyl chloride, gave β-nucleosides via Walden inversion. This was demonstrated in the preparation of 2-amino 2 desoxytubercidine and 2-aminotubercidine by condensation of 3,5-di-O-(ptolyl)-α-D-pentafuranosylchloride and 5-O-[(1,1-dimethylethyl)dimethylsilyl]2,3-O-(1-methylethyliden)-α-D-ribofuranosylchloride with the halopurine under Michael conditions. Final ammonia treatment provided the target deazanucleoside (Figure 3.16). The 7-deazapurine nucleoside Cadeguomycin isolated from strain of the actinomycete culture ﬁltrate Streptomyces hygroscopicus was also synthesized under this approach. Thus, coupling reaction between protected 7-deazapurine derivative with 1-chloro-2-deoxy-3,5-ditoluyl-α-D-erythro-pentofuranose was effected with preference for the β-isomer. Subsequent transformations provided the target molecule 2 -deoxycadeguomycin (Figure 3.17).31

NH2 Cl TsO

N

O

N

i,ii,iii

+

H2 N

N H

N

H2 N

Cl

N

N HO O

OTs

OH

NH2 Cl SiO

N

O i,iii,iv

N +

H2 N

N

N H

Cl O

O

N

N

H2 N HO

O

OH OH i) KOH, TBA/CH2 Cl2 . ii) MeONa/MeOH. iii) NH3/MeOH. iv) CF 3COOH/H2 O.

FIGURE 3.16. Synthesis of 2-aminotubercidine and 2-amino-2 -deoxytubercidine.

156

3. N-Glycosides O CO2Me MOM

N

O CO2Me MOM

N

O

TolO

Cl

N H

N

N R1

O

i

+ N R1

O

TolO

O

TolO ii

R 1 = BOM

TolO

R1 = H

(α:β; 1:5.5)

O CO2H

O CO2Me R3

HN

N

iii

R2

HO TolO

N

N

H2 N

vi

N

N

O

O

HO TolO iv

R2 = ArSO2O, R3 = CH2OCH3 R2 = PhCONH, R3 = CH2OCH 3

v

R2 = PhCONH, R3 = CH2OCH3

i) NaH, CH3CN, 25oC. ii) H2/Ph(OH)2, MeOH, 25oC. iii) NaH, THF, TIPBS-Cl. iv) PhCONH2, NaH, THF. v) TFA, CH2Cl2.

FIGURE 3.17. Synthesis of 7-deazapurine nucleoside 2-deoxycadeguomycin.

3.3.2 The Fischer-Helferich Reaction 3.3.2.1

General Figure and Conditions

O

N

promoter +

PO

X

O N H

PO

X = Cl, Br

Promoter

Conditions

Silver salts

xilene

This general procedure consists in the use of an acylfuranoside or acylpyranoside, which is reacted with the silver or mercury salts of a nitrogen base. The

3.3. General Methods

157 CH3

O H3C

OAc

N

N

CH3

O

N Ag H

N

O

AcO

i

OAc

Cl +

O

N

Cl

O

N

N

N

Br

OAc

O

OAc

OAc

CH3

OAc OAc

i) Xylene

FIGURE 3.18. The Fischer-Helferich method.

original reaction involves the condensation between silver salt of theophylline with acetobromoglucose in hot xylene, giving preferentially the N-7 regioisomer (Figure 3.18). The feasibility of this method is observed in the synthesis of adenosine and guanosine by condensation of tri-O-acetyl-α-D-ribofuranosyl chloride with the silver salt of 2,8-dichloroadenine to generate an intermediate which under the conditions described below can generate either adenosine or guanosine (Figure 3.19).32 The stereochemistry of this reaction can be predicted by applying the “trans rule” proposed by Tipson33 and extended by Baker. The rule establishes that the condensation between the purine or pyrimidine sal with the acyl-O-glycosyl halide will generate a nucleoside with C1-C2 trans conﬁguration regardless of the initial conﬁguration of C1-C2 of the sugar. The trans rule is demonstrated in the preparation of thymidine acetoglucopyranose and mannopyranose, where -OH at position 2 for the former is equatorial, and axial for the latter. By following the rule, the coupling reaction generates the β- and α-anomers, respectively, having both of them a trans disposition between substituents at positions 1 and 2 (Figure 3.20).

3.3.3 The Davol-Lowy Reaction 3.3.3.1

General Figure and Conditions

O

N

promoter +

PO

X

O N H

PO

X = Cl, Br

Promoter

Conditions

Hg(CN)2 Hg(CN)2

CH3 NO2 , reﬂux xilene

158

3. N-Glycosides NH2 NH2 N

N

AcO

Cl

O

i

Cl

Cl + Cl

N

N

N N AcO

Cl

N Ag H

N O

OAc OAc

OAc OAc ii,iii

NH2

N

N HO

ii,iv,v

N

N

O

O

N

HN

OH OH

H 2N

N

N HO

O

i) xylene. ii) MeONa/MeOH. iii) H2, Pd-C. iv) HNO2. v) NH3.

OH OH

FIGURE 3.19. Synthesis of adenosine and guanosine.

This method has been also considered a modiﬁed Fischer-Helferich procedure and involves the use of mercury chloride instead of silver salts. Under these conditions the useful intermediate chloropurine nucleoside has been prepared under mild conditions (Figure 3.21). The nature of the glycosyl halide is important for determine the regioselectivity of the glycosidic linkage. If the condensation reaction occurs between purines with acetobromoglucose the N-7 regioisomer is obtained preferentially. On the other hand, if acetoribosyl chloride is condensed with the same purine, the N-9 regioisomer is the major product observed (Figure 3.22). Another purine nucleoside prepared under these conditions is shown in Figure 3.23, consisting of the coupling reaction between the protected guanine with protected furanosyl chloride in nitromethane under reﬂuxing conditions produced by the corresponding N-glycoside in 50% yield.34

3.3. General Methods

159

O AcO AcO AcO

HN

O OAc

Br

O

OAc OAc

AcO

O HN O

OAc

N

O

AcO

N H

OAc

AcO O

OAc

OAc OAc

O

AcO AcO

N

O

Br NH O

FIGURE 3.20. Tipson’s trans rule.

Cl N

N Cl

BzO

O

N

N

i

+

O

Cl N

N HgCl H

N

N BzO

OBz OBz

i) xylene

OBz OBz

FIGURE 3.21. The Davol-Lowy method.

3.3.4 Silyl Coupling Reaction 3.3.4.1

General Figure and Conditions O

promoter

N

+

PO

OAc

N

O

OTMS PO

O

160

3. N-Glycosides OAc OAc

OAc

O

AcO

OAc OAc

OAc

N

N N

N-7 regioisomer i,ii

N

OAc

N

N

N(CH3)2 N

O

N(CH3)2

Br

N HgCl H

N(CH3)2 BzO

O

N

N Cl

OBz OBz

N

N AcO

O

i) xylene. ii) MeONa/MeOH.

OAc OAc N-9 regioisomer

FIGURE 3.22. Preparation of N-7 and N-9 regioisomers.

Promoter

Conditions

TMS-OTf TMS-OTf SnCl4 HMDS-TMDS (MeSi)2 NAc CF3 (CF2 )3 SO3 K/HMDS-TMSCl HMDS/(NH4 )2 SO4

CH3 CN, 0◦ C→r.t PhNO2 ,127◦ C CH3 CN

Various types of silyl agents have been tested as either protecting groups and/or N-glycoside promoters. Among them trimethylsilyl chloride (TMS-Cl), bis(trimethylsilyl) acetamide, trimethylsilyltriﬂate, and hexamethyldisilasane are representative examples. De Clercq et al.35 prepared purine and pyrimidine α-D-lixofuranosylnucleosides employing HMDS, TMS, and TMSF as silyl coupling agents. Nucleoside α-D-lyxofuranosyl thymine was prepared by condensation between 1,2,3,5-

3.3. General Methods

161 O

N

O EtHN

NH

O Cl

O

N

NH

OBz

N H

OBz

N

N

i

+

EtHN

N

NHAc

O O

NHAc

OBz

OBz

i) Hg(CN)2, CH3NO2, reflux, 16 h.

FIGURE 3.23. Glycosidation reaction for preparation of guanine derivative.

tetra-O-acetyl-α-D-lyxose with thymine in the presence of HMDS-TMSCl mixture (Figure 3.24). Likewise cytidine has been synthesized in 95% through condensation of silyl cytidine obtained from cytosine with bis [trimethylsilyl] acetamide, and sugar derivative 2,3,5-tri-O-benzoylribose, as represented in Figure 3.25. O

O

HN O AcO

N H

OAc OAc O OAc

O

N OAc OAc O OAc

O i

AcHN

N

N

HN

N SiMe3 H

O

+

N

HN AcHN

N

N

AcHN

i) HMDS-TMSCl

OAc OAc O OAc

O

N

HN

N

N OAc OAc O OAc

FIGURE 3.24. Preparation of α-D-lyxofuranosyl thymine and guanine protected nucleosides.

162

3. N-Glycosides NH2 NHTMS

BzO

O

N i,ii

N +

TMSO

OH

N

HO

N

O O

OBz OBz

OH OH

i) (CH3Si)2NAc. ii) Ba(OH)2/MeOH.

FIGURE 3.25. Silyl mediated coupling reaction.

Hilbert and Johnson36 developed a procedure for preparing nucleosides employing a mixture of hexamethyldisilane (HMDS), trimethylsilane chloride, and potassium nonaﬂate. According to this procedure, 5-methoxyuridine was prepared by condensing 5-methoxyuracil, with 1-O-acetyl-2,3,5-tri-O-benzoyl-βD-ribofuranose (Figure 3.26). A widespread silyl-based methodology was developed by Vorbr¨uggen37 based on the use of persilylated purines or pyrimidines, which are condensed with peracylated sugars in the presence of Lewis acid catalysis. Usually silylation of the base is achieved with hexamethyldisilazane (HMDS) or N,Obis(trimethylsilyl)acetamide, the latter less difﬁcult to remove during the workup process. Among the Lewis acids employed as catalysts, trimethylsilyl triﬂate (TMSOTf) has been the most suitable condensing agent for this reaction. AZT alkylthioanalogs have been synthesized under the method reported by Vorbr¨uggen. This condition requires hexamethyldisilane for activation of the anomeric center, and trimethylsilyltriﬂate as condensing agent (Figure 3.27). V¨orbruggen-type coupling reaction has been method of choice in the Nglycoside bond formation of various complex nucleosides such as octosyl acid A, tunicaminyl-uracil, sinefungin, and hikizimycin.Some of the general conditions O O

BzO OMe

HN

OMe

OAc

HN

O OBz

i,ii

O

N H

N

O

+

HO OBz

O OH

i) CF3(CF2)3SO3K/HMDS-TMSCl. ii) Ba(OH)2/MeOH.

FIGURE 3.26. Hilbert and Johnson approach.

OH

3.3. General Methods TMSO O R

R2S

O

O

OAc R

OTMS

HN

163

HN R

HN

i

N3

N H

R2S

N

R2S TMSO

ii

O

N H

i) HMDS/(NH4)2SO4. ii) TMSOTf/CH3CN.

N3

FIGURE 3.27. Vorbr¨uggen’s synthesis of AZT thioderivatives.

reported for the accomplishment of the mentioned synthesis are described in Figure 3.28.6a−b

3.3.5 Sulfur-mediated Reaction 3.3.5.1

General Figure and Conditions O

promoter

N

+

PO

SR

N

O

O

OTMS PO

R = Ph, (=O)Ph Promoter

Conditions

NIS-OTf TMS-OTf Br2

CH2 Cl2 DCE r.t. DMF

Derived from their extensive use in the preparation of O-glycosides, the sulfur glycosyl donors have become another standard procedure for N-glycosylations. The conditions reported for the coupling reactions involve the sulfur glycosyl donor, the silyl protected heterocycle acceptor, and usually N-iodosuccinimide, triﬂic acid as catalyst (Figure 3.29).38

3.3.6 Mitsunobu Reaction This reaction has been selected as another strategy for preparing N- and carboxyclic nucleosides. The mechanism involves a nucleophilic substitution displacement with inversion of the conﬁguration between species bearing poor leaving groups with nucleophiles. The reaction mechanism involves the initial reaction

164

3. N-Glycosides OMe OTMS

Cbz-HN

SnCl4

OAc

O

+

O

N

OAc

AcO

N

74.5%

OTMS

OAc OAc

AcO OMe Cbz-HN O

O

OAc O

N

AcO OAc

O OAc

AcO

OMs

CO2Me

OBn O

MeO

NH

OTMS OAc

+

N

TMS-OTf, CH3CN 0oC to r.t.

N 91 %

O OAc

AcO

OTMS CO2Me

OMs

O

OBn O

MeO

N

O

NH O

AcO

OAc

FIGURE 3.28. V¨orbruggen-type coupling reactions.

of triphenylphosphine (Ph3 P) with diethylazodicarboxylate (DEAD) to produce a dipolar intermediate that will react with an alcohol to form an alcoxyphosphonium salt and diimide. Then the nucleophile will displace triphenylphospine oxide to give the substitution product. (Figure 3.30).39 This procedure was used successfully for preparing the N-glycoside shown in Figure 3.31 by reacting 2,3,4,6-tetraacetyl glucose with the heterocyclic base under the Mitsunobu conditions.40

3.3.7 Palladium-mediated Reaction Palladium catalysis is a well-established and versatile methodology for the preparation of nucleosides. Also known as the Heck reaction, it was developed initially

3.3. General Methods BnO

BnO

OTMS O

SPh

AcO

N

O

i

N

O

N

AcO

+

NH O

OTMS

OPiv

MeO

165

OPiv

MeO

i) NIS, TfOH, CH2 Cl2, −20o C to 0o C, 10 min, 85%

Ph O O

Ph N

OPiv

O

OTMS

O O OPiv

O

SPh

O

N3

N

+

i

N3

O N

O

N Ac

TMS

N NHAc

i) NIS, TfOH, CH2 Cl2, 1h, 95%

FIGURE 3.29. N-glycoside formation via sulfur glycosyl donor.

for C-C bond formation and consists in the coupling of an aryl halide with activated oleﬁn in the presence of palladium (0) as catalyst (Figure 3.32).41 More recently, other palladium-mediated reaction have been developed with great potential for heterocycle coupling reaction with furanosides, to produce an interesting variety of nucleosides. The group of reactions includes the Suzuki X

EtO2C N N CO2 Et

Ph3 P HX

X +

PO

EtO2C X

PO

H N N PPh 3

EtO2 C

CO2 Et

H N N H

N

N H OPPh 3

OH X = CH2, O

X +

O

PPh3

PO

FIGURE 3.30. The Mitsunobu reaction for the construction of a glycosidic bond.

CO2Et

166

3. N-Glycosides OAc CH3 N

O

OAc

N

O

O

AcO AcO

O

CH3 O

OH N N Boc

i

N N Boc

N H AcO

N

O

i) DEAD, Ph3P, THF, −78oC, 61%

OAc

OAc OAc

FIGURE 3.31. The Mitsunobu reaction for preparation of N-glycosides.

(organoboranes),42 Stille (organostannanes),43 Negishi (zincated),44 Sonogashira (alkyne-CuI),45 Hiyama (organosilicon),46 and Tsuji-Trost47 (Figure 3.33). Early reports in the use of Heck-type reactions for the preparation of nucleosides were described by Bergstrom.48 More recently a comprehensive overview about palladium-mediated reactions for N -glycoside bond formation or modiﬁcations at the base or the sugar moieties were described. A general ﬁgure summarizing such possibilities is shown in Figure 3.34.49

3.3.8 Microbial/Enzymatic Approach The synthesis of nucleosides by enzymatic methods is another extended possibility, and for this purpose the enzyme nucleoside phosphorylase has been selected as one of the most appropriate one. Usually the conversion proceeds by the reversible formation of a purine or pyrimidine nucleoside and inorganic phosphate from ribose-1-phosphate (R-1-P) and a purine or pyrimidine base. The general approach consists of the reaction of R-1-P as the glycosyl donor, which is condensed with purine or pyrimidine analogs. Following this method any heterocycle recognized by this enzyme can be glycosilated (Figure 3.35). The enzyme synthetase phosphoribosyl pyrophosphate PRPP was used for nucleotide synthesis of UMP. The sequence involves the conversion of ribose-6-phosphate with PRPP synthetase to produce phosphoribosyl pyrophosphate which was condensed with orotate in the presence of O5P-pyrophosphorylase PdX

X

PdX

Pd

o

H 2C

CH2

FIGURE 3.32. The Heck reaction.

3.4. Oligonucleotide Synthesis

Br +

Ar

Ar

B(OH)2

167

Pd(0)

Ar

Ar

Suzuki reaction

R'

R

PdL4

+ OTf

R''

R'

R

SnR3

R'

Stille reaction

R Br + R2'

Pd(0)

Zn

R R'

Negishi reaction

R Br + R3'

Pd(0)

Si

R R'

Hiyama reaction

R

+

H

Pd(0)

R

R

CuI

R

X Sonogashira reaction

+

R

R

Nu

Pd(0)

X

Pd

R Nu

X

Tsuji-Trost reaction

FIGURE 3.33. Palladium-mediated coupling reactions.

to yield the nucleotide intermediate orotidine 5 -phosphate that, after descarboxylation produced by the action of O 5P-decarboxylase the nucleotide Uridine monophosphate (Figure 3.36).50

3.4 Oligonucleotide Synthesis Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are very important natural polymers responsible for the processing of the genetic information of all organisms.

168

3. N-Glycosides OP OAc

AcO

O

OP Heterocycle coupling "Pd"

Heck

Tsuji-Trost

O

O R

NH N

HO

"Pd"

O

various

O

N

HO

X

NH

X

Heck, Stille

OH OH

Negishi

OH OH Heterocycle modification

"Pd"

Tsuji-Trost "Pd"

Sonogashira

O

O NH NH HO

N

HO

O

N

O

X

X

Me Me

Sugar modification

R

FIGURE 3.34. Palladium-assisted modiﬁcations.

The basic repetitive unit known as nucleotide is composed by a nucleotide base, a sugar moiety, and a phosphate. The combinatorial pattern of the four different nucleosides constituted by the heterocyclic bases cytosine, thymine, guanine, and adenine are the base of the DNA structure. In RNA strands uracil replaces thymine and the furanoside is ribose instead of 2-deoxyribose. The phosphate group is attached at position 3 of one sugar unit and the 5 position of the next one, forming a 3 -5 elongation chain (Figure 3.37). Oligonucleotide synthesis does not involve N-glycoside bond formation, but it requires the design of nucleoside donors and nucleoside acceptors, following

3.4. Oligonucleotide Synthesis HO

HO

O B1

HO

O

i

OH X

O B2

i

O-PO32−

169

B2

OH X

OH X

X = H, OH

HO

O B1

ii

HO

OH X

O B2

OH X

i) nucleoside phosphorilase. ii) trasribosylase.

FIGURE 3.35. General ﬁgure for enzyme-mediated nucleoside synthesis.

the same principle that applies for glycoside coupling reactions where suitable protecting groups, glycosyl donors and acceptors are requested. Solid-phase procedures appear to be of great advantage for the coupling of nucleosides, and unlike for oligosaccharide solid-phase chemistry, the attachment positions are always the same (3 and 5 ). The sequence of reactions that occurs in oligonucleotide synthesis starts on the attachment of 3 -OH position of 5 -protected nucleoside to a resin. Next is deprotection of 5 -OH and subsequent attachment to a nucleoside donor, which contains a phosphate precursor, which in turn will be converted to a phosphate group. O O -O P O O−

NH

O ATP

O

AMP

OH OH OH

-O P O O−

PRPP synthetase

O O O O P O P O– O− O− OH OH

-O 2C

O NH

OH OH

NH O

O5P decarboxylase

O -O P O

O−

O

O5P pyrophosphorylase

O

O -O 2 C N -O P O O O−

N H

N O

OH OH UMP

FIGURE 3.36. Enzyme-catalyzed synthesis of nucleotide.

O

170

3. N-Glycosides NH2 N

N

N O

Adenine

N

O

NH2 N

Cytosine

O O

P

O−

N

O

O

O O N

NH

O O

P

O−

N

O

N

O

Guanine

NH2

O NH

Thymine

O O

P

O−

N

O

O

O O O

P

O−

O

FIGURE 3.37. Fragment of a single strand of DNA structure.

There are mainly two procedures for oligonucleotide synthesis: the phosphoramidite and the phosphonate methods.15,51

3.4.1 Phosphoramidite Method This methodology involves the use of the air-sensitive reagent 2-cyanoethyl tetraisopropylphosphorodiamidite {[(CH3 )2 CH]2 N}POCH2 CH2 CN or 2cyanoethyl N,N-diisopropylchlorophosphoramidite (iPr)2 NP(Cl)OCH2 CH2 CN for activation of nucleoside donor.52 This intermediate can be obtained by treatment of PCl3 with 2 eq of diisopropylamine, and 1 eq of cianoethylethanol. The general phosphoramidite approach is outlined in Figure 3.38 and begins with

3.4. Oligonucleotide Synthesis

171

HO2CCH2CH2CO2H

(EtO)2Si(CH2)NH2

(CH2)3NH2

CO2H

(CH2)3NHCOCH2CH2CO2H

B1

B1

OTr

O

OTr

O

CO2H

OH

O

O

OH

B2

B1 i

+

O

OTr

O

O

B2

O

OTr

O

O P

N

CN

O

O P B1

CN

O

O

O

iii

ii

O O B3 B2

OH

O

OTr

O

B3

OTr

O

O O

N P

P B1

O

O

O

O O

CN

CN

B2

O

O

P O

ii

O O

P O

B1

O

O

O

O O

FIGURE 3.38. The phosphoramidite oligonucleotide strategy.

CN

CN

172

3. N-Glycosides B3

OH

O

FIGURE 3.38. (continued)

O B2

O

O

P

O

OH

iii,iv

O P B1

O

O

O

OH

O O i) Cl3CCOOH. ii) tetrazol. iii) Cl3CCOOH. iv) a) I2/H2O. b) NH4OH

a nucleoside previously protected at the 5 -OH position with 4,4 -dimethoxytrityl group (Tr-), also attached to a silica support. The trityl group is then removed from the 5-OH position and allowed to react with a nucleoside donor protected at position 5-OH with Trityl group and activated at position 3 with 2-cyanoethyl diisopropylphophoroamidite. The coupling reaction being the critical step is catalyzed by tetrazol, and the process is repeated for the installation of subsequent nucleoside unit. Once the oligonucleotide chain is formed, the phosphoramidite group is transformed to phosphate with I2 -H2 O and released from resin with ammonia.

3.4.2 Phosphonate Method In this method the nucleoside donor functions as a phosphotriester sugar derivative that reacts with the nucleoside acceptor at the 5-OH position, which is available for linkage. An advantage of this method is the possibility of introducing substituents to the phosphate position giving place to the preparation of modiﬁed oligonucleotides (Figure 3.39).

3.4.3 Modiﬁed Oligonucleotides Modiﬁed oligonucleotides are another important application of solid-phase oligonucleotide synthesis. It is known that natural oligonucleotides used as therapeutic strategy against viral infections as antisense for targeting RNA sequences may undergo enzymatic hydrolysis by endonucleases. Series of modiﬁed oligonucleotides carrying the modiﬁcation either on the base, sugar, or phosphate moiety

3.4. Oligonucleotide Synthesis B2

B1

B2

OH

O

OTr

O

O

OTr

O

173

O

P

H

+

B1

O

O

P

O

O

H

O

O

B2 TrO

O

O

O

B1 O

O

O P

O

H

FIGURE 3.39. The phosphonate method.

provides ideally endonuclease resistance as well as high afﬁnity for complementary RNA sequences. Phosphodiester bond is the primary target for endonuclease breakage. Therefore, the effort has been focused mainly on the modiﬁcation of this segment of the chain. As a result of this, a ﬁrst generation of modiﬁed phosphorous oligonucleotides such as phosphothioates, methylphosphonates, phosphoramidates, phosphotriesters, and phosphodithioates was synthesized. Although these phosphorous derivatives showed increased resistance to endonuclease activity, the afﬁnity for complementary sequences was decreased.53-55 For instance, the

RO

B2

O

X

W

Y

Z

W

O

Y

Z

O

-P(O)S-

O

CH2

O

-P(O)CH3-

O

CH2

O

CH2

O

B1

X

-P(O)NHR-

O

-P(O)OR-

O

CH2

NH

-P(O)O-

O

CH2

OR

FIGURE 3.40. Modiﬁed oligonucleotides.

174

3. N-Glycosides O NH

O NH N

TrO

N

TrO

O

O

O O

NH O

O

N

SCN

O

i

NH

HN S

O

+

N

HN H2N

O

O

N3 N3

O

O

NH

NH O

N

TrO

O

N

TrO

O

O

O

O NH

HN

NH2

S O

N

HN

NH

HN ii,iii

N

HN

O

O

O

O

O NH

HN

NH2

S N

HN

NH

HN O

N

HN

N3

O

O

O

N3

i) DMF. ii) H2S. iii) 3´-azido-5´-isothiocyano-3´,5´-deoxythymidine. iv) a) TFA. b) H2NC(=NH)SO2H. c) NH4OH.

FIGURE 3.41. Preparation of guanidinium oligonucleotides.

synthesis of the antisense oligomer phosphorothioate analog of a 28-nucleotide homo-oligodeoxycytidine (S-dC28 ) was achieved, and tested as a potent inhibitor of HIV in vitro, showing signiﬁcant inhibition of reverse transcriptase activity and syncytium formation between HIV-1 producing cells and CD4+ .56

3.4. Oligonucleotide Synthesis

175

A second generation proposed the replacement of phosphodiester group by a bioisoster such as amides, urea, and carbamate (Figure 3.40). In general the observations reveal better enzymatic hydrolysis resistances, but again poor afﬁnity toward RNA complementary sequences. Alternatively, Dempcy et al.57 reported the synthesis of modiﬁed guanidinetimidine oligonucleotide following the procedure depicted in Figure 3.41. The reactions involved are the condensation between 3 -amino-5 -O-trityl-3 deoxythymidine and 3 -azido-5 -isothiocyano-3 ,5 -deoxythymidine, to generate 5 →3 thiourea-nucleoside dimer. Reduction followed by coupling reaction of dimer with the later nucleoside produced chain elongation reaction. Guanidine conversion was done with aminoiminosulfonic acid and ammonium hydroxide, affording guanidinium thymidyl pentamer. The unit assemble for oligoribonucleotide synthesis is to some extent similar to deoxyribonucleotides synthesis. However, an additional consideration should be taken into account, which is the suitable protection of position 2-OH of ribose. The use of the silyl protecting group is one of the best choices so far reported, in particular the hindered tert-butyldimethyl silyl (TBDS) group. The protection of tritylribonucleoside produced a mixture of isomers, being the 2-OH silyl derivative

TrO TrO

O

B

Si

B

O

TrO

O

B

Cl +

HO

O

HO

OH

Si

O Si

i

50-90 %

i

TrO

B O

NCH2CH2C

O

O P

Si

Ni-Pr

FIGURE 3.42. Ribose protecting groups for oligoribonucleotide synthesis.

OH

176

HO

3. N-Glycosides

O

B i

i

B

O

O

i

Pr2Si

ii

O

B

Pr2Si O

O i Pr Si 2

OH OH

O

i

O

OH

Pr2Si

O

O

OPh O

B = heterocyclic base HO

B O

iii,iv

OH i) (iPr)2SiCl O, Py. ii) ClC(S)OPh, DMAP, MeCN. iii) Bu3SnH, AlBN, PhCH3. 2 iv) Bu4NF, THF.

FIGURE 3.43. The Barton-McCombie procedure for the preparation of 2 deoxynucleosides.

generated in between 50–90% yield. Final removal of this protecting group is usually achieved with 1 M tetrabutylammonium ﬂuoride in THF (Figure 3.42). Some other choices for 2-OH protection are tertahydropyran-1-il, 4methoxytetrahydropyran-4-il, and modiﬁed ketal of 1-(2-ﬂuorophenyl)-4methoxypiperidin-4-il (Fpmp). However, it has been found that acid conditions for removal of these protecting groups are not compatible with the trityl protecting group. Simultaneous protection of positions 3 and 5 can be achieved by using the silyl protecting group tetraisopropyldisiloxychloride (TIPS-Cl) in pyridine. This type of protection has been useful in the conversion of adenosine to 2 -deoxyadenosine under the conditions reported by Barton and McCombie58 (Figure 3.43).

References 1. S. Nishimura, Progress Research Molecular Biology 12, 49 (1972). 2. T. Kondo, T. Ohgi, and T. Goto, Agric. Biol. Chem. 41, 1501 (1977). 3. H. Akimoto, E. Imayima, T. Hitaka, H. Nomura, and S. Nishimura, J. Chem Soc Perkin Trans. I 1637 (1988). 4. C.J. Barnett and L.M. Grubb, Tetrahedron 56, 9221 (2000). 5. T. Itaya, Chem. Pharm. Bull. 38, 2656 (1990). 6. (a) S. Knapp, Chem. Rev. 95, 1859 (1995). (b) M.T. Migawa, L.M. Risen, R.H. Griffey, and E.E. Swayze Org. Lett. 7, 3429 (2005). 7. N. Chida, K. Koizumi, Y. Kitada, C. Yokohama, and S. Ogawa, J. Chem. Soc. Chem. Comm. 11 (1994). 8. M.P. Maguire, P.L. Feldman, and H. Rapoport, J. Org. Chem. 55, 948 (1990). 9. L. Kalvoda, M. Prystas, and F. Sorm, Collect. Czech. Chem. Commun. 41, 788 (1976). 10. A.G. Myers, D.Y. Gin, and D.H. Rogers, J. Am. Chem. Soc. 116, 4697 (1994).

References

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11. H. Hahn, H. Heitsch, R. Rathmann, G. Zimmermann, C. Bormann, H. Zahner, and W. Konig, Liebigs Ann. Chem. 803 (1987). 12. S. Hanessian, J. Kloss, and T. Sugawara, J. Am. Chem. Soc. 108, 2758 (1986). 13. N. Ikemoto and S.L. Schreiber, J. Am. Chem. Soc. 114, 2524 (1992). 14. S. Knapp and S.R. Nandan, J. Org. Chem. 59, 281 (1994). 15. G.M. Blackburn and M. Gait, Nucleic Acids in Chemistry and Biology, IRL 77 (1990). 16. C.-H. Wong, S.T. Chen, W.J. Hennen, J.A. Bibbs, Y-F. Wang, J.L-C. Liu, M.W. Pantoliano, M. Whitlo, and P.N. Bryan, J. Am. Chem. Soc. 112, 945 (1990). 17. A. De la Cruz, J. Elguero, V. Gotor, P. Goya, A. Mart´ınez, and F. Moris, Synth. Commun. 21, 1477 (1991). V. Gotor, and F. Mor´ıs, Synthesis 626 (1992). 18. S. Ozaki, K. Yamashita, T. Konishi, T. Maekawa, M. Eshima, A. Uemura, and L. Ling, Nucleosides Nucleotides 14, 401 (1995). 19. H.K. Singh, G.L. Cote, and R.S. Sikirski, Tetrahedron Lett. 34, 5201 (1993). 20. D.L. Damkjaer, M. Petersen, and J. Wengel, Nucleosides Nucleotides 13, 1801 (1994). 21. Kawana et al., Chem Lett. 1541 (1981). 22. S. Kozai, T. Fuzikawa, K. Harumoto, T. Maruyama Nucleosides, Nucleotides & Nucleic Acids 22, 779 (2003). 23. V. Serebryany and L. Beigelman, Tetrahedron Lett. 43, 1983 (2002). 24. Taniguchi et al., Angew. Chem. Int. Ed. 37, 1136 (1988). 25. M.J. Robins, V. Samano, and M.D. Johnson, J. Org. Chem. 55, 410 (1990). 26. T. Kondo, T. Ohgi, and T. Goto, Chemistry Lett. 419 (1983). 27. B.K. Battacharya, T.S. Rao, and G.R. Revankar, J. Chem. Soc. 1543 (1995). 28. F. Seela, H. Steker,. H. Driller, and U. Bindig, Liebigs Ann. Chem.15 (1987). 29. F. Seela and A. Kehne, Liebigs Ann. Chem. 876 (1983). 30. Z. Kazimierczuk, H.B. Cottam, G.R. Revankar, and R.K. Robins, J. Am. Chem. Soc. 106, 6379 (1984). 31. E. Edstrom and Y. Wei, J. Org. Chem. 60, 5069 (1995). 32. J. Davoll, B. Lythgoe, and A.R. Todd, J. Chem. Soc. 967 (1948). 33. R. S. Tipson, J. Biol. Chem. 55, 130 (1939). 34. K.H. Jung, and R.R. Schmidt, Liebigs, Ann., Chem. 1013 (1988). 35. E. De Clercq, G. Gosselin, M.C. Bergogne, J. De Ruddes, and J.L. Imbach, J. Med. Chem. 30, 982 (1987). 36. G.E. Hilbert and T.B. Johnson, J. Am. Chem. Soc 52, 4489 (1930). 37. (a) H. Vorbr¨uggen, K. Krolikiewicz and B. Bennua, Chem. Ber. 114, 1234 (1981). (b) H. Vorbr¨uggen and G. H¨oﬂe, Chem. Ber. 114, 1256 (1981). 38. S. Maier, R. Preuss, and R.R. Schmidt, Liebigs Ann. Chem. 483 (1990). 39. O. Mitsunobu, Synthesis 1 (1981). 40. Marminon et al., J. Med. Chem. 46, 609 (2003). 41. R.F. Heck, Organic Rect. 27, 345 (1982). 42. N. Miyaura and A. Suzuki, Chem. Rev. 95, 2457 (1995). 43. W.J. Scott, G.T. Crisp, and J.K. Stille, J. Am. Chem. Soc. 106, 4630 (1984). 44. E.-I. Negishi, J. Organomet. Chem 653, 1 (2002). 45. K. Sonogashira, In Comprehensive Organic Chemistry, Trost, B. M., Fleming, I., Eds.; Pergamon Press: N.Y. 3, 521 (1991). 46. Y. Hatanaka and T. Hiyama, Synlett 845 (1991). 47. (a) B.M. Trost, and D.L. Van Vranken, Chem Rev. 96, 395 (1996). (b) Tsuji, J., Yamakawa, T., Tetrahedron Lett. 20, 613 (1979).

178

3. N-Glycosides

48. J.L. Ruth and D.E. Bergstrom, J. Org. Chem. 43, 2870 (1978). D.E. Bergstrom and J.L. Ruth, J. Am. Chem. Soc. 98, 1587 (1976). D.E. Bergstrom, J.L. Ruth, and P. Warwick, J. Org. Chem. 46, 1432 (1981). 49. L.A. Agrofolio, I. Gillaizeau, and Y. Saito, Chem. Rev. 103, 1877 (2003). 50. A. Gross, O. Abril, J.M. Lewis, S. Geresh, and G.M. Whitesides, J. Am. Chem. Soc. 105, 7428 (1983). 51. F. Eckstein, Oligonucleotides and Analog:- a Practical Approach, IRL (1991). 52. N.-S Li and J.A. Piccirilli, J. Org. Chem. 69, 4751 (2004). 53. A. De Mesmaeker, R. Haner, P. Martin, and H.E. Moser, Acc. Chem. Res. 28, 366 (1995). 54. M.J. Damha, P.A. Giannaris, P.A. Marfey, and L.S. Reid, Tetrahedon Lett. 32, 2573 (1991). 55. M. Sekine, H. Tsuruoka, S. Iimura, H. Kusuoku, and T. Wada,. J. Org. Chem. 61, 4087 (1996). 56. H. Mitsuya, R. Yarchoan, and S. Broder, Science 249, 1533 (1990). 57. R. Dempcy, K.A. Browne, and T.C. Bruice, J. Am. Chem. Soc. 117, 6140 (1995). 58. D.H.R. Barton and S.W. McCombie, J. Chem. Soc. Perkin Trans 1 1574 (1975).

4 Nucleoside Mimetics

Modiﬁed nucleosides are useful therapeutic agents being currently used as antitumor, antiviral, and antibiotic agents. Despite the fact that a signiﬁcant variety of modiﬁed nucleosides displays potent and selective action against the mentioned diseases, the challenge still attracts full attention since most of them do not discriminate between normal and tumor cell and in viral infection resistant strains usually appear during the course of the treatment. Synthetic acyclic, carbocyclic, C-nucleosides, and modiﬁed N-nucleosides have shown remarkable action against AIDS, hepatitis, and Herpes infections, among others. Some of the nucleosides used as approved drugs are acyclovir, carbovir being the treatment of choice against Herpes, AZT, ddI, ddC, ddG, Abacavir, which in combination with protease inhibitors is indicated in the treatment against HIV, and C-nucleoside Ribavirin in the treatment against hepatitis.1,2 Representative examples of chemotherapeutic agents modiﬁed at the heterocyclic base, the sugar fragment, L and Cnucleosides, carbocyclic and acyclic nucleosides are depicted in Figure 4.1. A signiﬁcant number of synthetically modiﬁed nucleosides were designed as antiretroviral drugs in the therapy of human immunodeﬁciency virus (HIV) infection. During retroviral infection, the viral RNA is used as a template for proviral DNA synthesis, a process mediated by the viral DNA polymerase better known as reverse transcriptase. Thus, the process involves the initial formation of an RNADNA hybrid, which is then degraded by an RNAse to release the DNA strand that will be the template for the synthesis of the doublestranded viral DNA, a process also catalyzed by the reverse transcriptase.3 The proposed mechanism of action of modiﬁed agents such as AZT during viral infection involves the interruption of the viral replication process that occurs between the virus and host, particularly the replication inhibition inside T cells, monocytes, and macrophages. When the modiﬁed nucleoside is introduced into the cell, a sequential 5 phosphorylation process mediated by kinases occurs on the furanoside ring, which is subsequently incorporated into the DNA as triphosphate (Figure 4.2).

179

180

4. Nucleoside Mimetics O

O F N

NH

NH

N N

N

N

NH2

HO

O

HO O

OH

O

OH

OH 2-Deoxyfluorouridine

8-Azainosine

O O

O

NH NH N

NH

O N

O

N

HO O

HO

HO O

N3

N3

AZT

O

F

FIGURE 4.1. Representative synthetically modiﬁed nucleosides.

An important collection of active nucleosides mimetics has been synthesized and classiﬁed for better understanding as follows:4 Modiﬁed Nnucleosides Lnucleosides (Disomers) Cnucleosides Carbocyclic nucleosides Acyclic nucleosides Thionucleosides

4.1 Modiﬁed Nucleosides A broad number of modiﬁed nucleosides have been developed and tested on clinical trials, some being highly promising. The chemical manipulations have been made at the heterocyclic base, the sugar of both. Some representative examples of chemical modiﬁcations leading to key intermediates or active nucleosides are

O

4.1. Modiﬁed Nucleosides NH2

181

NH2 N

N N

N

N

O

NH2

N

HO

HO

O

S O

HN O Thiazolidinone

DAPD

NH2

O

N

NH N

NH2

N

O

N O

N

HO

HO

HO O

O

O

O

O NH2 S

HN

NH

N

O HO

HO

O

O OH OH

OH

OH Pseudourdine

Tiazofurin

FIGURE 4.1. (Continued)

4.1.1 Heterocycle Modiﬁcations 4.1.1.1

C-5 Substituted Pyrimidines

Several nucleoside analogs bearing modiﬁcations at the 5-position have been found to be active as antiviral and anticancer drugs. Examples of this are BVDU, IDU, and FIAU (Figure 4.3) 5

O

182

4. Nucleoside Mimetics O NH2 H3C

F

NH N N N

O

O HO

HO O

F

O S

OH L-FMAU

L-FTC

NH2 N N

O N

N

N

NH

N

N

HO

O

N

N

HO O

ddA

ddI

O

ddG

O

N

NH2

HO

HN

N

NH N

NH2

N

HO

O

N

NH

N N

N

N

NH2

N

HO

N

N

H2N HO

OH Carbovir

OH Abacavir

Neplanocin A

O N N HO

O N

NH N

NH2

N HO

O

O

HO DHPG

Aciclovir

FIGURE 4.1. (Continued)

NH N

NH2

4.1. Modiﬁed Nucleosides O

183

O NH

N

NH O

HO

N

i

P

O

O

ii

O

O

N3

N3 O O NH NH N N

P

P

O

O

P

P

P

O

O

ii

O

O N3

N3 i) Timidinkinase. ii) Timidilatokinase. iii) Nucleosidediphosphatekinase.

FIGURE 4.2. Phosphorylation of AZT.

Palladium-mediated transformations is a suitable strategy for introducing substituents at C-5. Some of the reactions implemented for this purposes are the Sonogashira,6,7 Stille,8,9 Heck,10 and Hiyama11 (Figure 4.4). 4.1.1.2

C-6 Substituted Pyrimidines

By following palladium-mediated substitutions, a more limited number of C-6 substituted pyrimidines have been described in comparison with C-5. For instance, applying the Stille reaction allows one to prepare C-6 substituted aryl, vinyl, alkynyl derivatives (Figure 4.5). 12 O

O

O

I

Br

HO

N O

I

NH

NH O

HO

N O

NH O

HO

N

O

O F

OH BVDU

OH

OH IDU

FIGURE 4.3. Active C5-substituted pyrimidines.

FIAU

184

4. Nucleoside Mimetics O

O

R

TfO

NH

NH H N

AcO

R AcO

O

O

i

N

O

O

OAc OAc

OAc OAc

i) Pd(PPh3)4, 10%, CuI, 20%, Et3N 1.2 eq./DMF.

O

O

R

I

NH

NH H N

HO

R HO

O

O

i

N

O

O

OH

OH i) Pd(PPh3)4, CuI, 20%, iPrEtN 40-60%.

O

O I

CO2R

NH HO

N

HO

O

O

RO2C

i

OH

NH N

O

O

OH

i) Pd(OAc)2, PPh3, Et3N, dioxan, 40%.

FIGURE 4.4. Palladium-mediated substitutions at C-5 pyrimidine position.

4.1.1.3

Purine Formation

The conventional methods of preparation of C-C purines are based on heterocyclization.13,14 The classical procedures involve.

4.1. Modiﬁed Nucleosides

N

O

O

i

N

MOMO

O

O

G=

SnBu3

N

O

ii

MOMO

H Ph SiMe3

O

O

G

O

O

O

O

HN

HN

HN

MOMO

O

O

O

185

O

CH 2CH=CMe2

CH=C=CH2

N SEM

i) LDA, then Bu3SnCl, 98%, G-X (Pd), CuI, DMF, 60-90%.

FIGURE 4.5. Palladium-mediated substitution of 6-C substituted pyrimidines.

1. 2-C-Cpurines cyclization of 4-aminoimidazole-5-carboxamides or nitriles with carboxylic acid equivalents. 2. 8-C-Cpurines from 5,6-diaminopyrimidines and carboxylic acid derivatives; and for 6-C-C-purines from 4-alkyl or 4-aryl-substituted 5,6-diaminopyrimidines (Figure 4.6).15 Other explored methods involves radical16,17 or nucleophilic substitution,18 sulfur extrusion,19 and Wittig-type reactions.20,21 Despite their usefulness, other methods based on the use of organometallic complex are getting particular significance especially in the synthesis of substituted purines (Figure 4.7).15 Usually the cross-coupling reactions involving organometallic compounds include organolithium,22 magnesium,23 aluminum,24 cuprates,25 zinc,26 stannanes,27 and boron28 reagents, in the presence of palladium catalyst, and the purine base bearing a good leaving group is usually halides or tosyl (Figure 4.8). Deazapurines are pyrrolo[2,3]pyrimidines of natural or synthetic source with signiﬁcant antitumor, antiviral, and antibacterial activities. Some compounds included in this class are tubercidin, toyocamycin, sangivamycin, and the hypermodiﬁed nucleoside queuosine. A ﬂexible route for the preparation of pyrrolo[2,3]pyrimidines (7-deazapurines) has been developed, consisting of the condensation of protected uracil with ethyl N-(p-nitrophenethyl)glycinate and subsequent treatment with acetic anhydride and amine base with heating to afford 5(-acetyloxy)pyrrolo[2,3-d]pyrimidine 2,4-dione in 74% yield (Figure 4.9).29

4.1.2 Sugar Modiﬁcations 4.1.2.1

2 3 -Dideoxysugars

A signiﬁcant number of saturated and unsaturated dideoxysugars have been synthesized and tested as antiviral or anticancer drugs. Remarkably, ddI and ddC are

186

4. Nucleoside Mimetics Y

Y N

N

X R

N

N

HN

X

N

N

X

N

Z

R N

NH2

N X

N

YCOX'

NH Z

R

X

+

NH2

N X

N

RCOX'

Y N

N

+

Z

Y

X

RCOX'

N

H2 N

Z

N

+

N Z

X

N

NH Z

R = C-substituents X, Y, Z = other substituents

FIGURE 4.6. Conventional methods of preparation of C-C purines.

approved drugs for the treatment of AIDS,3 and others such as d4T being currently under clinical studies (Figure 4.10).30,31 A method for preparing ddC was described involving bromoacetylation with HBr in acetic acid of N4-acetylcytidine followed by reductive elimination with zinc-cooper couple in acetic acid to provide the corresponding 2 3 -unsaturated derivative. Final hydrogenation over 10% palladium on charcoal gave ddC in 95% accompanied by some N-C cleavage in 5% (Figure 4.11).32 Similar reaction conditions were used for preparing 2 3 -dideoxyadenosine in 81% yield from adenosine.33 The design and synthesis of potent inhibitors for Human Hepatitis B Virus (HBV) 2 ,3 -dideoxy-2 3 -didehydro-β-L-cytidine (β-L-d4C) and 2 ,3 -dideoxy2 3 -didehydro-β-L-5-ﬂuorocytidine (β-L-Fd4C) nucleosides were carried out according to the pathway shown in Figure 4.12.34 The key starting material 3 ,5 dibenzoyl-2 -deoxy-β-L-uridine was submitted to transglycosilation reaction with silylated 5-ﬂuorouracil using TMSOTf as catalyst, affording an anomeric mixture separated by chromatography. After benzoyl deprotection, the anomeric nucleosides were treated with mesyl chloride followed by base to form cyclic ethers.

4.1. Modiﬁed Nucleosides Y

Y R-M Pd(Ni) cat.

N

N N

X

N Z

R

N

LG

N

Y

N Z

X

N

Y

N

N Z

N

N

Y X

N Z

Y R-M Pd(Ni) cat.

N

N

N

N

Y X

N Z

R R-M Pd(Ni) cat.

N

N

N

N

X GL

187

R X

N

N Z

LG = Cl, Br, I, OTs R = alkyl, alkenyl, alkynyl, aryl, hetaryl M = MgBr, AlR 2, (Cu), ZnCl, SnR'3, B(OH)2 X, Y, Z = diverse substituents

FIGURE 4.7. General ﬁgure between purines and organometallic compounds.

Further transformation at the pyrimidine ring was followed by potassium tertbutoxide treatment to furnish β-L-d4C and β-L-Fd4C. Other methods designed for the preparation of 2 3 -unsaturated and saturated deoxyfuranosides are based on (a) the Corey-Winter reaction involving cyclic thionocarbonate,,35,37 (b) the Eastwood oleﬁnation process in which a ﬁvemembered cyclic orthoformate suffer a fragmentation to give in the presence of acetic anhydride the desired oleﬁn (successfully applied in the preparation of ddU),38,39 and (c) the Barton deoxygenation involving the cyclic thionocarbonate or the bisxantate, and then treated with tributyltin hydride,40,41 or alternatively diphenylsilane42 (Figure 4.13). The synthesis of modiﬁed nucleosides from natural nucleosides is another useful alternative for preparing pharmaceutically active dideoxy nucleosides.The potent antiviral inhibitors ddC, ddG, d4C, and d4G have been obtained from the corresponding protected natural nucleosides, as shown in Figure 4.14.43 The chemoenzymatic approach has been also explored for the synthesis of 2 ,3 dideoxynucleosides. Such is the case of the antiviral 2 ,3 -dideoxyguanosine, which was synthesized from guanosine in 40% overall yield using as a key step the commercially available mammalian adenosine deaminase (ADA) (Figure 4.15).44

188

4. Nucleoside Mimetics R' I

R

Li N

N

i

N

N

N THP

N

ii

OH N

N

N THP

N

N THP

N

i) BuLi, –130 oC. ii) RCOR'.

I

ZnI N

N

i

N

N

N Y

N

R ii

N

N

N Y

N

N Y

N

R = alkenyl, aryl Y = protected sugar i) Zn, THF. ii) R-X, Pd. cat.

Cl

Cl

Cl N

N

i

N Y

N

N

N Bu3Sn

ii

N Y

N

N

N R

N Y

N

i) a) LMPT. b) Bu3SnCl. ii) R-X, Pd, cat..

FIGURE 4.8. Cross-coupling reactions for purine modiﬁcation. O OAc

CO2Na

O

MOM MOM

N

i,ii

HN

N

+

N BOM

O O

Cl

N BOM

PNPE R = PNPE

iii R=H i) EtOH/H2O, ∆. ii) Ac2O, n-Pr3N, 100 C. iii) DBU, CH3CN, 25 C 81% o

o

FIGURE 4.9. Synthesis of 7-deazapurine analogs.

N R

4.1. Modiﬁed Nucleosides O

NH2

N

NH

N

HO

O

N

N

O

189

N

HO

NH O

ddI

O

N

HO

O

O

ddC

d4T

FIGURE 4.10. Anti-AIDS 2 3 -dideoxy nucleosides.

A strategy for preparing D- and L-2 -ﬂuoro-2 3 -unsaturated nucleosides has been described and their anti-HIV activity evaluated. This approach requires 1acetyl-5-O-benzoyl-2,3-dideoxy-3,3-diﬂuoro–D-ribofuranose as key starting material, which was condensed under V¨orbruggen’s conditions with purines and pyrimidines to provide the corresponding nucleosides. The resulting nucleosides were subjected to β-elimination to generate the ﬂuoro unsaturated nucleosides (Figure 4.16).45

HO

O

HO

NHAc

NHAc

NHAc

N

N

N

N

RO

i

O

OH

O

AcO

N

RO

O

O

+

Br

Br

N

O

OAc

R = Me2C(OAc)CO

NHAc

NH 2

N ii

RO

O

N

N O

iii,iv

HO

O

ddC i) Me2C(OAc)COBr. ii) Zn-Cu/AcOH. iii) H 2, 10% Pd-C. iv) Triton B.

FIGURE 4.11. Synthesis of anti-AIDS ddC.

N

O

190

4. Nucleoside Mimetics O

F

F

NH

NH

N O

N

BzO

O

OSiMe3

N

O

N

BzO

OSiMe3

O

O

i

OBz

OBz O

O R

N

R

NH

NH

BzO

O

R

ii

O

N

HO

NH O

O

O

N

iii,iv

O

O O

OH

R=H R=F

OBz

NH2

NH 2 R

R

N

N vii

v,vi

N O

O

N HO

O

O

O i) CF 3SO3 SiMe3 , CH3 CN. ii) NH 3 /MeOH. iii) MsCl, Py. iv) 1N NaOH, EtOH/H2 O. v) 1,2,4-triazole, p-ClC6 H 4 OPOCl2 , Py. vi) NH 4OH, dioxane. vii) t-BuOK, DMSO.

FIGURE 4.12. Synthesis of anti-hepatitis B virus β-L-d4C and β-L-Fd4C.

4.1.2.2

2 Deoxynucleosides

The Barton deoxygenation provides another useful method for preparing 2 - and 3 -deoxynucleosides (obtained as a mixture) and involves as a key step the hydride reduction of the cyclic thionocarbonate with tributyltin hydride.42 On the other hand, 2 -monotosylate nucleoside, when treated with excess of lithium triethylborohydride, produces the 2 -deoxy-3 β-hydroxy nucleoside in high yield (Figure 4.17).46 2 -deoxynucleosides have been obtained from starting materials of different composition such as α,β-unsaturated aldehydes47 chiral epoxy alcohols,48 butenolides,49,50 and polyfunctionalized acetals, among others.51

4.1. Modiﬁed Nucleosides FIGURE 4.13. Alternative procedures for preparing 2 3 -unsaturated nucleosides.

B

HO

O

B

HO

O

191

O

O S

i) P(OR)3 Corey-Winter reaction

HO

B

O

O

i

AcO

O

B

O OMe

i) Ac2O Eastwood olefination

RO

O

B i

O

MeS

O

B

SMe

O

S

RO

S

i) Bu3SnH or Ph2SiH2 Barton Deoxygenation

The remarkable 2 -deoxynucleoside AZT widely prescribed as an anti-AIDS drug was originally prepared from thymidine by Horwitz and coworkers,52 and since then, several other syntheses have been developed, some of them starting with either a nucleoside or a sugar derivative,5356 and others relaying on the use of noncarbohydrate starting materials.56,57 The procedure developed by Chu et al.50 consisted of the use of mannitol as staring material, which was subsequently transformed to provide the protected key intermediate 3 azide-2 deoxyribofuranose. The next step involved the coupling reaction with silylated thymine under V¨orbruggen’s conditions to produce an anomeric mixture of nucleosides in 66%. Final desilylation and separation by chromatography column provided AZT in overall yield of 25% from the furanoside intermediate (Figure 4.18).

192

RO

4. Nucleoside Mimetics

B

O

RO i

B=

HO

OH

O

i-PrOCHN

N

iii

RO

O

B

RO

O

B

RO v

iv

O

O HO

N

HN

N N

O

O

NH2

N

d4C

B

vi

NH2

O

O

O S

HO

N

N

O

ii

O

N

HN

N

O

OCSCH3

H3CSCO

O

NHAc

B

O

N

HO O

d2C

N

N

H2N

N

HN H2N

N

N HO

O

d4G

O

d2G

i) NaOH, CS2/CH3I. ii) (Im)2C=S. iii) Bu3SnH. iv) (EtO)3P. v) H2, Pd-C. vi) NH3/MeOH.

FIGURE 4.14. Antiviral modiﬁed nucleosides from natural sources.

Another possibility was described by Hager and Liotta involving the coupling reaction between the azido diol intermediate and silylated thymine under V¨orbruggen conditions to yield a diastereomeric mixture of azido diol nucleoside. Finally, when exposed to concentrated acidic conditions, the open form is converted into the βanomer of AZT in 67% yield (Figure 4.19).57 Transglycosidic reaction mediated by a deoxyribosyl transferase obtained from E.coli has been used in the synthesis of 3 -azido-2 ,3 -dideoxyguanosine. The enzymatic reaction occurs between AZT, which acts as glycosyl donor, with substituted 2-amino-6-purines to generate the desired purine nucleoside and thymine as byproduct (Figure 4.20).58 4.1.2.3

3 -Deoxynucleosides

These deoxynucleosides may be readily prepared from 3 -O-tosylate via a [1,2]hydride shift from C3 to C2 position with accompanying inversion of the C2 center

4.1. Modiﬁed Nucleosides Cl

O N

N HO

N

NH N

N

NH2

N N

HO O

O

OH

193

OH

OH

OH

O N

N HO

NH N

NH2

O

i) adnosine deaminase, phosphate buffer, pH 6.5.

FIGURE 4.15. Chemoenzymatic synthesis of 2 ,3 -dideoxyguanosine.

affording a 3 -ketone that was stereoselectively reduced by the hydride to produce 3 -deoxynucleoside (Figure 4.21).46,2 Also, 3 -deoxyguanosine was synthesized by an enzymatic transglycosylation of 2,6-diaminopurine using 3 -deoxycytidine as a donor of the sugar moiety. The diaminopurine nucleoside was transformed to 3 -deoxyguanosine by the action of adenosine deaminase (Figure 4.22).59 Lodenosine [9-(2,3-dideoxy-2-ﬂuoro-β-D-threo-pentofuranosyl)] adenine (FddA) is a reverse transcriptase inhibitor with activity against HIV. This purine analog was evaluated as one of the most selective inhibitors in a series of 2 3 -dideoxyadenosines, although less active than ddA. An efﬁcient method was developed starting from chloropurine riboside, which was tritylated and selectively benzoylated at 3 -position. Before ﬂuorination the 2 -hydroxyl group was converted to imidazolesulfonate or triﬂuoromethanesulfonate. Fluorination proceeds smoothly with 6 equiv. of Et3 N.3 HF at reﬂux in 88% yield. Simultaneous 6-amination and 3 -debenzoylation was done with ammonia in high yield. Elimination of the 3 -hydroxy group was carried out under the Barton-McCombie procedure involving the formation of the 3 -O-thiocarbonyl followed by silane treatment. Final removal of trityl group afforded FddA (Figure 4.23).60 4.1.2.4

4 -Substituted Nucleosides

4 -Substituted nucleosides have attracted much attention because of the discovery of potent anti-HIV agents 4 -azido- and 4 -cyano thymidine (Figure 4.24).

NH2

194

4. Nucleoside Mimetics BzO

O

OAc

F F

a

Cl

NHBz

O X

X

N

N

N

NH

BzO

O

O

O

X = CH 3 X=H

F

X=H X=F

F

e,h

N

N

O

F

F

f,g,h

f,g,h

O

X NH2

O

N

X

X

N

HO O

O

N

HO

O

O

NH

N

N

HO

O

N

NH2

O F

F

N

N

N

NH

HO

F

N

O F

e,h

N

N

N

BzO

F

F

F

Cl N

N

O BzO

N

BzO

d

c

b

F

F

i) silylated thymine, TMSOTf, MeCN. b) silylated N4-Bz-cytosine derivatives, TMSOTf, MeCN. c) silylated 6-chloropurine, TMSOTf, MeCN. d) silylated 6-Cl-2-F-purine, TMSOTf, MeCN. e) NH3/MeOH, r.t. f) NH3/MeOH, 90 oC. g) HSCH2CH2OH, MeONa, MeOH, reflux. h) t-BuOK, THF.

FIGURE 4.16. Preparation of D- and L-2 -ﬂuoro-2 3 -unsaturated nucleosides.

One procedure involves the epoxidation of the exoglycal with dimethyldioxirane and ring opening of the resulting 4 ,5 -epoxynucleosides to produce with high stereoselectivity the 4 -C-branched nucleosides (Figure 4.25).61 Likewise, other 4 -substituted nucleosides such as 4 -C-Ethynyl-β-D-arabino and 4 -C-Ethynyl-2 -deoxy-β-D-ribopentofuranosyl pyrimidines have been reported by a different approach outlined in Figure 4.26.62

4.1. Modiﬁed Nucleosides NH2

NH2 N

N

N

HO

i

N

N

O

O

HO

N

N

N

HO

195

HO

OTs i) LiEt3 BH, DMSO/THF

FIGURE 4.17. The Barton deoxygenation for preparing 2 -deoxynucleosides.

4.1.3 Complex Nucleosides The hypermodiﬁed Q base Queuine found in tRNA of plants and animals has been strongly associated with tumor growth inhibition. Three different approaches for preparing queuine have been described,63,65 the more recent in 11 steps from ribose. Completion of the synthesis involved the condensation of bromo aldehyde intermediate with 2,3-diamino-6-hydroxypyrimidine to give the desired heterocyclic product in 45%. Final removal of protecting groups provided Q base (Figure 4.27). Capuramycin is a complex nucleoside antibiotic isolated from Streptomyces griseus 446-S3, which exhibits antibacterial activity against Streptococcus pneumoniae and Mycobacterium smegmatis ATCC 607. The total synthesis was reported by Knapp and Nandan66 consisting of the glycosylation reaction between the key thioglycoside donor and silylated pyrimidine to produce the corresponding L-talo-uridine. The next glycosidic coupling reaction was carried out with L-talo-uridine and imidate glycosyl donor under TMSOTf conditions to afford

HO

CHO 2-steps D-Mannitol

i

O

CO2Et O

O

iii

TBDMSO

O

O

ii

O

O

iv

TBDMSO

O

O

N3

FIGURE 4.18. Synthesis of AZT from mannitol.

O

v

196

4. Nucleoside Mimetics OTMS CH3

N TBDMSO

N

TMSO

TBDMSO

O

O

vi

OH

OAc vii

N3

N3

O

TBDMSO CH3

HN O TBDMSO

O

vii

N

N3

O

N

+

O NH H3 C O

N3 O CH3

HN N

O HO

HO

O

N3 +

N

O

O NH H3 C O

N3

i) Ph3P=CHCO2Et, MeOH, 0oC. ii) HCl dil. iii) t-Bu(Me)2SiCl. imidazole, DMF. iv) LiN3, THF, AcOH, H2O. v) DIBAL, CH2Cl2, -78oC. vi) Ac2O, Py. vii) TMS-triflate, ClCH2CH2Cl. viii) n-Bu4NF, THF.

FIGURE 4.18. (Continued).

the disaccharide nucleoside. Further transformations led to the target molecule (Figure 4.28). Capuramycin has been also chemically transformed in an attempt to extend the antibacterial spectrum. Thus, radical oxygenation gave unexpected lactone

4.1. Modiﬁed Nucleosides

197

OTMS CH3

N TMSO

OR

O CH3

HN

N

OH

BnO

OR

N

O

OH

b,c

OBn

N3

OBn

N3

R=H a R = Bz

O CH 3

HN N

O d

HO

O

N3 i) PhCOCl (2.2 equiv.), NEt3 , DMAP, CH 2Cl2 . b) (CH 3) 3SiOTf, ClCH2 CH 2Cl. c) NaOH (2 equiv.), MeOH. b) 4.7 N H2 SO 4 in MeOH.

FIGURE 4.19. Synthesis of AZT from azido diol intermediate.

O

O

HN

HO

O

N

HN N

O

O

O

i

+ H2N

N

N3

N H

HN

+

N

HN

N

N

H2N HO

O

O

N3

i) glycosyltransferases, pH 6.0, 50oC.

FIGURE 4.20. Enzymatic synthesis of 3 -azido-2 ,3 -dideoxyguanosine.

N H

198

4. Nucleoside Mimetics NH2

NH2

HO

HO

O

N

N

N

N

N

N

N

N

N

N

NH2

N

N HO

O

LiEt3BH

O DMSO/THF

H OH

TsO

OTs O

O

H

NH2 N

N

N

N HO

O

OH

FIGURE 4.21. Method for preparation of -3 -deoxynucleoside.

in moderate yield via an intramolecular radical ArC glycosylationlactonization reaction (Figure 4.29).67 Synthestic studies of unique class Tunicamycin antibiotics leading to the preparation of (+)Tunicaminyluracil, (+)Tunicamycin-V, and 5 -epiTunicamacyn-V were described by Myers et al.68 The key features are the development and application of a silicon-mediated reductive coupling of aldehydes, the allylic alcohols to construct the undecose core of the natural product, and the development of an NH2

NH2 N

O N

NH

NH

N HO

N O

OH

O

HO i

N O

N

NH2 ii

HO

N

N

O

OH

OH

i) 2,6-diaminopurine, E. coli BM-11 and BMT-4D/1A, K-phosphate buffer, 52o C, 26 h, 64%. ii) Adenosine deaminase (ADase), r.t., 16 h, 68%.

FIGURE 4.22. Enzymatic synthesis of 3 -deoxyguanoside.

NH2

4.1. Modiﬁed Nucleosides Cl

Cl N

N

Cl N

N

ii

N

N TrO

HO

TrO

OH OH

Cl N

iii

N

N

N

TrO

TrO

N

N

N

N

O

N

N v

iv

N

O

OBz OH NH2

Cl

N

N

N

O

O

OH OH

N

N

i

N

N

199

TrO

O

O

F

F OBz OR

OH

OBz

R = SO 2-Imid R= SO 2CF3

N

N

N

N

vi

TrO

NH2

NH 2

NH2

O

N

N

N

N

vii

TrO

F

O

N

N

N

N

viii

HO

F

O F

OCSOPh i) TrCl-iPrNH, DMF, 79%. ii) a) BzCl-Py, toluene. b) cat. Et3N, toluene, 70%. iii) a) SO 2Cl2-Py, CH 2Cl2, b) imidazole. or CF3SO2Cl, DMAP, toluene. iv) Et3.3HF, Et3N, 70 and 78%. v) NH 3-MeOH, toluene 98%. vi) ClC(S)(OPh), DMAP, CH3CN, 92%. vii) Ph2SiH2, AlBN, dioxane, 81%. viii) 80% AcOH, 100oC, 85%.

FIGURE 4.23. Preparation of antiviral 2 3 -ﬂuoro dideoxyadenosine FddA.

efﬁcient procedure for the synthesis of the tetrahalose glycosidic bond within the antibiotic (Figure 4.30). 4.1.3.1

Fused Heterocyclic Nucleosides

Selective and potent antiVaricella Zoster Virus (VZV) bicyclic furanopyrimidine deoxynucleosides were synthesized. The bicyclic formation was performed by

200

4. Nucleoside Mimetics FIGURE 4.24. Structure of potent anti-HIV -4 -substituted nucleosides.

O NH N

HO

O

O

R OH R = N3, CN

palladium-catalyzed coupling of aryl acetylenes with 5-iodo-2 -deoxyridine affording the desired fused furanenucleoside (Figure 4.31).69 Triciribine is a tricyclic nucleoside with antineoplasic and antiviral properties, synthesized in an improved fashion from 6-Bromo-5-cyanopyrrolo [2,3-d] pyrimidin-4-one intermediate. Series of transformations including Nglycoside coupling reaction afforded 4-amino-5-cyano-7-[2,3,5-tri-O-benzoyl-βD-ribofuranosyl] pyrrolo [2,3-d] pyrimidine that was then converted to the desired tricyclic nucleoside (Figure 4.32).70

4.2 C-Nucleosides These modiﬁed nucleosides are structurally distinct to their counterparts Nnucleosides because of the presence of a C-C linkage instead of C-N between O

O

NH

NH N O

O

O

i

N O

NH

NH O

N

ii

O HO

TBDMSO

O

O

TBDMSO

TBDMS = tBuMe2Si

O

HO

N O

+

TBDMSO 64%

i) DMDO, CH 2Cl2, –30oC. ii) Me 3Al (3 eq.), CH 2Cl2, –30 oC, 2h.

FIGURE 4.25. Ring opening of 4 ,5 -epoxynucleosides.

TBDMSO 5%

O

4.2. C-Nucleosides BnO

BnO

O

BnO

O

i

HOH2C

BnO

O

ii

OHC

O

O

iii

Br2C

O

OBn O

201

C H

OBn O

HC

O

C

O OBn O

OBn O

O BnO

BnO

O

iv

O

OAc

v

Et3SiC C

HC

O

NH

vi

C

OBn O

BnO

N

BnO

OAc

O

O

HC C BnO

OAc

i) (COCl)2, DMSO, Et3N, CH2Cl2. ii) CBr4, PPh3, CH2Cl2. iii) n-BuLi, THF. iv) n-BuLi, THF, then Et3SiCl. v) a) 70% AcOH, TFA. b) Ac 2O, Py. vi) N,O-bis (trimethylsilyl)acetamide, thymine, CH2Cl2, reflux, 1h, 96%.

FIGURE 4.26. Synthesis of 4 -C-Ethynyl-β-Darabino and 4 -C-Ethynyl-2 -deoxy-βDribopentofuranosyl pyrimidines. Ns N

TBSO

i

O

Br O

iii

O

ii

O

O

Ns N

O

Ns N

HO

O

Ns N

iv

O

O

O

Ns O

H N

O

N v

HN O H2 N

N

N H

HN

O

OH H2 N

N

OH

N H

i) TBAF, THF, 87%. ii) TEMPO, NaOCl, KBr, CH 2Cl2, 88%. iii) TMSBr, DMSO, MeCN. iv) NaOAc, H2O/MeCN, 45%. v) a) HSCH2CH 2OH, DBU, DMF, 46%. b) HCl, MeOH, 84%.

FIGURE 4.27. Synthesis of hypermodiﬁed base Queuine.

202

4. Nucleoside Mimetics OTMS

O

N

BnO O

BnO

OTMS

N

SPh

NH N

AcO

O

O i

MeO

RO

OPiv

ii

R = Ac R=H

MeO

BnO AcO AcO

O OAc O

OAc O

AcO AcO

CCl3

O

O

O

BnO

OPiv

NH OBn

NH

O

O

N

O

iii

MeO

OPiv

OH OH

HN

O CONH 2

O H N

O O

N

NH

O

O

O MeO

OH

i) NIS, TfOH, CH 2Cl2, –20o C. ii) NaOMe, MeOH, 77%. iii) TMS-OTf, CH 2 Cl2, –25oC, 16 h, 85%.

FIGURE 4.28. Synthesis of Capuramycin.

the furanoside and the heterocyclic aglycon. Their source could be either naturally occurring (pyrazomycin, showdomycin, formycin) or synthetic (thiazofurin), having in either case signiﬁcant antitumor and antiviral activity. Also, some of them have been found in tRNA codons (pseudouridine) and others (tiazofurine and oxazofurine) designed as competitive inhibitors of cofactor nicotin adenin dinucleotide (Figure 4.33). An early approximation for the preparation of C-nucleosides proposed two basic possibilities depending on the nature of the atoms surrounding the C-C bond (Figure 4.34).71 1. If there is one heteroatom adjacent to the C-glycosidic bond, for example, tiazofurine, formicine, pyramicine.

4.2. C-Nucleosides

203

OR 3 OR 2

O CONH 2

O H N

HN

O O

N

NH

O

O

O MeO

OR 1

capuramycin R 1 = R 2 = R 3 = H i

R1 = R3 = TBDMS, R 2 = H

ii

R1 = R3 = TBDMS, R2 = C(S)OPh R1 = R3 = TBDMS, R2 = C(S)O-p-Tol iii

O O

Ph

O OR

O CONH 2

H N

O

HN

O

N

NH

O

O

O MeO

OR

R = TBDMS i) TBDMS-Cl, pyridine. ii) C 6H 5OC(S)Cl, DMAP, CH2 Cl2. iii) Bu3SnH, AlBN, PhMe, reflux

FIGURE 4.29. Chemical transformations of capuramycin

2. If there is no heteroatom adjacent to the C-glycosidic bond. Alternatively, other authors considers three general pathways for preparing Cnucleosides depending on the precursor employed as starting material.72 An early synthesis of modiﬁed C-nucleoside from naturally occurring pseudouridine was carried out via ring opening with ozone to generate an intermediate,

204

4. Nucleoside Mimetics O CH3

H 3C

Si O

BOMO

TBSO O O

NBoc

SePh H

O

O

N

O

CbzHN O AcHN

O CH2 TBSO

OTBS

O

i

O OH

BOMO CbzHN

OH

O

O TBSO O O

NPMB

H

O

N

O

AcHN O HO

OH

O OH

HO H2 N O

H O

O

AcHN HO HO HO

NH OH N

O

O HO

OH

i) triethyborate, Bu3 SnH, toluene, 0o C. 2 h. b) KF.H2 O, MeOH. 60%.

FIGURE 4.30. Key step for the synthesis of Tunicamycin antibiotic.

which was treated with thiosemicarbazone to afford 6-azathiopseudouridine. Treatment with iodomethane in acid medium produced the desired C-nucleoside as shown in Figure 4.35.73 The synthesis of the C-nucleoside pseudouridine was reported by Asburn and Binkley,74 involving the condensation between 5-O-acety-2,3-O-

4.2. C-Nucleosides

205 R

O I HN

H

O

R HN

HO

N

O

i

O

HO

N

O O

OH OH

R

O N

ii

HO

O

N O

OH i) Pd(PPh3)4, iPr2EtN, CuI, DMF, r.t, 19 h. ii) Et3N/MeOH. CuI, ∆ , 4h.

FIGURE 4.31. Synthesis of bicyclic furano pyrimidine.

isopropylidene-D-ribonolactone with 2,4-dibenzyloxypirimidin-5-il lithium to provide the condensation product, which was subjected to hydride reduction and hydrogenolysis to yield pseudouridine (Figure 4.36). Antitumor C-nucleoside Tiazofurine was synthesized by Robins et al.75 from 2,3,5-tri-O-benzoyl-β–Dribofuranosyl cyanide, which undergoes ring closure under conditions described in Figure 4.37. A new report for the synthesis of Tiazofurine is described, avoiding the use of H2 S gas, which is unsafe on large-scale production. The synthesis initiates with the preparation of 1-cyano-2,3-O-isopropylidene-5-O-benzoyl-β-Dribofuranose, which was reacted with cysteine ethyl ester hydrochloride to give thiazoline derivative in 90%. Further steps including oxidative aromatization under MnO2 in benzene and acetonide deprotection with iodide in methanol produced the desired C-nucleoside (Figure 4.38).76

206 NH2

4. Nucleoside Mimetics O

CN i

N

HN

N H

Cl

Br

N H

N

H 3C

CN

N iii

Br

v

Br

N

BzO

O

OBz OBz

H3C

NH2 CN

N

vi

O

OBz OBz

N

N

NH2

N

N

N

N

N

O

OBz OBz

BzO

CN

N

iv

N H

NH2

N

N

N

H3C

N

ii

N

BzO

CN

Br

Br N

Cl

CN

N

N HO

O

OH OH

i) NaNO2, AcOH, H2O, ii) POCl3. iii) BSA, CH3CN then 1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-ribofuranoside, TMSOTf. iv) NH2NHCH3, EtOH, CHCl3. v) HCO2NH4, 10% Pd-C, EtOH, reflux. vi) NaOMe, MeOH, reflux.

FIGURE 4.32. Synthesis of tricyclic nucleoside Triciribine.

Another biologically important C-nucleoside known as showdomicine was prepared by Trumnlitz and Moffat.77 The aldehyde used as starting material was converted ﬁrst to an α–hidroxyacid and then to α–ketoacid. Wittig reaction on this intermediate and Lewis acid catalysis produced ring closure (Figure 4.39). Pyrazine riboside derivative was synthesized by treatment of glycine riboside with formaldehyde and cyanide (Strecker conditions) to generate cyanide intermediate as a mixture of isomers. Sulfenylation and sodium methoxide treatment produce the C-nucleoside (Figure 4.40).78

4.2. C-Nucleosides O HN

O

O

O

O

O

O

NH2

NH

NH HO

HO

S

HO

OH

HO

tiazofurin

showdimicine

pseudouridine

O H N

NH2 H N

NH

N HO

N

O

OH

HO OH

HO

N

N N H

O

HO

O

HO

OH

N

O

HO

oxoformicine B

OH formicine

CO2H O

O

H2N

O OH

CO2H O

OH

MeO O

OH

NH

H 2N O

O

O

NH

H2N

207

O

HN

NH

O HN

NH

O

O Ezomycin B2

Malayamycin A

FIGURE 4.33. Biologically active C-nucleosides.

Analogs of antiviral C-nucleoside Formicine have been synthesized by using the palladium-mediated glycosidic reaction between the furanoid glycal and the iodinated heterocycle. Similar conditions were used for preparing the pyrimidine analogs (Figure 4.41).79 Radical cyclization of ribo-phenylselenoglycoside tethered with propargyl moieties on C-5 hydroxyl group afforded cyclic intermediates potentially useful for the

208

4. Nucleoside Mimetics NH

C HO

C

HO

O

O

C

HO

C

OH

a

HO

OH

b

O

O HN

FIGURE 4.34. C-nucleosides partial representations, with and without heteroatom attached to the Cglycosidic bond.

C

C

C

S H2N

NH

NH HN

O AcO

O

O

AcO

O

HO

i

O

HO

OAc

ii

NH

N HO

O

O

OH HO

OH

O HN iii

NH

N HO

O

O

HO

OH

i) O3. ii) NH2NHCNNH2=S. iii) MeI/H3O+

FIGURE 4.35. Preparation of 6azapseudouridine.

synthesis of C-nucleoside derivatives. Propargyl intermediate was prepared from ribo-phenylselenoglycoside via two-step sequence and then under radical reaction conditions (Bu3 SnH/AIBN) transformed to the cyclic intermediates in high yields. Further ring opening produced an aldehyde intermediate, which was subjected to a coupling reaction with 1,2-phenylenediamine to produce the pyrazine C-glycoside (Figure 4.42).80 Polyhalogenated quinoline C-nucleosides were synthesized as potential antiviral agents. The key step reaction for quinolin-2-one ring formation consisted in the condensation between 2-aminophenoneallose derivative with keteneylidene(triphenyl)-phosphorane in benzene under reﬂux to provide the desired 6,7-dichloroquinolin-2-one nucleoside in 50% yield (Figure 4.43).81

4.3. Carbocyclic Nucleosides

209

OBn OBn

HO

O

+ O

N

N

O N

N

OBn

HO

O

OBn

O

OH

Li O

O

O HN

NH

i,ii

O

HO

O

OH OH i) NaBH4. ii) a) H2, Pd-C. b) H3O+

FIGURE 4.36. Preparation of pseudouridine.

The Novel bicyclic C-nucleoside Malayamycin A from Streptomyces malaysiensis was elegantly synthesized from D-Ribonolactone, which was transformed to the target molecule according to the pathway indicated in Figure 4.44.82

4.3 Carbocyclic Nucleosides This class of modiﬁed nucleosides in which the furanose ring has been replaced by a cycloalcane ring (mainly cyclopentane) has been prepared by chemical or O NH2

NH2 BzO

CN O

OBz OBz

BzO i

C O

S

ii,iii

S

HO

N

O

OBz OBz HO

i) H2S, 4-DMAP. ii) ethylbromopyruvate. iii) NH3/MeOH.

FIGURE 4.37. Synthesis of tiazofurine.

OH

210

4. Nucleoside Mimetics O

EtO

BzO O

N

N

CN

S

S i

ii

BzO O

O

EtO

BzO O

O

O O

O

O

O

O

H2N N

S iii-iv

HO O

OH

OH

i) Cysteine ether ester hydrochloride/TEA. ii) MnO2/Ph, reflux. iii) 90% TFA. iv) MeOH/NH3

FIGURE 4.38. A new synthetic methodology for tiazofurine.

enzymatic methods. Besides their potent antitumor and antiviral activity for some of them, they have also shown high resistance to phosphorylases. The use of enzymes, particularly lipases, for protections and deprotections is an important strategy for preparing carbocyclic nucleosides. This approach has been advantageous especially for the resolution of enantiomeric forms, leading to high enantiomeric purity. Constrained three83 and four84 member ring carbocyclic nucleosides have been obtained by applying chemoenzymatic methodologies involving lipase for enantiomeric resolution and stereoselective deprotections. In the case of more abundant ﬁve-member rings, the use of lipases for enzymatic resolution and regioselective deprotections has been under intense study. Special attention has been paid to cyclopentenyl diacetates, which have been used as building blocks for the preparation of important carbocyclic nucleosides such as Neplanocin and Aristeromycin. To achieve this goal, the hydrolase enzyme acetylcholinesterase (EEAC)85 showed high efﬁciency for obtaining the desired enantiomer (1R,4S)-4-hydroxy-2-cyclopentenyl derivative in enantiomeric excess (ee) up to 96% (Figure 4.45).86,87 Racemic cyclopentenyl derivatives have been used as starting material in the preparation of the antiviral carboxyclic nucleoside (-)-5 -Deoxyaristeromycin. The

4.3. Carbocyclic Nucleosides CO2CH3 BzO

CHO O

BzO i,ii

OBz OBz

CO2CH3

CHOH O

C

BzO

iii

OBz OBz

O

O

OBz OBz O

CO2H CO2H

HO

211

O

iv

O

AcO

O

O

OH

HO

AcO

OAc

O NH v

HO

O

O

HO

OH

i) NaCN. ii) a) MeOH-H3O+. b) Me2SO-DCC. iii) Ph3P=CHCONH2. iv) Ac2O. v) a) NH3. b) EPP. v) H +.

FIGURE 4.39. Preparation of showdomicine.

key step reaction was the enzymatic resolution with Pseudomona sp lipase (PSL) of the racemic mixture affording the (+)-enantiomer, which was transformed chemically to the desired carbocyclic nucleoside (Figure 4.46). The separation of racemic carbocyclic nucleosides by enzymatic means has been reported as an alternative approach. Thus, racemic aristeromycin was treated with adenosine deaminase (ADA) to give (−)-carbocyclic inosine and pure destrorotatory enantiomer (Figure 4.47).88

4.3.1 Cyclopropane Carbocyclic Nucleosides Conformationally constrained cyclopropane nucleosides have been prepared following a chemoenzymatic approach.83 Thus, the racemic resolution of trans-1-(diethoxyphosphyl)diﬂuoromethyl-2-hydroxymethylcyclopropane followed by acetate hydrolysis was carried out with porcine pancreas lipase (PPL) to yield (+)- and (−)-cyclopropanes in high enantiomeric excess. Further

212

4. Nucleoside Mimetics CN O H2N

TBDMSO

HN

NH2

O

O

O

i

TBDMSO

O

NH2

O

O

ii

O

TBDMS = tBuMe2Si

NH2 NO2

CN NH

O S

iii

N

TBDMSO

NH2

O

O

N O

TBDMSO

O

O

O

O

i) CH 2O, HCN. ii) 2-NO2C6H4SCl. iii) NaOMe. MeOH.

FIGURE 4.40. Synthesis of C-nucleoside by pyrazine ring formation.

transformation led to the preparation of the target cyclopropane nucleoside (Figure 4.48).

4.3.2 Cyclobutane Carbocyclic Nucleosides Lubocavir is a synthetic potent inhibitor of DNA polymerase, active against cytomegalovirus89 (Figure 4.49). The carboxyclic four-membered C-nucleoside Cyclobut-A was prepared following the Barton decarboxylation method. The method is based on the reaction between carboxylic acids with heteroaromatic compounds (Figure 4.50).90 Other carbocyclic oxetanocin analogs have been prepared from oxetanocin A91 3,3-diethoxy-1,2-cyclobutanedicarboxylate-92 and enantiomeric cyclobutanone intermediates93 as starting materials.

4.3.3 Cyclopentane Carbocyclic Nucleosides The Mitsunobu reaction has become a valuable alternative approach for preparing cyclopentane carbocyclic nucleosides. This has been demonstrated in the preparation of conformationally locked carbocyclic AZT triphosphate analogs under this

4.3. Carbocyclic Nucleosides

213

OTHP OTHP HO

O

N

THP N

N

N i

N

N

+

THP N

HO N

O

I

t-Bu(Ph)2SiO

t-Bu(Ph)2SiO i) Pd(dba)2, AsPh3, MeCN.

OMe OMe

OMe N

N N

N

N

N

OMe TrO

O

OMe

I

TrO

+

OMe TrO

O

O

i

53 %

30 %

OMe N

N

ii

OMe TrO

O

i) Pd(OAc)2, NaOAc, n-Bu4NCl, Et3N, DMF. ii) H2, Pd/C, ammonium formate, ETOH.

FIGURE 4.41. Palladium-mediated synthesis of C-nucleoside formycin analogs.

versatile condition.94 The standard procedure usually takes place with diethyl or diisipropylazocarboxylate (DEAD or DIAD) with triphenylphosphine (Ph)3 P in THF to yield carbocyclic purines or pyrimidines nucleosides in high yield (Figure 4.51).95

214

4. Nucleoside Mimetics O R

O

HO

SePh

O

O

i,ii

R

O

SePh

iii

+

Br O

R = Me R = TMS

O

O O

O

O

O H HO

N

O

iv

R

v

HO

O N

O

O O

O

i) NaH, ii) n-BuLi, TMSI or MeI. iii) n-Bu3 SnH. iv) SeO2-AcOH, 1,4-Dioxane. v) a) O3. b) DMS. 3) 1,2-phenylenediamine.

FIGURE 4.42. C-nucleoside derivative formation via radical cyclization.

Another example on the applicability of this method was observed in the preparation of the carbocyclic thymidine nucleoside. It is worth mentioning that the desired stereochemistry of the hydroxyl group is obtained also through the Mitsunobu reaction (Figure 4.52).96

4.3.4 Palladium Mediated Based on the widespread palladium-coupling methodologies, several dideoxy, carbocyclic and C-nucleosides have been efﬁciently prepared. For instance, the Cl

NH2

O Cl

O

i,ii

Cl O

O

TBDMSO

H N

Cl

O

O

O HO

O

i) Ph3P=C=C=O, PhH, reflux. ii) TBAF, THF, rt.

FIGURE 4.43. Quinolin-2-one C-nucleoside formation via Wittig reaction.

4.3. Carbocyclic Nucleosides

215 OMe N

OMe

O

O

O

HO

OMe

O

O

a

MeO O

O HO

N

b

OH

OH

O

O

O

O

OMe N O

OMe

c

OH

OMe

O

O

N

OMe

OH N

N

MeO

d

O

O

OMe

O

O

OMe OMe

N

N N

MeO e,f,g

h

O O

O

O

HO OH Si

Si O

N

MeO

OPMB

O

O

OPMB

Si

Si

FIGURE 4.44. Total synthesis of C-nucleoside Malayamycin A.

antiviral C-nucleosides 2 -deoxyformycin B was prepared by condensation reaction between the heterocycle iodide intermediate with the glycal, under Pd(dba)2 as palladium catalyst in 62% yield (Figure 4.53).97 Solid-phase synthesis of carbovir analogs under palladium catalysis was recently reported.98 The carbocyclic derivative was linked to the Wang resin and then coupled with chloropurines under Pd(0) catalyst (Figure 4.54). The Tsuji-Trost approach was used to prepare (−)-neplanocin A and its analog.99 This synthesis proceeds via an allylic rearrangement of the hydroxyl group from the (+)-allylic alcohol to the (−)-allylic acetate (Figure 4.55).

216

4. Nucleoside Mimetics OMe

OMe

N

N N

MeO i,j

N

MeO

l

k

O

O

OPMB

OPMB

HO

O

Br

MeO

H

MeO

H HO

N

O

N

O

m

n

OMe

OMe

N

O H

O

H

OPMB

N3

MeO

H

N

O

OPMB

HO

N

O

MeO

H

N

O

o

OMe

OMe N

O H

H

OPMB

N3

MeO

H

MeO

N

O

OPMB

N3

MeO

H

O

N

O

N

O

q,r

p

OMe

OMe O

N

O H

N3

N H

OPMB

OPMB

MeO

O

s,t

N3

MeO

H

N

u

O

H

MeO

O

NH O

OMe N

O H

NH

O H

OPiv

FIGURE 4.44. (Continued)

OPiv

4.3. Carbocyclic Nucleosides

217

O H2 N

NH

O

H

MeO

O

v,w,x

NH O NH

O H

OH

a) 2,2-dimethoxypropane, Na 2SO4, PPTS, 94%. b) 2,4-dimethoxy-5-iodopyrimidine, t-BuLi, 75%. c) L-Selectride, ZnCl 2, DCM, 86%. d) DIAD, Ph3P, THF, 91%. e) 70% AcOH, 85%. f) 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, pyridine, 89%. g) NaH, PMBBr, DMF/THF, 84%. h) 1 N HCl, Dioxane, 88%. i) DMSO, (COCl)2, i-Pr2 NEt, DCM. j) Ph3PCH 3Br, NaHMDS, THF (36%, 2 steps). k) allyl bromide, NaH, DMF, 93%. l) Cl2 RuCHPh(PCy 3)2, DCM, reflux (89%). m) NBS, H 2O, THF. n) NaOH, THF. o) NaN 3, methoxyethanol (5:1, 41%, 3 steps for 14). p) Dess-Martin periodinate, DCM. q) NaBH 4, MeOH. r) NaH, MeI, DMF (93%, 3 steps). s) DDQ, H2 O, DCM (84%). t) Piv-Cl, DMAP, NEt3, pyridine. u) TMS-Cl, NaI, actonitrile (42%, 2 steps). v) PMe 3, H2 O, THF. w) trichloroacetylisocyanate, DCM. x) MeNH 2 , MeOH, H 2O (60%, 3 steps).

FIGURE 4.44. (Continued) HO

OAc

OAc

OH

OAc

i

A

OH

OH

(–)-Aristeromycin

OH

(+)

A

i) acetylcholinesterase (EEAC), buffer.

OH

OH

(–)-Neplanocin A

FIGURE 4.45. Enantiomeric resolution of prochiral cyclopentene diacetate. Me OPh

OAc

OPh i

(±)

A

OAc

(+)

OH

OH

(–)-5-Deoxyaristeromycin i) PSL, buffer.

FIGURE 4.46. Enzymatic resolution of racemic cyclopentene building blocks.

NH2 N HO

N

N

N

OH

NH2

N

i

HO

OH

N

N

N

OH

O

N

HO

+

OH

N

OH

OH

(+)

(±)

FIGURE 4.47. Enzymatic resolution of carbocyclic nucleoside. (EtO)2

P O

PPL

THF, 37oC

OAc

(EtO)2

OAc

CF2

P O

OH

CF2

+

(EtO)2

P O

OAc

CF2

PPL 30% iPr2O-buffer pH 7.3

(EtO)2

P O

OH

CF2

(EtO)2

P O

OH

CF2

O

O N

N N

(EtO)2

P O

CF2

NH

NH N

N

NH2

(EtO)2

P O

N

NH2

CF2

FIGURE 4.48. Chemoenzymatic syntheses of cyclopropane nucleosides.

NH N

4.3. Carbocyclic Nucleosides

219

FIGURE 4.49. Structures of carboxetan carbocyclic nucleoside.

O N

NH

N

N

OH

NH2

OH Structure of Lubocavir

Carbocyclic nucleoside Aristeromycin with antitumor and antiviral activity was prepared by condensation of the carbocyclic diacetate intermediate with the sodium salt of adenosine base under Pd(0) in 75% yield (Figure 4.56).100 Palladium-mediated coupling of purine base with carbocyclic acetates, carbonates, or benzoates led to a mixture of N-7 and N-9 isomers. The regioselectivity of purine alkylations depends on the size and nature of the ligands, being the most typical Ph3 P, BINAP, P(OMe)3 , P(OiPr)3 , P(OPh)3 . (Figure 4.57).101 Another straightforward methodology for preparing carbocyclic nucleosides involves the direct condensation of mesylated carboxyclic intermediate with the HON RO

O

RO S

C O N

CO2H DCC, THF r.t. dark

OR

S

OR

NH2 NH2

N

N

N

N RO

N

N H N

W-hv, 30-40oC

N

RO

OR OR

FIGURE 4.50. The Barton decarboxylation method for the preparation of carbocyclic Cnucleosides.

220

4. Nucleoside Mimetics Cl Cl N N

NH

+

BnO

OH OBn

i

N N H

NH N

NH2

NH2

N

BnO OBn i) DIAD, Ph3P, THF, r.t.

O

O I

PhOCN

I PhOCN

BnO O

N H

O

N

OH i,ii

BnO

HO

OH

i) Ph3P, DEAD, THF. ii) BCl3, CH2Cl2.

FIGURE 4.51. Synthesis of conformationally locked carbocyclic purine and pyrimidines under the Mitsunobu approach.

heterocyclic base in the presence of potassium carbonate and crown ethers as coupling conditions (Figure 4.58).102

4.3.5 Enzymatic Synthesis Likewise, carbocyclic nucleosides aristeromycin and neplanocin A can be biosynthetically prepared by using a mutant strain of S. citricolor as is observed in Figure 4.59. The cyclopropylamino carbocyclic nucleosides (−)-Abacavir is a potent antiHIV drug with promising results on clinical trials.103 An improved synthesis has been described by Crimmins et al.104 involving the treatment of key carbocyclic 2-amino-6-cloropurine intermediate with cyclopropilamine producing Abacavir along its parent anti-HIV carbocyclic nucleoside (−)-Carbovir (Figure 4.60). 4.3.5.1

Base Ring Formation

Another useful strategy used for preparing carbocyclic nucleosides involves the use of intermediates in which the amino group is already attached to the sugar

4.3. Carbocyclic Nucleosides

221 O NBz

BnO

OH

BnO

BnO i

N H

ii

OH OH

OH

O iii-iv

OH

O NH N

HO

O

OH

i) NaH, BnBr. ii) 9-BBN, H 2O 2/NaOH, 87 %. iii) PPh3, DIAD. iv) a) NaOH/MeOH. b) H 2, Pd/C, 90%

FIGURE 4.52. The Mitsunobu reaction for preparation of the carbocyclic thymidine nucleoside.

moiety and once the coupling reaction is achieved, a ring closure process takes place to generate the expected nucleoside. According to this procedure Roberts et al.105 prepared the potent antiviral inhibitor (−)-carbovir, which possesses similar activity than AZT against HIV in MT-4 cells. Thus, the starting material (±)2-azabiciclo [2.2.1] hept-5-en-3-one was submitted to microbial treatment with Pseudomona solanacearum to provide enantiomerically pure (−) isomer. The enantiomerically pure carbocyclic amine was then conjugated to 2-amino-4,6dichloropirimidine to produce the carbocyclic precursor which was ultimately cyclized to afford the desired (−)-carbovir (Figure 4.61). Anti-leukemia carboxylic nucleoside Neplanocin A has been synthesized by Marquez et al., under the ring closure approach mentioned above. Thus, condensation of pyrimidine intermediate with isopropylideneaminocyclopentenediol furnished intermediate, which was further cyclized to the purine base with

222

4. Nucleoside Mimetics OTHP OTHP THP N

N

N

HO

O

N

i

+

HO

N I

THP N

N N

OSi(Ph)2t-Bu

O

t-Bu(Ph)2SiO

O H N

HN

N HO

N O

R

R= OH R=H

i) Pd(dba)2, AsPh3, CH 3CN.

FIGURE 4.53. Palladium-mediated 2 -deoxyformycin B and 2 ,3 -dideoxyformycin B.

triethylortoformiate. Final conversion to adenine ring with ammonia and protecting group removal gave place to Neplanocin A (Figure 4.62).106 Likewise, this procedure was applied for the preparation of the close related pyrimidine analog by condensation of the previous carbocyclic amine with the unsaturated ether to produce the pyrimidine precursor who was transformed to thiopyrimidine and then to carbocyclic cytosine, as Figure 4.63 shows. This compound has been found to be active against leukemia type L1210 in vivo.107 An antiviral carbocyclic purine nucleoside was also reported108 by following a ring closure step for purine formation. Condensation between pyrimidine intermediate with carbocyclic amine afforded condensation product, which is activated with diazonium salt for amino introduction. Ring closure was achieved with triethyl orthoformate in acid medium (Figure 4.64).

4.4. Acyclic Nucleosides

223

HO O O OAr

O

BzO

X N

N

O O O

N H

O

Y

N

Pd(0) BzO X N

N

O O O

O

N

N

Y

FIGURE 4.54. Solid-phase synthesis of carbocyclic nucleosides under palladium catalysis.

4.3.6 Carbocyclic C-Nucleosides This class of C-nucleosides in which a methylene group replaces the furan oxygen ring does not show signiﬁcant biological activity so far; however, there is an interest to synthesize C-nucleoside with natural heterocycle moieties in a stereocontrolled fashion. A recent stereocontrolled synthesis of carbocyclic C-nucleosides has been proposed involving as key starting material the cyano carboxyclic intermediate which was condensed to 9-deazapurine to produce saturated and unsaturated carbocyclic 9-deazapurine nucleosides (Figure 4.65).109

4.4 Acyclic Nucleosides Since the discovery of Acyclovir as an anti-herpes drug, important efforts have been made toward the synthesis of analogs of acyclovir and other acyclic nucleosides. A comprehensive review made by Chu and Cutler110 summarizes the major

224

4. Nucleoside Mimetics BnO

BnO

BnO

i

ii

AcO

HO O

OAc O

O

O

O (–)

(+)

(+)

O

X N HO

N

O

O

N N

Y

X = NH 2, Y = H X = OH, Y = H

i) Ac2O, DMAP. ii) PdCl2(MeCN)2, pBQ, THF.

FIGURE 4.55. Tsuji-Trost reaction for the preparation of neplanocin A.

achievements carried out for preparing acyclonucleosides deﬁned as those heterocyclic compounds containing one or more hydroxyl groups on the alkyl side chain. At least three representative synthesis of acyclovir have been made, the ﬁrst by Schaeffer et al.111 involving a condensation reaction of dichloropurine with ether chloride intermediate, and further purine transformation to generate 9-(2hydroxyethoxymethyl)guanine (acyclovir) (Figure 4.66). An improved version introduced by Barrio et al.112,113 consists of the initial reaction of 1,3-dioxolane with trimethylsilyl iodide to produce the side chain, NH 2 NH2 AcO

OAc

N N

N

+

i

AcO N Na

N

N

N

i) Pd(PPh3)4 (0.005 eq.), Et3N, THF, reflux

FIGURE 4.56. Palladium catalyzed synthesis of aristeromycin.

N

4.4. Acyclic Nucleosides

225

X N

N

X PO N

NH2

N

N

OP OP

N

N H

+

NH2

N i

NH2

N

N P = Ac, CO 2Me, CO P = Ac, X = Cl N9/N7 = 86:14

PO

P = CO2Me, X = Cl N9/N7 = 74:26

N

N

P = H, X = Cl N9/N7 = 85:15

X

P = Ac, X = NHcyclopropyl N9/N7 = 95:5 i) Pd(PPh3)4, THF/DMSO, 45oC 50-70%.

FIGURE 4.57. Palladium-catalyzed coupling with purine base.

which was then condensed with the halogenated purine to yield after hydrolysis and ammonolysis the target acyclovir (Figure 4.67). Robins and Hatﬁeld114 employed a chemoenzymatic approach for preparing acyclovir consisting initially in the use of mercury salts and hexamethyldisilane (HMDS) and in the ﬁnal step an enzymatic conversion. Thus, the procedure NH2

OBn

N

N

+

OMs O

N

NH 2

F

O

N H

i

F

N

N N

OBn N

O

O

i) K2CO3, 18-Crown-8.

FIGURE 4.58. Preparation of carbocyclic nucleosides with mesylated carboxyclic intermediates.

OP

OP OH HO OHC

O

HO

NH2 N N H

OH

OH

HO

OH

OH

NH2

NH2

N

N

N

OH

N

HO

N

N OH

N

N

HO

OH

N N

OH

Aristeromicin

Neplanocin A

FIGURE 4.59. Biosynthetic pathway of neplanocin A and aristeromicin. Cl N

N OAc

H2 N

N H

N

AcO

NH2

Cl N

N

+

N

N

H2 N

Cl

11%

65%

ii, iii

iii

O HN

H2 N

N

N

N

N

N

N

N

N AcO

AcO

i

N

N

H2 N

N

N

HO

HO

i) NaH, Pd(PPh3 )4 , 1:1 THF:DMSO. ii) cyclopropylamine, EtOH. iii) NaOH, H 2 O.

FIGURE 4.60. Synthesis of anti-HIV (-)-Abacavir and (-)-Carbovir.

4.4. Acyclic Nucleosides O

HO

O

NH

227

NH i

NH2 ii-vi

(+) –

(–)

Cl

O H2N

Cl

N

HN

vii-xi

H 2N

N

N

HO

(–)-carabovir

i) P. solanacearum NCIB 40249. ii)HCl-H2O. iii) (MeO)2CMe2. iv) Ac2O/Py. v) Ca(BH4)2/THF. vi) HCl-H2O/EtOH. vii) PrNEt, nBuOH. viii) 4-Cl-C6H4N2+ Cl-AcOH, AcONa/H2O. ix) Zn, AcOH/EtOH-H2O. x) (EtO)3CH/HCl. xi) NaOH/H2O.

FIGURE 4.61. Synthesis of (−)-Carbovir.

involves the condensation between 2,6-dichloropurine with the bromoether, affording regioisomer N-7 shown in Figure 4.68. Further amination and ﬁnal transformation to guanine with the enzyme adenosine-deaminase produces the desired antiviral compound. The phosphonate acyclic nucleoside 9(2-phosphonomethoxyethyl)adenine (PMEA) was founded as a good antiviral analog with prolongated action.115 A regio-deﬁned synthesis base on the purine ring formation was described involving the initial attachment of the phosphonate amine intermediate by nucleophilic substitution to the 5-amino-4,6-dichoropyrimidine base, and then ring formation followed by amination to produce the desired phosphonate acyclic adenine PMEA (Figure 4.69).116 The effectiveness of acyclovir as an antiviral drug encouraged different groups to synthesize more potent acyclic analogs. As a result of these efforts, the acyclic nucleoside 9-[(1,3-Dihydroxy-2-prpoxy)methyl]guanine (DHPG)117 was prepared and tested as antiviral nucleoside, showing similar potency as acyclovir against simple herpes but stronger against encephalitis and vaginitis herpes. Various report of DHPG were described, one of them involving the use of hexamethyldisilylasane (HMDS) as condensing agent (Figure 4.70).110 An alternative route for preparing DHPG involved the condensation reaction of acetylguanine base and triacetate derivative in the presence of ethanesulfonic

228

4. Nucleoside Mimetics Cl Cl

NH2

BzO NH2

N

i

ii

+

N O

Cl

N

NH2

N

NH

BzO

O

O

O NH2

NH2

Cl N

N

iv

iii

N

N

N

N

N

N

N

N

N

N

HO

BzO

BzO

OH OH O

O

O

O

i) EtN3/EtOH. ii) HC(OEt)3, Ac2O. ii) NH3/MeOH. iii) BCl3/CH2Cl2-MeOH

FIGURE 4.62. Synthesis of neplanocin A.

acid, at temperatures ranging from 155 to 160◦ C. As result two regioisomers were obtained from which one of those was converted to the desired antiviral compound Figure 4.71.110

4.5 Thionucleosides Nucleosides having the sugar ring oxygen replaced by sulfur are known as thionucleosides. The synthesis and therapeutic evaluation mainly as antiviral and anticancer drugs of these nucleoside mimics have been reviewed.118 A comparative analysis of thionucleosides and nucleosides showed that sulfur replacement in some cases produced equivalent or higher potency119,120 and do not undergo enzymatic cleavage of the glycosidic bond, although it has been also observed increased toxicity as in the case of β-4 -thiothymidine.121 Some thionucleosides displaying antiviral and/or anticancer activity are shown in Figure 4.72.

Cl NH2

BzO

EtO

N

i

ii,iii

+

NH

O

CONCO O

O

S

NH2

HN N

HN N

O

BzO

HO

iv

O

O

N

O

BzO

iv

O

O

NH2

HN O

OEt

BzO

O

O

OH OH

i) PhH. ii) DMF, NH4OH. iii) Lawesson. iv) NH3 liq. v) a) BCl3 /CH2Cl2-MeOH. b) Dowex H+

FIGURE 4.63. Synthesis of carbocyclic pyrimidine nucleoside. Cl

Cl NH2

HO

ii

+

N

H2N

Cl

N

H2N

N

i

N

NH

HO

OH

OH Cl

Cl N

N

Cl

NC6H4-Cl

NH2

N iii

N

H2N

NH

HO

H2N HO

N H

NH H2N HO

OH

N

N

iv

N H

N

OH OH

i) Et3N/EtOH. ii) 4-Cl-C6H4N2+Cl– , Na2CO3, AcOH/H2O. iii) Zn/AcOH. iv) CH(OEt)3-HCl/DMF.

FIGURE 4.64. Ring closure approach for preparation of carbocyclic purine.

230

4. Nucleoside Mimetics NH2 H N

N N

HO

HO t-BuO

EtO2C

OH

CN O

O H N

H N O

N

HO

N

t-BuO

O

NH

NH

O

OH OH

O

R = H, NH2

O H N

HO

NH N

R

R

R=H

FIGURE 4.65. Stereocontrolled syntheses of carbocyclic 9-deazapurine nucleosides.

Based on their structural features N-thionucleosides deﬁned also as thioribosyl sugars are classiﬁed into four groups (Figure 4.73):

4.5.1 Preparation of Thioribofuranosyl Intermediates A number of approaches oriented to replace or insert a sulfur atom instead or besides the cyclic oxygen into the ribose ring have been described. One of the

4.5. Thionucleosides

231

Cl Cl i

C6H5CO 2CH2CH 2OCH2Cl

+

Cl

N

Cl N C6H5OO

N H

N

N

N

N

N

O

O N

HN

ii

H2 N

N

N HO

O

Acyclovir

i) Et3N. ii) NH 3.

FIGURE 4.66. First synthesis of Acyclovir.

I N

N

O

O

+

Me3SiO

Me3SiI

O

I

O

I N

N N Cl Me3SiO

N H

N

Cl

N

HN i,ii

N

HO

O

N

N

H2 N

O

Acyclovir i) K2CO 3. ii) NH 3.

FIGURE 4.67. Improved synthesis of acyclovir.

232

4. Nucleoside Mimetics Cl Cl AcO

N

N

O

N

N

i

+

Cl

N H

N

Cl

Br

N

N

AcO O O ii,iii

N

HN H2N

N

N

AcO O

i) Hg(CN)2-HMDS. ii) NH3. iii) adenosine-deaminase.

FIGURE 4.68. Acyclovir synthesis.

Cl NH2

N

Cl O

NH2

N

EtO

+

Cl

N

P

NH2

i

N

NH

O

O EtO

EtO

P

O

EtO

Cl

NH 2 N

N

N

N iii,iv

ii

N

N

N

O EtO EtO

P

O O

HO

P

O

HO

i) Et3N, EtOH, reflux. ii) diethoxymethyl acetate, 120oC. iii) NH3. iv) TMSBr, MeCN.

FIGURE 4.69. Synthesis of phosphonate acyclic adenine PMEA.

N

4.5. Thionucleosides

233

Cl Cl

N

N BnO

N

N

O

Cl

i

+

N H

N

H2 N

H2 N

N

N BnO

O

OBn

OBn O O

N

HN N

HN

ii

iii

H2 N N

N

H2 N BnO

N

N HO

O

O OH OBn DHPG

i) HMDS. ii) NaOMe, HSCH2CH 2OH. iii) H2, Pd-C

FIGURE 4.70. Synthesis of antiviral acyclic nucleoside DHPG. O N

HN AcHN

i,ii +

AcO

N H

N

OAc OCH2OAc

O N

N

HN

+

N

N

H2N HO

NH

N HO

O

NH2

N

O

O

OH OH DHPG i) EtSO3H, 155-160oC. ii) MeONa/MeOH

FIGURE 4.71. Alternative synthesis of DHPG.

NH2

NH.HCl

CN

N

HO

N N

N

O

N

HO

S

S HO

OH OH

OH

Thiotoyocamycin

Thioarabinofuranosylcytosine

Leukemia growth inhibitor

KB cell growth inhibitor

O

O N

N

N

N

H2 N HO

CH3

HN N

O HO

S

S

OH

OH

Thiothymidine

2'-Deoxy thioguanosine

Carcinoma growth inhibitor

Antiviral against HBV and HCMV

FIGURE 4.72. Some active N-thionucleosides. HO

S

B

HO

S

B

HO

B

HO

S OH OH

B

S

OH N-Isothionucleosides N-Thionucleosides

S

B

HO

S

B

HO

B

O

HO

O

HO S OH OH

S

OH

N-L-Thionucleosides

N-Thiooxonucleosides

FIGURE 4.73. Classiﬁcation of N-thionucleosides.

B

4.5. Thionucleosides FIGURE 4.74. Early synthesis of thioribofuranosyl derivatives.

AcO

O OMe

S

235

OAc

i

BzS OH OH

OAc OAc

AcO

O OMe

S

OAc

i

OBz

OBz

BzS OBz

OBz

i) Ac2O/AcOH, conc. H 2SO4, 97%

earliest methods for preparing thioribosyl acetates was described by Reinst et al.122,123 involving as key steps the conversion of the 4-thiobenzoyl pyranoside into the thioribofuranosyl acetate (Figure 4.74). A short time later, another report introduced the use of sodium in liquid ammonia followed by benzoylation to yield tribenzoylated thioribofuranoside as a mixture of anomers (α:β, 1:3) (Figure 4.75).124 The thioribosyl derivative benzyl 3,5-di-O-benzyl-2-deoxy-1,4-dithio-Derythro-pentofuranoside has been prepared and used as glycosyl donor in various thionucleoside synthesis.125,126,127 The synthesis started from 2-deoxy-ribose, which was transformed to the methylbenzyl derivative by following a standard procedure and then treated with benzylmercaptan in acid to produce the dithiobenzylated derivative. The next step was to invert the hydroxyl group at 4position by using the Mitsunobu protocol to generate the intermediate with the desired stereochemistry. Finally, tosyl protection and NaI-BaCO3 treatment afforded the desired thiosugar (Figure 4.76).125

BzO

O

S

OBz

i,ii

BzS

OBz OBz

FIGURE 4.75. Preparation of benzoylated thioribofuranoside.

i) Na/aq. NH3. ii) BzCl/Py

OBz

236 HO

4. Nucleoside Mimetics HO

O OH

OMe

OH

O

ii

OMe

OH

BnS

iii

BnO

O

i

H

SBn

OBn

BnS

H

SBn

H

iv

H

H

v

OBn

OBn

OH

H

HO CH2OBn

BnS

SBn

OBn

BzO

CH2OBn

CH2OBn

SBn BnO

H

vi

BnS

H

O SBn

vii

OBn MsO

OBn CH2OBn

i) MeOH, HCl. ii) NaH, Bu4 NI, BnBr/THF. iii) BnSH, HCl. iv) PPh3 , PhCO 2 H, DEAD/THF. v) NaOMe/MeOH. vi) MsCl/Py. vii) NaI, BaCO3 , acetone

FIGURE 4.76. Synthesis pentofuranoside.

of

benzyl

3,5-di-O-benzyl-2-deoxy-1,4-dithio-Derythro-

4.5.2 Glycosidic Bond Formation The general methods for preparing N-thionucleosides are similar as for Nnucleosides. However, variations from slight to signiﬁcant can be found especially in the preparation of four-ring thietanocin or thiolane analogs.126,127 Thus, according to a comprehensive review,118 the earliest reports for N-thionucleoside formation used chloromercury salt of purine and chlorine or benzoyl thioriboside as glycosyl donor, while more recently the silyl approach has been preferred (Figure 4.77).

4.5. Thionucleosides

237

NHBz N

N NHBz AcO

S Cl

+

N

N

AcO N

OAc OAc

N

N

i

S

N HgCl OAc OAc

i) toluene

Ref.122

Cl Cl

AcO

S Cl

+

N

N

N

N N

OAc OAc

N

N

N

AcO

S

HgCl

i) toluene, heat, 37%.

OAc OAc

Ref.128 FIGURE 4.77. Common glycosylation reactions for the preparation of thionucleosides.

4.5.3 Chloromercuration Promoted Coupling Reactions 4.5.3.1

Silylmediated Coupling Reactions

The preparation of potential anti-HIV N-Isothionucleosides was described starting from glucose. The key coupling reaction proceeds in low yield between the pyrimidine base and the mesyl tetrahydrothiophene derivative under potassium conditions (Figure 4.78).132 N-Thioxonucleosides are another class of N-thionucleosides tested as antiHIV agents. The conditions employed for performing the coupling reaction were TMSOTf as Lewis acid catalyst, affording a mixture of anomers (α:β, 1:2) in 64% (Figure 4.79).133 Thietane nucleoside was synthesized starting from the benzoyl thietane derivative, which prior to the coupling reaction was treated with peroxide to produce the

238

4. Nucleoside Mimetics NHR N

N NHR BzO

S OBz

N

N

+

N

N

i

BzO N

S

N HgCl

OBz

OBz i) TiCl4, EtCl2, heat.

Fig.129 NHOAc N N(TMS)OAc AcO

N

O

S OAc +

AcO

S

N

TMSO

OAc

AcO

i

N

OAc

AcO i) SnCl4, EtCl2, heat, 65%.

Ref.128 O R HN O TolO2CO

R

S OAc

TolO2CO

+

O TolO2CO

O

i

HN N H

TolO2CO i) a) HMDS, TMSCl, MeCN. b) TfOTMS.

Ref.130 FIGURE 4.77. (Continued)

N S

4.5. Thionucleosides

239 O

HN OTMS BnO

S SBn

N

O

i

N

+

BnO

S

N

TMSO BnO

BnO i) HgBr2, CdCO3, toluene.

Ref.121 Cl Cl TBDPSO

S

N

N OAc

N

N i

TBDPSO

S

N H

N

i) Et3AlCl.

Ref.131 FIGURE 4.77. (Continued) O O NH

BnO i

NH

+

N

BnO S

OMs

i) K2CO 3, DMSO, ∆, 15%

N H

N

N

+

O S

FIGURE 4.78. Preparation of N-Isothionucleoside.

O

240

4. Nucleoside Mimetics

O OAc TBDPSO

O

NHAc

NHAc N

TBDPSO

N

i

S

+

S

N

OTMS

N

N

O

+

O

O TBDPSO

N

S

NHAc

i) TMSOTf, CH2Cl2, 64%

FIGURE 4.79. Preparation of N-thioxonucleosides. O

O NH

NH

O BzO

S

BzO

i

N H

S

O

O BzO

N

O

S

ii

OBz

OBz

OBz i) TMSOTf, Et3N, ZnI2, toluene, 30 %.

FIGURE 4.80. Synthesis of thymidine thietane nucleoside. OTMS O N TBDMSO

NH S

N

OTMS TBDMSO

i

N

O

S

TBDMSO TBDMSO

SePh

i) PhSeCl 88 %.

FIGURE 4.81. Synthesis β-4 -thionucleosides based on electrophilic glycosidation of 4thiofuranoid glycols.

References

241

O O HN AcHN

N

i

SCH2OAc

+

N Ac

N

HN

OBn

N

N

N

AcHN BnO

OBn

+ 7 isomer

S

OBn

O

O N

HN ii

N

HN iii

AcHN AcO

N

N

H 2N

S

OAc

HO

N

N S

OH

i) (p-NO2C6H4)2P(O)OH. ii) (C 2H5)O-BF 3, Ac2O. iii) NH 3.

FIGURE 4.82. Synthesis of thio analog of DHPG.

sulfoxide derivative. Then under Lewis acid conditions a Pummerer rearrangement process takes place to produce in the presence of thymine the expected thietane nucleoside (Figure 4.80).127 More recently the stereoselective synthesis β-4 -thionucleosides based on electrophilic glycosilation of 4-thiofuranoid glycals was described. Thus, the condensation of TBDMS-4-thioglycal with silylated uracil in the presence of PhSeCl as electrophile furnished thionucleosides in 88% as a mixture of anomers (α:β; 1:4) (Figure 4.81).134 The thio analog of antiviral DHPG with comparable activity to DHPG against HSV1 and human cytomegalovirus was synthesized according to the ﬁgure shown below (Figure 4.82).110

References 1. 2. 3. 4.

H. Mitsuya, R. Yarchoan, and S. Broder, Science, 249, 1533 (1990). D.M. Huryn and M. Okabe, Chem. Rev., 92, 1745 (1992). H.Mitsuya and S. Broder, S. Proc. Natl. Acad. Sci. USA, 83, 1911 (1986). C. Simons, Nucleoside Mimetics, Ed. Gordon and Breach Science Publishers, (2001).

242

4. Nucleoside Mimetics

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5 C-Glycosides

These types of glycosides have attracted much attention, considering that many of them have demonstrated their effectiveness as therapeutic agents. The increasing signiﬁcance of C-glycosides is that the conformational differences compared to O- or N -glycosides are minimal and that they are resistant to enzymatic or acidic hydrolysis since the anomeric center has been transformed from acetal to ether.1 A glycoside is deﬁned as C-glycoside when what it is suppose to be the anomeric carbon of a sugar is interconnected to the aglycon, generating a new C-C bond. According to Levy and Tang2 the term C-glycoside describes those structures in which a common structural motifs the presence of carbon functionality at what would otherwise be the anomeric position of a sugar or derivative. Structurally C-glycosides can be constituted by aliphatic, or aromatic aglycon, and the sugar can be pyranose or furanose. A variety of natural product C-glycosides has been described. Examples of C-glycosides isolated from different plant genus and characterized spectroscopically are: Carminic acid, Aloin, Scoparin, Saponarin, and more recently Cucumerins (ﬂavonoid phytoalexins)3 and C-glucosylxanthones4 and complex benzoquinone Altromycin B5 among others (Figure 5.1). Moreover, much effort and creativity have been devoted to the preparation of complex C-glycosides with potent antibiotics activity. That is the case of Aurodox6 , Lasalocid A,7 Herbicidin,8 and the hyperfunctionalized molecules Spongistatin9 and Palytoxin10 (Figure 5.2).

5.1 Synthetic Approaches for the Preparation of C-Glycosides Accordinng to comprehensive studies,2,11,12,13 the general strategies for C-glycosides can be overviewed as follows: r r r r

Electrophilic glycosyl donors Concerted reactions Wittig aproximation Palladium mediated reactions 247

OH

OH

O

OH

HO O

OH

O

CH3

OH COOH

HO

OH O

OH HO

OH OH

HO

OH

O OH Aloin

Carminic acid OH

OH

OH HO

HO

OH

O O

OH

HO

O

O

HO O

OH O

OCH3 HO

OH OH

O

OH

HO

O

OH

Saponarin

Scoparin

OH H3C

OH

HO

HO

O

OH

O O

OH

HO

O

OH

HO OH

O

OH

HO

O

OH

HO OH

OH

C-Glucosylxanthones

Cucumerin B

CH3

HO H3CO O

O OH

OH

H3CO2C CH3 O O

O

OH

O

O (H3C)2N

O CH3

H3C O

CH3

Altromycin B

FIGURE 5.1. Some naturally ocurring C-glycosides.

OCH3

OH CH3

5.1. Synthetic Approaches for the Preparation of C-Glycosides

Me Me

OH

OMe

H N

249

O

CH3 N

O

OH

O

O OH

O OH OH Aurodox CO2H

Me

Me

HO OH

CH2OH

O

O

Me O OH Me

Et

Lasalocid A O OH O

O

OH

OH

OH

HO OH

OH

O

H2N

OH

OH Me

HO

OH OH

OH

OH OH

OH HO

R

OH OH

OH

O

OH

HO

CH3 H3C

O OH

HO OH O

OH

O O

HO

OH

Me

OH

Me OH O OH

OH HO

OH

OH

OH OH

FIGURE 5.2. Complex C-glycoside antibiotics.

250

5. C-Glycosides OH

Me

OH

Me

O

R=

O N H

OH

N H

OH

Me Palytoxin

O HO OAc

OMe

O

O

O

O

AcO

HO O

O

HO

O

OH

OH

O Cl

OH OH

Spongistatin 1

FIGURE 5.2. (continued)

r r r r r r r r

Mitsunobu reaction Nucleophilic sugars or anomeric anions intermediates. Cross-metathesis reaction Samarium promoted reaction Ramberg-B¨acklund reaction Free radical approaches Exoglycals The tether approach

5.1.1 Electrophilic Glycosyl Donors 5.1.1.1

Glycosyl Donors Bearing Good Leaving Groups

A general approach for the C-glycosidic bond formation is based on nucleophilic carbon addition on the electrophilic center of a glycosyl donor. The most extensively used glycosyl donors divided in four main groups (good leaving groups, sugar lactones, glycals, and 1,2 anhydrosugars) are used as electrophilic donors

5.1. Synthetic Approaches for the Preparation of C-Glycosides O

O

R-M

251

R

PO

PO X P = protecting group M = Li, MgBr,

SnBu3

X = leaving group (I, Cl, Br, imidate, acetate and Lewis acid)

to generate C-glycosidic bonds when reacted with organocuprates, organotin, organozinc, cyanide, allylic Grignard, vinyl silyl reagents, and activated aromatic compounds. among others.11 Some of the reactions described that have been used for preparing useful intermediates or C-glycosides are shown in Figure 5.3. It is worth mentioning that for aryl C-glycosylations, there is a dependence on the electron density of the aromatic ring and the protecting groups at the glycosyl moiety.15 Moreover, depending on the reaction conditions, there is a competing parallel process that ultimately will drive the reaction either to the O- or to the C-glycoside formation. This afﬁrmation was demonstrated in the preparation of C- and O-ﬂavonoid glycosides by Oyama et al., which treated glycosyl ﬂuoride with ﬂavan under Lewis acid conditions. It was observed that BF3 .ET2 O and 2,6di-tert-butyl-4-methyl pyridine (DTBMP) resulted predominantly in the formation of the 5-O-β-glycoside, while if the reaction is carried out only with BF3 -Et2 O, the C-glycoside is obtained (Figure 5.4). 5.1.1.2

Other Electrophilic Glycosyl Donors

Additionally, the introduction of other electrophilic centers at the anometic position has extended the possibilities for preparation of C-glycosides by using electrophilic sugars. Some of these electrophilic sugars are lactols, anomeric esters, glycals, anhydrides, and lactones. O

OR

O

O PO

PO

Lewis acid R = TMS

R-M

O

O R

PO

PO

Lewis acid

OH

O R = TMS

PO

O

Nucleophile

O

Lewis acid

O

hydride

Nu

PO OH

O PO

Nu

OR2

OR2 RMgBr

O

R2O R 2O

O

R 2O

R

R2O

OR1

OR2

R 2O

OR1

Br

O

R2O

+

OR1 R

R1 = R2 = Bn, Me, silyl; α -selectivity

R = allyl, vinyl, benzyl, alkynyl; α -selectivity

R1 = Ac, or Bz, R2 = Bn, Me, silyl; β-selectivity

CO2Et R2

R2

OBn CO2Et

O

R1 BnO

OBn

O

R1 BnO

NaHMDS 5-crown-15

I

R2

OBn

OBn EtO2C

OBn CO2Et

O

R1 BnO

+

CO2Et OBn

CO2Et

R1 = OBn, R2 = H R1 = H, R2 = OBn

Ref.14 OBn

OBn

OBn O

O

OC(NH)CCl3

OBn

CH2NH2

LiAlH4

TMSCN

BnO

O

CN

BnO

BnO

OBn

OBn

OBn OBn

OBn

OBn

OBn O

BnO

OC(NH)CCl3

OBn

O

TMS ZnCl2 BnO

OBn

OBn

OBn OMe

OBn

OMe

OBn O

OC(NH)CCl3

O

OMe

OMe BnO

OBn OBn

BnO

OBn OBn

FIGURE 5.3. Preparation of C-glycosides or intermediates from electrophilic glycosyl donor with good leaving groups.

5.1. Synthetic Approaches for the Preparation of C-Glycosides

OBn

OBn

O + BnO

BnO

OBn

O

O

i

NH

O

253

BnO BnO

O

OBn

CCl3

i) ZnCl2.

OBn

OBn

OBn O

O

OAc

O

CN

CN

TMSCN +

BnO

BnO

OBn

40:50 OSiR3

OAc

O

OBn OBn

OBn

OBn AcO

BnO

OBn

OSiR3

AcO

OAc

AcO

O

O

i

Et

O

O

Et

OAc

AcO

O

i) SnCl4.

FIGURE 5.3. (continued) OAc

OAc O

H3 CO

OAc O

AcO AcO AcO i

OH

O

H3 CO F

AcO AcO AcO

O OH

OAc

+

O

H3CO

C- vs Oglycoside i) BF3-Et2O (4 equiv.) 1 h

94/6

i) BF3-Et2O (4 equiv.), DTBMP 1 h

8/92

AcO AcO AcO

O

OAc

O

OAc

FIGURE 5.4. C-glycosylations involving glycosyl donors with leaving group.

254

5. C-Glycosides OBn

OBn O

O

1) Me 2CuLi

Me

O 2) Ac2O/Py

BnO

BnO OBn

OAc OBn 60 %

OBn

OBn O

O

O

OH

MgBr

BnO

OBn

BnO

OBn

OBn

OBn

OBn

OBn

OBn O

O

O

RLi

Et3SiH BF3-Et2O

R

O

R

OH BnO

BnO

OBn

BnO

OBn

OBn

R = Me, alkynyl

16

Ref.

OMgBr

HO

O AcO

i +

O AcO

t-Bu

AcO OAc

AcO t-Bu

i ) ultrasound

FIGURE 5.5. C-glycoside formation with electrophilic sugars.

Some of the reactions carried out for preparing C-glycoside intermediates involving these alternative glycosyl donors are shown in Figure 5.5. In 1,2anhydrosugars the stereoselectivity is 1,2-trans type and involves a typical SN 2 process. On the other hand, glycals exhibit high stereoselectivity, and in glycosyl acetates the stereocontrol relies on the electronic and steric properties of the nucleophiles.

5.1. Synthetic Approaches for the Preparation of C-Glycosides

255 OMOM

OH OMOM

Me O O BnO

O

Me

CO2H i,ii,iii

CO2H

Me

CO2Me

O

BnO Me

HO OH

O

CH2OH

O

Me O OH i) (COCl)2, ii) n-BuLi. iii) LDA

Me

Et

FIGURE 5.6. Synthesis of Lasalocid A.

5.1.2 Concerted Reaction and Ring Formation This type of reactions includes sigmatropic rearrangements and cycloadition transformations. As an example of the applicability of the sigmatropic rearrangment for preparation o C-glycosydes, Ireland7 reported the synthesis of Lasalocid A, consisting in the coupling of acid derivative with protected glycal as a result of enolate addition and Claisen rearrangement. Series of transformation of this precursor will give place to Lasalocid A (Figure 5.6). To exemplify the effectiveness of cycloadditions for preparation of C-glycosides, Schmidt et al.17 prepared p-methoxyphenyl 2,3,4,6-tetraacetyl C-glucopyranose, by following a Diels-Alder approach. The reaction between heterodiene and dienophile produced cycloadduct that was successively transformed to give the desired product (Figure 5.7). Protected monosaccharide is reacted with Wittig ilide to produce a ring opening unsaturated intermediate, which was cyclized to produce a mixture of α,β C-glycosides. The α form could be converted to the β form under sodium methoxide conditions (Figure 5.8).18 Cation-mediated cyclization reactions of silyl enols ether-containing thioglycosides give bicyclic ketotetrahydrofurans. Treatment with sodium amalgam in buffered methanol yields the expected dihydropyran, which was transformed to the diol intermediate, and after separation converted to the bis-acetonides (Figure 5.9).19 Ring closure of polyalcohol has been proposed as a suitable strategy for preparing C-glycosides.20 Condensation between iodine pyranoside intermediate with

256

5. C-Glycosides OBn

BnO SPh PhS

O

OMe

+

O AcO AcO OMe OMe

OAc O

AcO AcO

OAc

FIGURE 5.7. The Diels-Alder reaction for C-glycoside formation. O O HO

H3C

i

O

O

OH ii

H3C

O O HO

OH

O

H 3C

OH

CO2Me HO OH

O

CO2Me

H3C

+

O O HO

OH

O OH

CO2Me

iii i) Ph2P=CHCO2Me. ii) KOH/MeOH. iii) MeONa/MeOH.

FIGURE 5.8. Wittig reaction for C-glycoside formation.

an aldohexose will result in the condensation product which undergoes ciclyzation to give the mixture of C-disaccharides shown in Figure 5.10.

5.1.3 Palladium-Mediated Reactions Heck-type reactions have been successfully assayed for preparing interesting C-glycosides. Such is the case of Vineomicinone B2 prepared by palladium catalyzed condensation between TBS protected glycal with antracene derivative.21 Further transformations will generate C-glycoside Vineomcinone B2 (Figure 5.11). Other palladium-mediated coupling includes Stille (palladium-catalyzed vinyl substitution),22 and Suzuki cross-coupling reactions. 23

5.1.4 Mitsunobu Reaction A Mitsunobu reaction is an additional useful reaction for preparing C-glycosides (Figure 5.12). When tetra-O-methyl glucopyranose is reacted with 1-naphtol in the

5.1. Synthetic Approaches for the Preparation of C-Glycosides SO2Ph

SO2Ph

SO2Ph O

OH

O i

O

257

ii

O

SPy

SPy

O

Ph

OTBDPS

OTBDPS

OTBDPS Ph

SO2 Ph O v

iii,iv

OH

vi

O H

O H OTBDPS

OTBDPS

OH

OH

OH

OH

OH

OH +

OH

OH O

O H OTBDPS

H OH

OTBDPS

OH

i) PhCH=CHCH2Br, CH2Cl2, 50% aq. NaOH, r.t. ii) AgOSO2CF3, MS, CH2Cl2, r.t. then DBU. iii) O 3, CH2Cl2, –78oC, then PPh3, –78oC to r.t. iv) NaBH4, MeOH, 0oC. v) 6% Na(Hg), Na2HPO 4, MeOH, 0oC. vi) OsO4, NMO, 9:1 acetone-H2O, r.t.

FIGURE 5.9. Formation of tetrahydrofurans and application to the synthesis of 2-octulopyranosides.

presence of Mitsunobu conditons (diethylazidodicarboxylate and triphenylphosphine), the resulting product is the O-glycoside, which is rearranged with BF3 Et2 O to the corresponding C-glycoside.24

5.1.5 Nucleophilic Sugars Anomeric carbons are considered electrophilic sites by nature; however, it is possible to invert this reactivity by using metallic bases. The resulting carbanion character is known as umpolung reactivity and allows the species to behave as nucleophiles. A variety of glycosyl donors have been converted to lithium or stannane glycosyl anions (Figure 5.13).11

258

5. C-Glycosides CHO

I

OR

OR

OR i

O

+

RO

OR OR

OR OR

O

RO

RO

OR

OR

OR

OR

OR

RO

OR

OTBDMS OR OR

OR

O RO RO

RO

O

OR

OR +

RO

OR OR

O

O RO OR

OR OR

RO i) n-BuLi, THF

FIGURE 5.10. C-disaccharide formation from aldohexoses.

OH RO OH

O

Me

Me

OR CO2Me

O

i) Pd(Ph3)2Cl2/DIBAL/THF

OH

OH RO OH

O

Me

Me

OR CO2Me

i) Pd(Ph3)2Cl2/DIBAL/THF

O

OH

FIGURE 5.11. Synthesis of Vineomicinone B2 methyl esther.

5.1. Synthetic Approaches for the Preparation of C-Glycosides

259

OH OMe MeO MeO

O

OMe

i

OH +

O

MeO MeO

OMe

O

OMe OMe ii

MeO MeO

O OMe

OH

i) DEAD, Ph3P. ii) BF3.Et 2O.

FIGURE 5.12. Mitsunobu reaction for aromatic C-glycoside formation.

By using this possibility, the synthesis of the C-glycosyl asparagine analog has been completed by Kessler and coworkers.25 The transformation of the stannane to the lithium donor was followed by the coupling reaction with the aldehyde glutamic acid derivative to provide the β-D-linked C-glycoside. Removal of Boc protecting group and dehydroxylation reaction under Barton-McCombie condition provided the target molecule (Figure 5.14). Another accomplishment following this umpolung strategy was the preparation of the aromatic C-glycoside shown in Figure 5.15. Hence, lithium glycal (obtained from glycal treatment with lithium diisopropylamide) was reacted with quinolic ketal to yield addition product, which was transformed to the aromatic C-glycal.26,12 Aldol condensations between glycosyl donors containing active methylene carbons with glycosyl acceptors have been also proposed as suitable approaches for preparing C-disaccharides. Martin et al,27 described a procedure for preparing

BnO

BnO BnO BnO

i

BnO BnO

O OLi

O

Li

OH Cl

ii

BnO BnO BnO

O

SnBu3

OH i) BuLi-LiN. ii) Bu 3SnLi.

FIGURE 5.13. Preparation of lithium and stannane glycosyl anions.

260

5. C-Glycosides OBn O

BnO BnO

OBn i

SnBu3

O

BnO BnO

NHAc

Li

NHLi

NBoc2 OBn OH OHC

CO2Me 73%

O

BnO BnO

NBoc2

NHAc

CO2Me

OBn OH ii 67%

BnO BnO

O

NHAc

NBoc2 CO2Me

i) a) MeLi. b) BuLi. ii) a) MgClO4. b) deoxygenation.

FIGURE 5.14. Preparation of C-analogs of glycosyl asparagines from anionic glycosyl donors.

(1,6)- and (1,1)-linked C-disaccharides based on the nitroaldol condensation between the glycosylnitromethane peracetate with the galactose-derived aldehyde to provide after dehydration, reduction of the double bond, and and radical denitration the desired C-disaccharide (Figure 5.16).

5.1.6 Cross-Metathesis Reaction Cross-metathesis reaction is an emerging methodology for C-C bond formation. The air stable Grubbs ruthenium complex28 has become an attractive catalyst for the oleﬁn cross methatesis reactions and has been also applied successfully for the preparation of pseudosaccharides. The coupling reaction between C-allyl α-D-galactopyranoside with 4-acetoxystyrene led to the formation of the crossmetathesis product (Figure 5.17).29

5.1.7 Samarium Promoted Reaction The synthesis of a C-glycoside analog of α-1,3-mannobiose has been reported via SmI2 -promoted C-glycosilation. The general approach is based on the Barbier-type

5.1. Synthetic Approaches for the Preparation of C-Glycosides MeO MeO Me

O

261

OMe

OMe

Li +

Me

O OH

TBSO OTBS

O

TBSO OTBS

HO

OMe OMe

i

Me

O

Me

ii

O

OH TBSO

TBSO

OTBS

OTBS i) DIBAL/CH2Cl2. ii) POCl3/Py.

FIGURE 5.15. C-glycoside formation using lithium glycal nucleophilic donor. CHO OAc O

O

O O

+

O

AcO AcO

OH O

i,ii

HO HO

NO2

OAc

OH

O

OH

OH HO

O

OH

i) KF, MeCN, DCH-18-crown-6. ii) a) Ac2O, Py, CHCl3. b) NaBH4, MeOH, CH2Cl2, 0oC. c) Bu3SnH, AlBN, reflux. d) NaOMe, MeOH. e) H3+O.

FIGURE 5.16. C-disaccharide formation with glycosyl donors containing active methylene carbons. OAc

Ph

Ru

Cl

OAc OAc O AcO

PCy3 Cl

PCy3

+

OAc OAc O AcO

CH2Cl2, reflux

OAc

OAc

OAc

FIGURE 5.17. Cross-metathesis reaction for C-glycoside formation.

262

BnO

5. C-Glycosides

OBn O

OBn O

AcO

i

BnO BnO

+ BnO

BnO BnO

SO2(2-Pyr)

OMe

O

OBn O

BnO

OAc OBn O

BnO OMe

(Imid)SCO BnO ii

BnO BnO

OBn O

OAc OBn O

BnO OMe

i) a) SmI2 (2.8 eq), THF, 20oC. b) (Imid)2CS (15 eq), CH3CN, reflux, 35% ii) F5PhOH, Ph3SnH, AlBN, toluene, reflux, 65%.

O

O

SO2Ph

BnO

OMe

HO O

BnO

O

O

BnO

OMe

O

OBn

i

Si OBn

OBn

BnO

OBn

OH

OBn

OBn

i) SmI2, PhH, HMPA, 60oC. b) aq. HF.

FIGURE 5.18. Samarium promoted C-glycosylation.

coupling30 and involves the use of pyridyl sulfone glycosyl donor with a sugar aldehyde in the presence of SmI2 as catalyst. This procedure has been exploited successfully for the preparation of disaccharides under the tether approach (Figure 5.18).31

5.1.8 The Ramberg-B¨acklund Reaction This novel procedure introduced by Franck et al. is becoming a practical and versatile approach for the preparation of biologically active C-glycosides such as aromatic,5 aminoacids,32,33 or glycerolipids.34 The reaction sequence for C-glycoside formation consists of the initial S-glycoside formation, transformation to the sulfone derivative, Ramberg-B¨acklund rearrangement involving sulfone extrusion, and hydrogenolysis (Figure 5.19). Another C-disaccharide was prepared by transformation of benzylated exoglycal to the iodide derivative, which in turn was coupled with the sulfur glycosyl donor. Further transformation to the sulfone and Ramberg-B¨acklund rearrangement produced the unsaturated disaccharide, which was ﬁnally reduced under Perlman conditions to provide disaccharide in 70% yield (Figure 5.20).35

5.1. Synthetic Approaches for the Preparation of C-Glycosides

263

RO

RO O

RO RO

i

S

R

OR

ii

O

RO RO

S R O O

OR

RO

RO O

RO RO

iii

OR

O

RO RO

R

R

OR

H

i) mCPBA, Na 2HPO 4/CH 2Cl2, 77%. ii) KOH/Al2O 3, CBrF2CBrF2/tBuOH, 50oC. iii) H 2, Pd(OH) 2/EtOAc.

FIGURE 5.19. The Ramberg-B¨acklund approach for C-glycoside formation.

BnO

O

BnO BnO

I ii

OBn

OBn

BnO

BnO

BnO BnO BnO

O

SH OBn

O

BnO BnO

i

O

BnO BnO

BnO

BnO

OBn

BnO Z

O

iii

OBn

BnO

O

O

BnO BnO

OBn

OBn

BnO

OBn

OBn

HO iv

HO HO HO

HO O

OH O

OH

OH i)a) 9-BBN. b) Ph3 P, I2, Imid. 81%. ii) K 2CO 3, then oxidation. iii) KOH, CCl4 , tBuOH, 60o C. iv) H2, Pd(OH) 2, 70%.

FIGURE 5.20. Synthesis of C-isotrehalose.

5.1.9 Free Radical Approach This approach is based on the generation of free radical at the anomeric carbon by using glycosyl donors, which are subjected to stannous treatment of free radical conditions that in turn will react with mainly exoglycals to produce a C-glycosidic linkage. The general methods leading to anomeric radicals formation are summarized in Figure 5.21:36

264

5. C-Glycosides O

O

X RO m

RO m

n

a) Bu3SnH (Hg-hν or

n

∆)

X = NO2, I, Br, Cl, SeAr, SAr b) Bu3SnSnBu3H (Hg-hν) X = NO2, I, Br, Cl, SeAr, SAr

c) (C2H5)3B (air) or N-acetoxy-2-thiopyridone (W-hν) X = TeAr d) Ester of N-Hydroxy-2-thiopyridone (W-hν) X = COOH e) (Diacyloxyiodo)arenes (Hg-hν or ∆) X = COOH f) Hg-hν X = COBu-t etc g) W-hν X = Co(dmgH)2Py

FIGURE 5.21. General methods leading to anomeric radicals formation.

The coupling reaction between acetobromoglucose and the unsaturated lactone shown in Figure 5.22 will result in the C-disaccharide formation, where a free radical mechanism promoted by a mixture of AlBN-Bu3 SnH is involved.37 Anomer radicals may also generate rearranged products as a result of 1,2migration particularly for the case of acetoxy and phosphate groups. This feature has been exploited successfully for preparing 2-deoxy sugars from commercially available sugars Figure 5.23.38 OAc

HO

OAc

OH

HO

OH

O i

Br +

AcO

AcO O

AcO

OAc

O

O AcO

i) AlBN, Bu3SnH.

FIGURE 5.22. Free radical coupling reaction.

OAc O

5.1. Synthetic Approaches for the Preparation of C-Glycosides OR

OR

O

O

RO

X

RO

265

RO

OR

OR

RO 70-92 %

X = Br R = OAc, OBz, OPO3(Ph)2

FIGURE 5.23. Synthesis of 2-deoxy sugars by reductive 1,2-migration of glycosyl bromide.

5.1.10 Exoglycals Exoglycals have been described as another possibility for preparing C-glycosyl derivatives. The term exo-glycal is given to those unsaturated sugars with exocyclic double bonds. The most representative of these compounds are 1,2- and 5-6unsaturated sugars (Figure 5.24).39 They were ﬁrst prepared by reacting lactones with ethyl isocyanoacetate and subsequent hydrogenolysis40,41 (Figure5.25). This reaction has not been exploited extensively due to sugar oxazole formation. More recently two methods were reported for direct oleﬁnation of lactones. One is based on phosphorous Wittig-type reaction42 and the other by direct methylenation using the Tebbe reagent43 (Figure 5.26). Alternative methods for the preparation of exoglycals includes β-elimination of halides44,45 dehydration, Grignard nucleophilic addition, sulfone extrusion (Ramberg-B¨acklunnd oleﬁnation),46 and tosyl hydrazones (Bamford-Stevens conditions)47 among others (Figure 5.27). The synthesis of several C-disaccharides by using exoglycals has been described. Such is the case of the preparation of C-disaccharide by reaction of two molecules of the C-methylene intermediate under Lewis acid conditions (Figure 5.28). The reaction was proposed to proceed via oxonium cation.48 A 1,3-dipolar cycloaddition of exomethylene sugar with glycosyl nitrone has been proposed as an approach for the formation of amino-C-ketosyl disaccharides (Figure 5.29).49

OR O

R

O OR

FIGURE 5.24. 1,2- and 5,6- unsaturated sugars.

R

266

5. C-Glycosides O

O O

O

O

ii

O

NHCHO

O

O

COOEt

O

i

O

O

O COOEt

O O

NHCHO O

O

i) a) EtOOCCH 2NC, KH. b) AcOH. ii) a) H 2, Pd/C b) H 2O.

FIGURE 5.25. First synthesis of 1,2-unsaturated sugar.

Cl

O

R

O

O

R

i

Cl O

O

O

O

i) P(NMe2 )3 , CCl4 , −30-0oC.

O

TBSO

O

Al

+

O

O

H2 C

Me Me

O

TBSO Cp

i

Ti Cl

Cp

i) THF, Tol, −40oC.

FIGURE 5.26. Early methods for preparation of exoglycals.

O

O

5.1. Synthetic Approaches for the Preparation of C-Glycosides

267

OAc OAc

OAc OAc AgF, Py

O CH2I

AcO

O AcO OAc

OAc

OAc OAc

OAc OAc Br

O

AgF, Py

O AcO

AcO

CN

OAc

OAc OAc O

O

CN

OAc

OAc OAc

1) NaBH 4 2) TsCl, Py

O AcO

AcO

OAc

OAc

Z isomer

BnO

BnO O

BnO BnO R = H, Ph

O S OBn O

i

O

BnO BnO

R

R

OBn R = H (72%) R = Ph Z/E 88:12 (56%)

i) KOH, CCl4, tBuOH, H 2O.

AcO

AcO AcO AcO

O CHNNHTs NHPht

i

AcO AcO

O NHPht

CH2

NaH, dioxane, reflux 74%.

FIGURE 5.27. Alternative methods for the preparation of exoglycals.

5.1.11 The Tether Approach Various approaches for C-glycoside construction are comprehensively reviewed, focusing mainly on the methylene formation.50 The strategies presented are based on the concept that a nucleophilic anomeric donor is condensed with an exomethylene sugar to produce a C-disaccharide linkage.51 According to this strategy methyl

268

5. C-Glycosides BnO O

BnO BnO

BnO BnO BnO

CH3

i

O

OBn BnO

OBn

O

BnO BnO

OH

OBn

BF3.Et2O, CH 3CN, 66%.

FIGURE 5.28. Preparation of C-disaccharide from exoglycals.

R3 R4 BnO

Me

OBn R1 O R2

+ +

BnO BnO

N

i

R4 BnO

O OBn

Glc, Gal, Man

R3

O−

OBn R1 O

O NMe

R2

OMe

O BnO

OBn OBn OMe

i) PhCH3 , reflux.

FIGURE 5.29. Dipolar cycloaddition of exoglycals.

OH

OBn BnO BnO

O O

OBn

SePh Si

O

i

HO HO

O OH OH

O

OBn

O HO

OMe

OH OMe

i) a) Bu3SnH, AlBN, PhH, 60oC. b) HF, THF. c) H2,Pd/C, MeOH, AcOEt.

FIGURE 5.30. C-glycoside construction under the tether approach.

α-C-isomaltoside was prepared from the silaketal connected precursor as shown in Figure 5.30. The tether approach considers the preliminary formation of a temporary attachment usually involving a silyl protecting group, as tether which is cleaved after formation of the desired C-C bond. The general conditions involve the use of selenoglucopyranosides52 or phenylsulfoxides37,53 as glycosyl donors. An important application of this methodology can be seen in the preparation of O-C mixed sulfated trisaccharide (Figure 5.31).13

5.1. Synthetic Approaches for the Preparation of C-Glycosides O

Ph SePh OH +

O O

O

Ph

O

O

O

O

i

O

269

O

OH

O

OMe O

O

OMe

SePh Si

O

OBn O

Ph

O

O

ii

iii,iv

OH O O

BnO

OMe

O

OH

O

O

BzO

O

OBz

BzO

OBn

O

OBn

O

OBn O

O

OMe

OBn

O

HO

OBn

OH

OH

O

O

O

O

HO

vii, viii, ix

O

O

NaO 3SO OH

OH

OBn OMe O

O

vi

OBz

v

OH

O

O

BzO

OBz Br

O

OMe

OBz

BzO

HO

O

HO

OH OMe O

OBn

OH

HO OH

i) a) BuLi; Me2SiCl2 (4.4 equiv.), THF, −78oC to 20oC. b) imidazole, THF, r.t. ii) Bu3SnH (2 equiv.), AIBN, PhMe, 110oC, 17 h. then Bu4NF, THF, r.t., 60%. iii) BnBr, NaH, DMF, 100%. iv) NaBH 3CN, HCl, 70%. v) AgOTf, collidine, MS. 80%. vi) MeONa/MeOH, 97%. vii) Bu 2SnO, MeOH. viii) SO3/ Me3N, 70%, 3 steps. ix) H2, Pd/C, 100%.

FIGURE 5.31. Preparation of sulfated C-trisaccharide under the tether methodology.

270

5. C-Glycosides

References 1. P.S. Belica, and R.W. Franck, Tetrahedron Lett. 39, 8225 (1998). 2. D.E. Levy, and C. Tang, The Chemistry of C-Glycosides, Pergamon Press: Oxford (1995). 3. D.J. McNally, K.V. Wurms, C. Labb´e, S. Quideau, and R.R. Belanger, J. Nat. Prod. 66, 1280 (2003). 4. P.M. Pauletti, I. Castro-Gamboa, D.H. Siqueira-Silva, M.C. Marx-Young, D.M. Tomazela, M.N. Eberlin, V. da Silva-Bolzani, J. Nat. Prod. 66, 1384 (2003). 5. P. Pasetto and W. Franck, J. Org. Chem. 68, 8042 (2003). 6. R.E. Dolle and K.C. Niclolaou, J. Am. Chem. Soc. 107, 1691 (1985). 7. R.E. Ireland, R.C. Anderson, R. Badoub, B. Fitzsimmons, S. McGarvey, S. Thaissivongs, and C.S. Wicox, J. Am. Chem. Soc., 105, 1983 (1983). 8. F. Emery and P. Vogel, Tetrahedron Lett. 4209 (1993). 9. L. Paterson and L.E. Keown, Tetrahedron Lett. 38, 5727 (1997). 10. M.D. Lewis, J.K. Cha, and Y. Kishi, J. Am. Chem. Soc. 104, 4976 (1982). 11. Y. Du, R.J. Linhardt, and I. Vlahov, Tetrahedron 54, 9913 (1998). 12. (a) M.H.D. Postema, C-glycoside Synthesis; CRC Press: Boca Rat´on (1995); b) M.H.D. Postema Tetrahedron 48, 8545 (1992). 13. P. Sina¨y, Pure & Appl. Chem. 69, 459 (1997). 14. J. Gervay, and M.J. Hadd, J. Org. Chem. 62, 6961 (1997). 15. K. Oyama and T.J. Kondo, Org. Chem. 69, 5240 (2004). 16. E. Calzada, C.A. Clarke, C. Roussin-Bouchard, and R.H. Wightman, J. Chem. Soc. Perkin Trans 1, 717 (1995). 17. R.W. Schmidt, B. Frick, B. Haag-Zeino and S. Apparao, Tetrahedron Lett. 28, 4045 (1987). 18. J.M. Lancelin, J.R. Pougny, and P. Sina¨y, Carbohydr. Res. 136, 369 (1985). 19. D. Craig, M.W. Pennington, and P. Warner, Tetrahedron 55, 13495 (1999). 20. R.R. Schmidt and P. Shorma, Carbohydr. Res. 14, 1353 (1985). 21. M.A. Tius, J. Gomez-Galano, X. Gu, and J.H. Zaidi, J. Am. Chem. Soc. 113, 5775 (1991). 22. A. Abas, R.L. Beddoes, J.C. Conway, P. Quayle, and C.J. Urch, Synlett, 1264 (1995). 23. B.A. Johns, Y.T. Pan, A.D. Elbein, and C.R. Johnson, Carbohydr. Res. 136, 369 (1985). 24. T. Kometani, H. Kondo, and Y. Fujimori, Synthesis 1005 (1988). 25. F. Burkhart, M. Hoffmann, and H. Kessler, Angew. Chem. Int. Ed. Engl. 36, 1191 (1997). 26. K.A. Parker and C.A. Coburn, J. Am. Chem. Soc. 113, 8516 (1991). 27. O.R. Martin and W. Lai, J. Org. Chem. 58, 176 (1993). 28. (a) P. Schawab, M.B. France, J.W. Ziller, and R.H. Grubbs, Angew. Chem., Int, Ed. Engl. 34, 2039 (1995). (b) M.H.D Postema, J.L. Piper, and R.L. Betts, J. Org. Chem. 70, 829 (2005). 29. R. Roy, R. Dominique, and S.K. Das, J. Org. Chem. 64, 5408 (1999). 30. T. Maz´eas, J.M. Skrydstrup, and J.M. Beau, Angew. Chem, 107, 990 (1995). 31. S.L. Krintel, J. Jim´enez-Barbero, and T. Skrydstrup, Tetrahedron Lett., 40, 7565 (1999). 32. A. Dondoni and A. Marra, Chem. Rev. 100, 4395 (2000). 33. Y. Ohnishi and Y. Ichikawa, Bioorg. Med. Chem. Lett. 12, 997 (2002). 34. G. Yang, R.W. Franck, R. Bittman, P. Samadder, and G. Arthur, Org. Lett. 3, 197 (2001). 35. F.K. Grifﬁn, D.E. Paterson, and R.J. Taylor, Angew. Chem., Int. Ed. 38, 2939 (1999). 36. H. Togo, W. He, and Y. Waki, and M. Yokohama, Synlett 700 (1998). 37. A. Chenede, E. Perrin, E.D. Rekai, and P. Sina¨y, Synlett 420 (1994).

References

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38. B. Giese, K.S. Gr¨oninger, T. Witzel, H.G. Korth, and R. Sustmann, Angew. Chem. Int. Ed. Engl., 26, 233 (1987). 39. C. Taillefumier and Y. Chapleur, Chem. Rev. 104, 263 (2004). 40. K. Bischofberger, R.H. Hall, and A.J. Jordaan, Chem. Soc. Chem. Commun. 806 (1975). 41. R.H. Hall, K. Bischofberger, S.J. Eitelman, and A.J. Jordaan, Chem. Soc. Perkin Trans. 1, 743 (1977). 42. Y. Chapleur J. Chem. Soc. Chem. Commun. 449 (1984). 43. C.S. Wilcox, G.W. Long, and H. Suh, Tetrahedron Lett. 25, 395 (1984). 44. M. Brockhaus and J. Lehmann, Carbohydr. Res. 53, 21 (1977). 45. S.J. Eitelman, R.H. Hall, and A.J. Jordaan, Chem. Soc., Chem. Commun. 923 (1976). 46. F.K. Grifﬁn, D.E. Paterson, P.V. Murphy, and R.J.K. Taylor, Eur. J. Org. Chem. 1305 (2002). 47. M. Toth and L. Somsak, J. Chem. Soc., Perkin Trans. 1, 942 (2001). 48. L. Lay, F. Nicotra, L. Panza, G. Russo, E. Caneva, J. Org. Chem. 57, 1304 (1992). 49. X. Li, H. Takahasi, H. Ohtake, and S. Ikegami, Tetrahedron Lett, 45, 3981 (2004). 50. G. Casiraghi, F. Zanardi, G. Rassu, and P. Spanu, Chem. Rev. 95, 1677 (1995). 51. B. Vauzeilles, D. Cravo, J.-M. Mallet, and P. Sina¨y, Synlett, 522 (1993). 52. A.J. Fairbanks, E. Perrin, and P. Sina¨y, Synlett 679 (1996). 53. D.S.T. Mazeas, O. Doumeix, J.-M. Beau, Angew. Chem. Int. Ed. Engl. 33, 1383 (1994).

6 Glycoconjugates

Carbohydrates covalently attached to proteins and lipids produce three types of glycoconjugates: proteoglycans, glycoproteins, and glycolipids. Although in the ﬁrst two cases the types of linkages are the same, chemically proteoglycans behave as polysaccharides and glycoproteins having much less carbohydrate content as proteins. The third important class of glycoconjugates, constituted by a carbohydrate residue covalently attached to a lipidic component, has been classiﬁed into four types depending on the lipidic nature: glycoglycerol, glycosyl polyisoprenol pyrophosphates, fatty acid esthers, and glycosphingolipids.1 The most common monosaccharides residues found in glycoconjugates are D-galactose, D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, Lfucose, D-xylose, and sialic acids (Figure 6.1).

6.1 Biological Function and Structural Information Glycoproteins and glycolipids are major components of the outer surface of mammalian cells. The former have been implicated in several essential events such as immune defense, viral replication, cell-cell adhesion, inﬂammation and cell growth, while the latter in cell-cell recognition, growth, differentiation, and interaction with proteins of viral and bacterial pathogens. It is attributed to the hepatic Gal2 /GalNAc-binding receptor as the ﬁrst recognition discovery of carbohydrates as biological signals.2 Subsequently, Man6-phosphate receptor for lysosomal enzymes and Man-receptor from alveolar macrophages were reported and investigated.3,4 In the cellular immune system, some speciﬁc glycoproteins are implicated in the folding, quality control, and assembly of peptide-loaded major histocompatibility complex antigens and the T cells receptor complex. Furthermore, the oligosaccharides linked to glycoproteins provide protease protection, ER-associated retrograde transport of misfolded proteins, loading of antigenic peptides into MHC I class I, and inﬂuence the range of antigenic peptides generated in the endosomal pathway for presentation by MHC class II.5 272

6.1. Biological Function and Structural Information OH OH

OH OH

OH OH O

O OH

HO

273

O OH

HO

HO HO

OH

OH

NHAc β-D-mannose

β-D-galactose

OH OH

N-acetyl-β-D-glucosamine

OH

OH O

O OH

HO

O

H 3C

OH

OH

HO OH

HO OH

NHAc

β-D-xylose

α-L-fucose

N-acetyl-β-D-galactosamine

OH

OH OH

HO

O H2 N

COOH

HO α-L-sialic acids

FIGURE 6.1. Monosaccharides residues of glycoproteins.

In addition, enveloped viruses such as human immunodeﬁciency virus (HIV) evades immune response by exploiting the host glycosylation machinery to protect potential antigenic epitopes.6 They also use the host secretory pathway to fold and assemble their often heavily glycosylated coat proteins. Another important fact to mention is that normal cells and tumor cells have evident differences in glycoprotein content on their cell membranes. Altered glycoproteins of the tumor membranes such as Thomsen-Friedenreich (T antigen) are tumor-associated antigen and belong to the class of O-glycoproteins.7−9

6.1.1 Classiﬁcation of Glycocoproteins Based on the type of the glycosidic bond formed between the sugar and the protein residues, glycoproteins are divided in N - and O-glycans. The ﬁrst type involved the glycosidic linkage between asparagine and N -acetylglucosamine and the second involves an O-glycosidic linkage between the sugar residue (fucose, galactose, N acetylgalactosamine, and N -acetylglucosamine) and the oxygen in the side chain serine, threonine, or hydroxyl lysine.

274

6. Glycoconjugates

It is known that N-linked glycans contain the pentasaccharide Manα1-6(Manα13)Manβ1-4GlcNAcβ1-4GlcNAc as a common core, and they have been classiﬁed into four main groups on the basis of the structure and the location of glycan residues added to the trimannosyl core: oligomannose, complex, hybrid, poly-Nacetylglucosamine (Figure 6.2).10 First type Manα1−2Manα1 6

Manα1

3

6 Manβ1−4GlcNAcβ1 4GlcNAc Asn 3

Manα1−2Manα1 Manα1−2Manα1 2 Manα1

Second type NeuNAcα2−6Galβ1−4GlcNAcβ1 NeuNAcα2−6Galβ1−4GlcNAcβ1 NeuNAcα2−6Galβ1−4GlcNAcβ1 NeuNAcα2−3Galβ1−4GlcNAcβ1 NeuNAcα2−6Galβ1−4GlcNAcβ1

Fucα1 GlcNAcβ1 6 4 Manα1 2 6 6 4 Manβ1−4GlcNAcβ1 4GlcNAc Asn 3 4 Manα1 2

Third type Manα1 6

Manα1

3 Manα1

6 Manβ1−4GlcNAcβ1 4GlcNAc Asn 3 6 4

NeuNAcα2-3Galβ1-4GlcNAcβ1

GlcNAcβ1

4 2 Manα1 GlcNAcβ1

Fucα1

Fourth type GlcNAcβ1

Fucα1

NeuNAcα2−3(Galβ1−4GlcNAcβ1-3)oGalβ1-4GlcNAcβ1 6

Manα1

2 6 4 6 Manβ1−4GlcNAcβ1 4GlcNAcβ1 Asn 3

NeuNAcα2−3(Galβ1−4GlcNAcβ1-3)mGalβ1-4GlcNAcβ1 NeuNAcα2−3(Galβ1−4GlcNAcβ1-3)nGalβ1-4GlcNAcβ1 4 2 Manα1 NeuNAcα2−6Galβ1-4GlcNAcβ1

FIGURE 6.2. The four groups of N-linked glycans.

6.1. Biological Function and Structural Information Core 1

GalNAcβ1−3GalNAcα

Core 2

GlcNAcβ1−6GalNAcα

Galβ1−4 GlcNAcβ1 Core 3 Galβ1−4 GlcNAcβ1

275

6 GalNAcα−Ser(Thr) 3

Galβ1−4GlcNAcβ1 Core 4 Galβ1−4GlcNAcβ1

6 Galβ1−4GlcNAcβ1−3GalNAcα−Ser(Thr) 3

Galβ1−4(GlcNAcβ1−3Galβ1−4)n-GlcNAcβ1 Core 5 Galβ1

6 GalNAcα−Ser(Thr) 3

Galβ1−4GlcNAcβ1 Core 6 Galβ1−3GlcNAcβ1

6 Galβ1−3GlcNAcβ1−3Galβ1−3GalNAcα-Ser(Thr) 3

FIGURE 6.3. Core structures in O-linked glycans.

O-glycans do not present common core structures and until now they have been classiﬁed in at least six groups according to different core structures (Figure 6.3).

6.1.2 Recognition Sites There are two main classes of glycosidic linkage depending on the type of glycosidic bond formed between the sugar residue and the protein: the O-linked glycans involving the amino acids serine, threonine, and hydroxyl lysine, and N -linked glycans involving the amino acid asparagine in the form of tripeptide with sequence AsnXSer (where X is any amino acid except proline). Thorough studies with sugar analogs indicate that presumably the most important of the substituents is the equatorial OH-group on carbon 3. Also important is the OH-group on carbon 4 which can be either axial or equatorial depending on the glycoprotein. Regarding C-2, there is certain tolerance; however, the size of the group should not be too large. Finally, C-6 and the anomeric carbon apparently do not play signiﬁcant roles in the binding (Table 6.1).11

276

6. Glycoconjugates

TABLE 6.1. Sugar requirements for three different glycoproteins. Rat hepatic

Chicken hepatic

Detrimental

α ≈ β large substituents tolerated, negative group Enhancing

Tolerable

Eq. N-Ac enhance binding

Eq. N-Ac enhance binding

No effect by N-Ac

1

2

MBP-A

3

Eq. OH required

4

Axial OH required

Eq. OH required

5

Eq. OH required

Large subst. accepted

6.1.3 Structural Information of Glycoproteins A better understanding about the conformation of glycoproteins has been reached by using NMR, molecular dynamics (MD), and in some cases X-ray diffraction techniques. The high motion of oligosaccharides mainly across the glycosidic linkage (Figure 6.4) has limited the unambiguous conformational determinations in glycoproteins. However, the conformations from the MD simulations are in good agreement with the values from NMR studies. It has been observed that ω-angle prefers Gauche conformation by solvation effects with φ-angle largely determined by the anomeric effect, and the ψ-angle highly inﬂuenced by nonbonded interactions.12 The linkage between the sugar residue and the amino acid asparagine (N-linked glycans) is planar along the C1-NH-C=O glycosidic linkage and ﬂexible along the CO-CH2 -CH- bonds (Figure 6.5). Based on the considerations that N-glycosidic linkages are rigid for the amide group and ﬂexible for the side chain angles, the conformational motion of the glycoproteins depends on the ﬂexibility of the asparagine side chain. This ﬂexibility will have considerable effect on the volume occupied by the sugar and the shielding effects of the carbohydrate over the protein surface. OH O

HO HO HO

ω

O O

HO HO

ψ

HO

φ

OH O

O HO HO

OH

FIGURE 6.4. Angles of rotation of carbohydrates.

6.2. Carbohydrate Binding Proteins

FIGURE 6.5. Planarity of the Asn-GlcNAc glycosidic linkage.

OH O

HO

277

H N

HO NHAc

O

Hydrogen bond and van der Waals interactions showed for some cases stacked conformations, and distances across a carbohydrate residue (from O-1 to O-4) of ˚ and for the ﬁrst three residues of the core of an N-linked oligosaccharide 5.4 A ˚ from head to tail.12 extend to approximately 16 A

6.2 Carbohydrate Binding Proteins Carbohydrate binding proteins are deﬁned as those proteins that interact through noncovalent bonds with carbohydrates. Of particular interest are Lectins. which bind reversibly to mono- and oligosaccharides with high speciﬁcity, and are apparently devoid of catalytic activity.13 Carbohydrate binding proteins are widespread macromolecules found in virus, bacteria, plants, and animals and act as recognition determinants including clearance of glycoproteins from the circulatory system, control of intracellular trafﬁc of glycoproteins, recruitment of leukocytes to inﬂammatory sites, adhesion of infectious agents to host cells, and cell interactions in the immune system in malignancy and metastasis.13 Depending on the afﬁnity shown toward the type of monosaccharide, they can be classiﬁed in mannose, galactose/N-acetylgalactosamine, N-acetylglucosamine, L-fucose, and N-acetylneuraminic acid. Due to their high speciﬁcity, lectins speciﬁc for galactose do not recognize glucose or mannose, nor N-acetylglucosamine with N-acetylgalactosamine, but mannose-speciﬁc animals lectins do recognize fucose. Lectins also exhibit high speciﬁcity for di-, tri-, and tetrasacharides and some interact only with oligosaccharides. Moreover, different lectins speciﬁc for the same oligosaccharide may recognize different regions of its surface. Some of the lectins and their afﬁnity ligands are shown in Table 6.2. High-resolution studies involving the protein sequence determination and threedimensional analysis gave insight about the structure and molecular interaction between the sugar ligands and the proteins. As result of this structural analysis, it was observed on the basis of common structural features that lectins fall into three main categories: 1. simple, 2. mosaic or multidomain, 3. macromolecular assemblies.

278

6. Glycoconjugates

TABLE 6.2. Lectines and afﬁnity ligands. Family Legume (plant lectins)

Lectin concanavalin

Abbrev ConA

Ligand MeαMan, MeαGlc Manα3(Manα6)Man

Erithina corallodendron

EcorL

Galβ4Glc

Fava bean

Favin

MeαMan

Griffonia simplicifolia

GSIV

Fucα2Galβ3(Fucα4)GlcNAc

Red kidney bean

PHA

Complex pentasaccharide

Lathyrus ochrus

LOL I,II

Manα3Manβ4GlcNAc, complex octasaccharide

lentil

LCL

MeαMan, MeαGlc

pea

PSL

Manα3(Manα6)Man

Peanut

PNA

Galβ4Glc

Soybean

SBA

Biantennary pentasccharide

cereal

Wheat germ

WGA

NeuAc(α2-3)Galβ4Glc GlcNAcβ4GlcNAc sialoglycopeptide

Amaryllidaceae

Snow drop

GNA

MeαMann mannopentaose

Moraceae

Artocarpus integrifolia

Jacalin

MeαGal

Galectins (animal lectins)

Human heart

Galectin 1

Galβ4GlcNAc octasaccharide

Rat liver

Galectin 2

Galβ4Glc

Simple lectins are most of known plant lectins (Legume, Cereal, Amaryllidaceae, Moraceae, Euphorbiaceae), animal lectins (galectins or formely S-lectins), and Pentraxins, and contain a nonidentical small number of subunits of molecular weight below 40 kDa. Mosaic or multidomain include viral hemagglutinins and animal lectins C- (endocytic lectins, collectins, selectins), P-, and I type. Their molecular weight is variable and is formed by different protein domains, only one of them having the carbohydrate binding site. Macromolecular assemblies are common in bacteria and usually present in the form of ﬁmbriae, which are ﬁlamentous, heteropolymeric organelles present on the surface of the bacteria.14 Most plants lectins recognize and interact with terminal nonreducing units of oligo- and polysaccharides, glycoproteins, and glycolipids. Anomeric preference is an important ﬁnding observed for different carbohydrate binding proteins, for instance, all mannose/glucose binding lectins display great preference for the α-anomeric forms;15 however, lectins from Ricinus communis bind preferentially to β-galactosidases while other lectins make no difference in binding to anomers of GalNAc and GlcNAc. A considerable amount of structural information about carbohydrate binding proteins such as the complete amino acid sequences for various lectins is available.13,16

6.2. Carbohydrate Binding Proteins

279

Asp 80 Ser 211

OH

Asp 83

Gly 213

O HO

O

OH Gly 104

W2

OH O OH

OH

OH

W1

Asn 127

NH O

W3

Glu 129

W4 Leu 212

Ile 101 Asn 41

FIGURE 6.6. Gal(β1→3)GalNAc in the combining site of peanut agglutinin.

6.2.1 Combining Sites Lectins combine with carbohydrates mainly through weak forces such as hydrogen bonding, coordination with metal ions, and hydrophobic interactions. The hydrogen bridge interaction is established between the carbohydrate hydroxyl groups and the amino groups. Additionally, contacts between the carbohydrate and the protein are mediated by water bridges (Figure 6.6).17 Although carbohydrates are essentially polar molecules, there is a signiﬁcant nonpolar or hydrophobic interaction that occurs between the N-acetyl group of amino sugars and the glycerol moiety of neuraminic acid, with the aromatic amino acids phenylalanine, tyrosine, and tryptophan. In the combining site of wheat germ agglutinin with sialyllactose, several van der Waals contacts stabilize the orientation of the sugar ring through nonpolar stacking interactions with the aromatic side chain of Tyr64, and Tyr66 that interacts through nonpolar with the glycerol tail of the N-acetyl neuraminic acid (Figure 6.7).18 Several classes of lectins are ion-dependent for their functional interaction with the ligands. Divalent ions such as calcium and magnesium participate in the stabilization of the amino acid positions that interact with the sugars. The Ca2+ ion establishes a coordination bond with the carbonyl group of asparagine and with one carboxylate oxygen of an acidic amino acid. The Mn2+ does not coordinate any residues that interact directly with the protein, but is involved in ﬁxing the Ca2+ .position (Figure 6.8).19,20 In the interaction of Concanavalin A with the branched trisaccharide Man(α16)[Man(α1-3)]Man, several hydrogen bond contacts between the hydroxyl group of the sugar and the amino acid residues are observed. Some of these interactions are

280

6. Glycoconjugates Tyr 66

OH Tyr 64

OH HO

OH O

O Ser 62

OH

OH

O

OH

O

N

OH

H

OH

Tyr 73

H

O H

HN

Ser 114

O O

O

OH Ser 43

Glu 115

FIGURE 6.7. Sialyllactose in the combining site of wheat germ agglutinin.

bifurcated or involve water and contribute importantly in the recognition process (Figure 6.9).13,21 Carbohydrate binding proteins are classiﬁed in two types: calcium-dependent (C-type glycoproteins), and thiol reagent-dependent (S-type). The former are structurally more diverse (although the binding region known as carbohydrate recognition domain CRD is highly conserved) and more speciﬁc to organs and tissues, while the latter are structurally more conserved and are more widespread among the organs and tissue examined.22 Other carbohydrate binding proteins that do not Asp

NH2 O

Glu

O OH

O Ca

OHO

H O

2+

O As

O H

n

OH NH2

O

O

Glu

FIGURE 6.8. Mannose binding protein C with bound mannose.

6.3. Glycopeptide Synthesis

281

O Thr 15 Tyr 12

CH3

N H

O H H

Tyr 100

O

HO

O H H

O

H O

O

O

O

OH

O

N

Asp 16

OH

O

O H H

O

H

O

OH Leu 99

N H

O

O HO

O

O H O

H

O

H

H 2N

N H

H

Asp 14

NH2 NH

O Asp 208

O

Arg 228

H N O

FIGURE 6.9. Trimannoside binding site of Concanavalin A.

fall into this two categories are ﬁbronectin and laminin, serum immunoglobulins, mannose-phosphate receptor, viral hemagglutinins, and serum amyloid protein. Another important class of carbohydrate binding proteins are known as Selectins (classiﬁed as E-, P-, and L-selectins) and are deﬁned as nonenzymatic and nonimmune proteins involved in the leukocyte recruitment to sites of inﬂammation.2324 It has been found that the tetrasaccharide sialyl Lewis x is the recognition molecule and the use of synthetic sialyl Lewis x conﬁrmed the hypothesis that sulfation increases the afﬁnity for L-selectins.25

6.3 Glycopeptide Synthesis The design of glycopeptides requires a combination of sugar and peptide chemistry, being a substantial part the installation of the O- or N -glycosidic bond26,27 The synthetic approach is in principle designed on the basis of the glycosidic bond required. Thus, while in the case of O-glycopeptides, the synthetic methods relies on the common strategies for the preparation of O-glycosides, for the preparation

282

6. Glycoconjugates O

Fmoc

H H Asn Leu N

H Asn Leu N

COOBzl

O O

BzO BzO

COOBzl

i

CH2 O

CH2

O

BzO BzO

O OBz

OBz i) Morpholine

FIGURE 6.10. Peptide protecting group Fmoc and removal conditions.

of N -glycopeptides the strategy of choice involves the coupling between the amino glycosyl donor with aspartate in the presence of a condensing agent or by enzymatic catalysis. Compatibility between the protecting groups and the glycosidic bond when they are subjected to different reaction conditions such as acid or base conditions is a sensitive issue. For instance, it is known that the glycosidic bond in acetals is acid–sensitive; however, in the case of O-glycosyl serine and threonine, they conversely present base-sensitivity. The introduction of selective protecting groups for amino acid functionalities that can be cleaved under mild conditions without affecting the glycoside bond or protecting groups attached to the sugar moiety is a feasible approach. Widely employed protecting groups used for this purpose is the Fmoc protecting group (9-ﬂuorenyl)methoxycarbonyl), Pyroc (2(pyridyl)ethoxycarbonyl), and Aloc (allyloxycarbonyl) for the peptide and Mpm (4-methoxy-benzyl ether) for the sugar region. The conditions needed for the cleavage of the mentioned protecting group in the presence of other functionalities are indicated in Figure 6.10.28,29 The synthesis of N-α-FMOC amino acid glycosides was carried out from O’Donnell Schiff bases or from N-α-FMOC amino protected serine or threonine and the appropriate glycosyl bromides under Koenigs-Knorr modiﬁed conditions.30 The α-FMOC-protected glycosides were incorporated into 22 encephalin glycopeptides analogs (Figure 6.11).

RO RO RO

O O O OR

R = Ac, Bz

RO RO RO

OH N-FMOC O

O O OR

OH N-FMOC

AcO O

AcO AcO

OAc

AcO O AcO

O OAc

AcO AcO AcO

O O

O O O OAc

FIGURE 6.11. N-α-FMOC-amino acid glycosides.

OH N-FMOC

OH N-FMOC

6.3. Glycopeptide Synthesis Mpm

OMpm OMpm

OMpm OMpm

OMpm

O

H3 C

O MeO OAc OAc HO O O AcO OAc

N3

OMpm

O

H3 C

Br

O

283

OAc OAc O O O AcO OAc

i

NHAc

OMpm O N3 NHAc

Teoc

Cl3C

OAc OAc OAc

O

COOH

OAc v

O

OAc OAc O O O AcO OAc

NH2 NHAc CCl3

OAc OAc H 3C

O

O

ii-iv

H3 C

O

H N

O

O

O OAc

OAc OAc O O O AcO OAc

O

OAc O

NH H N

NHAc

C O

O O

i) NEt4Br. ii) CAN. iii) Ac2 O, Py. iv) H2 , Raney-Ni. v) carbodiimide-HOBt, CHCl3.

FIGURE 6.12. Synthesis of glycopeptide Lewisa .

Pyroc is another protecting group useful in peptide chemistry. It is stable to acids, bases, and hydrogenolysis, but sensitive to morpholine. The allylic protecting group Aloc is also stable to acid, base, and can be removed under of Pd(0) catalysis or weak base as morpholine.29 A tumor-associated antigen Lewisa was synthesized by applying a combination of compatible sugar and peptide protecting groups. For this method the azide group was used as anomeric amine precursor (Figure 6.12).30 Enzymes has been useful for peptide elongation using an engineered subtilisin and disaccharide bond formation with glycosyltransferase as shown in Figure 6.13.31 A novel chemoenzymatic synthesis of Ee1 Calcitonine glycopeptide analog having natural N-linked oligosaccharides such as disialo biantennary complex

284

6. Glycoconjugates Cbz-Ala-Ser-OCH3

HO HO

Cbz-Ala-Ser-Gly-Ala-NH2

O

i

O

+

HO HO

NH2-Gly-Ala-NH2

OH

O O OH

OH ii,iii

OH O

NH2-Ala-Ser-Gly-Ala-NH2

O HO

HO OH

O O OH

i) Thiosubtilisin mutant, pH = 9, 50oC. ii) H2, Pd/C. iii) UDP-Gal β1-4-GalTase.

FIGURE 6.13. Chemo-enzymatic synthesis of glycopeptide.

type as model compound for glycoprotein has been described. Natural oligosaccharides were next added by a transglycosylation reaction using endo-β-Nacetylglucosaminidase from Mucor hiemalis (Figure 6.14).32 According to Sears & Wong33 there are three basic approaches for preparing glycopeptides with complex glycans: (1) a converged method consisting in the independent preparation of the sugar and peptide components, and ﬁnal assemble, (2) the preparation of the sugar attached to an amino acid using glycopeptide chemistry and simultaneous peptide-linked to a glycal, (3) solid-phase synthesis of the glycopeptide and chemoenzymatic elaboration of the glycal (Figure 6.15).

GlcNAc H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-ThrAsp-Val-Gly-Ala-Gly-Thr-Pro-NH2

Asn(GlcNAc)3 -eCT NeuAc-Gal-GlcNAc-Man NeuAc-Gal-GlcNAc-Man

Man-GlcNAc-GlcNAc H-Asn-OH

GlcNAc

Endo-M phosphate buffer pH 6.25 8.5 %

H-Asn-OH

NeuAc-Gal-GlcNAc-Man NeuAc-Gal-GlcNAc-Man

Man-GlcNAc-GlcNAc H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-ThrAsp-Val-Gly-Ala-Gly-Thr-Pro-NH2

FIGURE 6.14. A transglycosylation reaction for preparation of glycopeptide.

6.4. Glycoprotein Synthesis

285

A) +

HO

Activated oligosaccharide

B)

Glycopeptide

Peptide

NHP CO2H Oligosaccharideamino acid

SPPS Glycopeptide

C)

NHP CO2H

SPPS

Oligosaccharideamino acid

Enzymatic elaboration Glycopeptide

FIGURE 6.15. Proposed general approaches for glycopeptide synthesis.

6.4 Glycoprotein Synthesis Glycoproteins are essential macromolecules involved in a wide range of functions related to cellular recognition processes. Natural glycoproteins usually exist as a mixture of glycoforms and are found difﬁcult to isolate for their structural characterization and for understanding more about their function.34,35 As mentioned, glycoproteins can be obtained by fermentation process, however this natural approximation produce a population of many different glycoforms as result of the participation of many glycosidases and transferases for a given protein, although the mixture can be useful for preparing homogeneous core which in turn might be reelaborated enzymatically.33 The synthetic preparation of glycoproteins can be considered to some extend glycopeptide chemistry, although the complexity is undoubtedly superior. The synthesis of glycoproteins has received considerable attention, most of it involving a combination of chemical and enzymatic methods.35,36−41 A general strategy proposed by Duus et al.42 considers the assembly of glycosylated amino acid building blocks in solid-phase peptide synthesis according to the general ﬁgure shown in Figure 6.16.

286

6. Glycoconjugates

A OH O PGO

O Aan Aa6Aa5Aa4Aa3Aa2Aa1

linker

OH

1)

O PGO

LG

+ promoter

1)

deprotection

2) 2)

O

deprotection +

HO

+ enzyme

Nu

O HO O HO

O

O O HO

O Aan Aa6Aa5Aa4Aa3Aa2Aa1

linker

B O PGO

1) Activation

O

X

PGO

X

2) Aa5-Aan

PGHN +

HN Aa3Aa2Aa1

O

2)

Aan

Aa6Aa5 OCHN

Y

O HN Aa3Aa2Aa1

linker

linker

FIGURE 6.16. Strategies for glycopeptide synthesis.

According to a comprehensive review the strategies described so far for chemical glycoprotein synthesis are (a) indiscriminate glycosylation, (b) chemoselective and site-speciﬁc glycosylation (c) site-selective glycosylation.43

6.4.1 Indiscriminate Glycosylation This nonselective approach consists of the preparation of sugars bearing functionalities that under proper conditions may react with a protein. Some of the sugar derivatives used for this purpose are shown in Figure 6.17.

AcO

O

a)

(i) (H2N) 2CS

AcO

O

AcO

MeO–

S

(ii) ClCH2CN

NH

O

CN

S

OMe

Br

Lys

H2N

(CH2)4 Protein

AcO

Lys

NH

O S

N H

(CH2)4 Protein

Ref.44 Lys

b)

HO

O

H2N

(CH2)4

Lys

Protein

HO

OH

OH

NaBH3CN

Protein

(CH2)4

N H

Ref.45

c)

HO

HO

O

O

HNO2

O

+

NH2

O

N2

Tyr Protein Tyr Protein

HO

HO

O N

O

N HO

Ref.46 d)

HO

O

HO

(Im)2CS

O

NH2

O

NCS

O

Lys

H2N

(CH2)4

Protein

S HO

Lys

O O

N H

N H

(CH2)4

Ref.47 FIGURE 6.17. Indiscriminate glycoprotein syntheses.

Protein

O O

HO

e)

HO

O

L-Glu-pNA

O

HO

H N

pNA

O

O N

(i) BOP, imd (ii) Pd, H2

OH

(iii) as for (d)

HO

NC6H4NCS

O

O Lys

HO

(CH2)4

H2N

O

Protein

N S

Lys

N H

NC6H4NH

O

(CH2)4

Protein

Ref.48 N2H4

HO

f)

HO

R = OMe

O O

O

HNO2

O

R = NHNH2

(CH2)8COR

(CH2)8CON3

Lys

H2N

(CH2)4

Protein

HO

O O

H N

(CH2)8

Lys

(CH2)4

Protein

O

Ref.

49

O (i) Hal oxidation

HO

g)

O HO

(ii) NaOH(aq.)

O

OH

Lys

Cl H2N

O-Na+

(CH2)4

Et3N

OH O Lys

HO

OH

NH

(CH2)4

Protein

O Lys

(CH2)4

HO HO

h)

Protein

HO O

O NH2

OH

EDC

O HN

OH

O

Ref.50,51 FIGURE 6.17. (Continued)

Asp, Glu

(CH2)1,2

Protein

Protein

6.4. Glycoprotein Synthesis O

O HO

i)

Lys

NH2(CH2)2NH2

O

R = OMe

O

(CH2)8COR

OEt

EtO

H2N

(CH2)4

Protein

R = NH(CH2)2NH2

O HO

289

O Lys

O O

NH(CH2)2 HN

(CH2)8

N H

(CH2)4

Protein

O 52

Ref.

FIGURE 6.17. (Continued)

6.4.2 Chemoselective and Site-Speciﬁc Glycosylation This approach intends to direct selectively the glycosidic linkage by using chemical and enzymatic tools. Such selectivity has been attempted under a strategy coined with the term chemoselective ligation, and some enzymes involved in this strategy are galactose oxidase,53 horseradish peroxidase Examples of these step reactions are indicated in Figure 6.18.

6.4.3 Site-Selective Glycosylation This possibility implies the choice of site selectivity on the glycan. In order to reach this goal a combined site-directed mutagenesis and chemical modiﬁcation has been performed.62,63 This strategy involves the introduction of cysteine as chemoselective tag at preselected positions within a given protein and then reaction of its thiol group with glycomethanethiosulfonate (Figure 6.19).

6.4.4 Enzymatic Synthesis Three basic strategies are considered for obtaining glycoproteins following an enzymatic approach: the elaboration of glycans through the use of glycosyltransferases,64−67 the trimming of glycans by puriﬁcation of glycoform mixtures through selective enzymatic degradation,68 and alteration of glycans or glycoprotein remodelation, consisting in combined trimming of existing glycan structures followed by elaboration to alternative ones. Theses methods were used for preparing an unnatural glycoform of ribonuclease B by using endoH degradation and elaboration with galactosyltransferase, fucosyltransferase, and sialyltransferase system to construct an sLex glycoform.69 Other approaches for the assembling of peptides are the “native peptide ligation”70 and endoglycosidasecatalyzed transglycosylation.32 Recent advances on glycoprotein synthesis proposes and in vitro approaches involve the following sequential steps: (a) remodeling of recombinant glycoproteins

290

6. Glycoconjugates Protein

O

a)

HO

R O

HO

Protein

O

X

X

NH 2

N R

X = O or N

OH

e.g.

O O

HO AcHN

O

54-56

Ref.

O I

b)

HO O

HO

HO

PtO2, H2

O

N3

HO H N

O

I

NH2

EEDQ

O Cys Protein

HO

HS

O

Cys

H N

Protein

S O

Ref.57,58 Cys

S

NO2

Protein

2

N

c)

HS

HO

HO O

O

SH

S

S

NO2 N

Cys

HO O

Protein

S

S

Ref.59 FIGURE 6.18. Chemoselective and site-speciﬁc glycoprotein syntheses.

by using glycosidases and glycosyltransferases: (b) ligation of synthetic glycopeptides by enzymatic or chemical methods: (c) intein-mediated coupling of glycopeptides to larger proteins expressed as intein-fusion proteins: (d) ligation of glycopeptides to larger proteins containing N-terminal cysteine expressed as TEV

O

O

O

(i)

PO

PO

Pd, H2

O

d)

O

N3

O HO

then HMDS, ZnCl2

NH2

N

(ii) deprotect

O

Cys

O

Cys

O

Protein

Protein

HO

HS

S

O

N O

60

Ref.

Cys Protein

HO

e)

O

HO Y

NaSSO 2CH 3

O

O

X

HS

S

CH3

O X = S or O(CH2)2S

Cys

HO O

Protein

X

S

Ref.61-62 FIGURE 6.18. (Continued) HO

O

O

HO

X

S O

or

O

H N

I

Cys

site directed mutagenesis

O

Protein

Protein

HS Cys

HO O

Protein

X

S

X = NHC(O)CH2 or S or O(CH 2)2S

FIGURE 6.19. Site-selective glycoprotein syntheses.

CH3

292

6. Glycoconjugates

FIGURE 6.20. Strategies for glycoprotein synthesis in vitro.

protease cleavable fusion proteins: (e) in vitro translation; and (f) pathway re-engineering in yeast system to produce human–type N-lynked glycoforms (Figure 6.20).71

6.5 Synthesis of Antigenic Glycoconjugates The preparation of complex glycoconjugates has been a current strategy for the design of synthetic vaccines and usually involves the preparation of the oligosaccharide moiety, which provides the immune speciﬁcity by chemical or enzymatic methods, and further attachment through a linker with an immunogenic protein. There has been a continuous effort for developing glycoconjugates containing antigens such as MBr1 antigen Globo-H, the blood group determinant and ovarian cancer antigen Lewis y , N3 antigens associated with gastrointestinal cancer, the adenocarcinoma antigen KH-1, and the small cell lung carcinoma antigen fucosyl GM1, among others (Figure 6.21), as promising alternatives to develop potentially useful carbohydrate-based anticancer vaccines accessible for clinical program. The synthetic approach becomes justiﬁed if we consider that cancer and normal cell growing in tissue culture generally show minimal level of expression of such antigens.72 The chemical synthesis of most of these complex oligosaccharides represents a formidable challenge and requires a convenient combination of strategies that

OH

OH O

OH

HO

OH O

O O

OH

OH O O

O NHAc

O H3C

OH

OH

OH

O

OH O

HO

OH

O OH

OH O

HN

HO

C15H31 C13H27

O OH OH

MBr1 antigen / Globo-H

OH CH3

O

OH CH3

O OH

OH

OH OH O

OH O

HO

O

O

O H3C OH

O

OH OH

OH O

O NHAc

O

OH OH O O

OH

O NHAc

OH

OH

OH O

OH O

O HO

O

OH

KH-1 antigen

HN

C15H31 C13H27

R=

OH

OH CH3 O O

OH OH

OH OH O

OH O

HO

O

O

O H3C

O

OH

OH

HN

C15H31 C13H27

O NHAc OH

OH

OH Lewisy blood group determinant

OH O

HO

O OH H3C

O OH

OH OR OR'

OH O O NHAc

O

OH OH O O NHAc

OH O OH

O HO

OR OH

OH

OH O OH OH

major N3 antigen (R = α-fucose, R' = β-Gal) minor N3 antigen (R' = α-fucose, R = β-Gal)

FIGURE 6.21. Carbohydrate structures of tumor-associated antigens.

294

6. Glycoconjugates

OH

OH O

HO O

OH

OH O

O

O O

H3C

OH

OH

O NHAc

OH

OH O

O

O OH

HO2C

OH O HO

HN

C15H31 C13H27

O OH OH

O OH HO HO

Fucosyl GM1

NHAc

HO

FIGURE 6.21. (Continued)

allowed suitable manipulations using appropriate protecting groups, glycosyl donors, acceptors, and coupling reactions conditions. For instance, the synthesis of glycolipid KH-1 was achieved by Desphande et al.72 based on the glycal methodology (Figure 6.22). Likewise, the synthesis of the water-soluble galactosphingolipid analog that binds speciﬁcally to recombinant gp 120 was prepared by condensation of Cglucosyl aldehyde with Wittig reagent affording the oxazolidone which was transformed into the C-glycosylamino acid. By following a subsequent standard protocol represented in Figure 6.23 the target glycolipid was constructed.73 Glycoside ceramides are important molecules involved in apoptosis or active cell death. In leukemia cell lines C2 ceramide induces apoptosis via the sphingomyelin pathway. It has been observed that α-galactosylceramides having more than 10 carbons in fatty acid chain have immune stimulatory activities. Thus, the α-Gal-C2 was synthesized by direct glycosylation of C2-Cer with galactosyl ﬂuoride donor in the presence of silver perchlorate as condensing agent (Figure 6.24).74 The convergent synthesis is a procedure consisting in the parallel preparation of fragments or building block that will be connected through a coupling reaction, prior deprotection. This procedure was applied successfully for preparation of glycosylphosphatydil inositols (GPI), which are involved in the attachment of glycoproteins with eukaryotic cells (Figure 6.25).75 The potential of carbohydrates as antibiotics, antiviral, and anticancer substances has been established.76,77 Besides, it has been demonstrated their involvement in fertilization, embryogenesis, regulation of the immune system tissue repair, neuronal development, intracellular pathways, and cancer transformation, among others.12 There is an increasing understanding of how carbohydrates behave biologically between normal and disease states and with this accurate information, novel carbohydrates and therapeutic approaches are developed.78 For instance, novel glycoside sulfates have been reported as novel potentially useful drugs (Figure 6.26).76,77

O

OBn O

O O

O OH

OAc OBn O O OAc NHSO2Ph

OBn O HO

O HO

OBn O

OAc OBn O

OBn O

O NHSO2Ph

O BnO

OAc

F H3C

O

OBz OBz CH3

OBz CH3

O

O OBn

O O O

O O

H3C

O

OH

OBn

OBn OBn O

OBn O

OAc OBn O O OAc NHSO2Ph

O

OH

O

OBn OBn O O

OBz CH3

O NHSO2Ph

OBn

O O

H3C

O

OH

OBn

OAc OBn O O OAc NHSO2Ph

O

OH

O

OBn OBn O O

O NHSO2Ph

OBz CH3 OBn

O O

H3C OH

O

SEt

OAc

OAc

OAc OBn O

OBn O

O OBn

OBn OBn O

OBn O

O

O BnO

OBz CH3

O

O

OBn O

OAc OBn O

iii

OH

O

OAc

O OBn OBn O

OBn O

O

O BnO

OBz CH3

O

O

OBn O

OAc OBn O

ii

OH

O

i

OBn

OBn

OAc OBn O O OAc NHSO2Ph

O

O

OBn OBn O O

O NHSO2Ph

OH

O BnO

OAc

R OAc

NHC(O)C15H27

OH N3

R=

C13H27

HO OBn

C13H27

O

iv,v

OBn

KH-1

i) Sn(OTf), PhMe/THF (10:1), 4 Å MS. ii) a) DMDO, CH2Cl2. b) EtSH, CH2Cl2, H+ (cat.). c) Ac2O, Py, CH2Cl2. iii) MeOTf, Et2O/CH2Cl2 (2:1) 4 Å M.S. iv) a) H2/Pd-CaCO3, palmitic anh. EtOAc. v) a) Na/NH3, THF, then MeOH. b) Ac2O, Et3N, DMAP, CH2Cl2. c) MeONa, MeOH.

FIGURE 6.22. Synthesis of KH-1 antigen.

296

6. Glycoconjugates PPh3+

OBn OBn O

O

BnO

OBn OBn O

i

HN

+

O

O OBn OBn O

O

iii

O

BnO

OBn OBn O

iv

BnO

BnO

N H

OBn

N H

OBn OBn OBn O

OBn BocHN

OBn BocHN

v

OH vi

O

BnO

OH O O

HO

OBn BocHN

NH(CH2)13CH3

OH +H N 3

O

NH(CH2)13CH3 O

i) BULi, THF. ii) TsNHNH, NaOAc, DME, H 2/cat. iii) a) Boc2O, Et3N, DMAP. b) CSCO 3, MeOH. iv) Jones. v) EDC, HOBt, tetradecylamine. vi) a) TFA. b) H2/Pd-C.

FIGURE 6.23. Synthesis of galactosphingolipid analog.

O OBn OBn

HN HO

(CH2)12CH3

+

O BnO OBn

OH i,ii

OH

OH

O

O HO

HN HO

O

OH O

OH

OBn OBn O

ii

O

BnO

H

OBn

O

(CH2)12CH3 OH

i) AgClO4, THF, MS, 2h. ii) Na/NH3, 2h.

FIGURE 6.24. Synthesis of glycosylceramide.

F

6.5. Synthesis of Antigenic Glycoconjugates

297

O

N H

H N O

O

O

P

O

OH O

O HO HO OH

HO

O O

HO HO

O HO HO

OH O HO O HO

O H3 N

O O HO

HO O

C15H31 OH OH

O H2NH2CH2CO

P

BnO

N H

H N O

O

BnO BnO

O OH OH

+

OBn O

BnO BnO BnO

O O O BnO BnO

OBn O

BnO O BnO

O BzCHN

O MBnO

O BnO O

C15H31 OBn OBn

FIGURE 6.25. Retrosynthesis for the preparation of GPI-anchored peptide using convergent synthesis.

It has been mentioned that carbohydrate-based agents such as glycoproteins and polysaccharides obtained from synthetic routes is an emerging and promising strategy for the preparation of vaccines.79−81 This possibility has become available due the remarkable progress for the chemical and enzymatic preparation of oligosaccharides.

298

6. Glycoconjugates O

N H

H N O

O

O

P

O

OH O

O HO HO OH

HO

O O

HO HO

O HO HO

OH O HO O HO

O H3 N

O O HO

HO O

C15H31 OH OH

O H2NH2CH2CO

P

BnO

N H

H N O

O

BnO BnO

O OH OH

+

OBn O

BnO BnO BnO

O O O BnO BnO

OBn O

BnO O BnO

O BzCHN

O MBnO

O BnO O

C15H31 OBn OBn

FIGURE 6.25. (Continued)

Recent developments on carbohydrate chemistry made possible the design and escalation of new immunogenic carbohydrates. A newly developed synthetic carbohydrate attached to a protein carrier was reported by Verez-Bencomo and Fern´andez-Santana, and currently administrated against Haemophilus inﬂuenzae type b disease. The chemical synthesis leading to the oligomeric polyribosylribitol phosphate is described in Figure 6.27.82

SO3–

-O2C

O

HO HO

SO3– O

SO3–

-O2C

O

O NHAc HO

O OH

SO3– O O

O OH

NHAc

Chondroitin sulfates

O N

OH

NH

O

HO HO

HO3SO

HO

N

O

SO3– O

N

NH2

OH

O

OH

O

OH OH O

tetrahalose-2-sulfate

OH OH

OH

ribonucleoside monosulfate

OH

O O

HO HO

HN

(CH2)24CH3 OH

SO3–

(CH2)13CH3 OH C-glycoside KRN7000 Me

HO HO HO

O O

O O HO AcHN CO OH HN

O

O

HO

OH HO

CONH2 O O O O P O OH O NHAc

HO HO

Me O O

OH

OH

Menomycin A

SO3– Na+ O

HO R3O R3O

OR2 O OR1

R1 = oleoyl R1 = linoleoyl R1 = palmitoyl R1 = linoleoyl

R2 = palmitoyl R2 = palmitoyl R2 = palmitoyl R2 = palmitoyl

R3 = H R3 = H R3 = H R3 = palmitoyl

FIGURE 6.26. Novel glycoside sulfates and phosphates as potential drugs.

AcO

OAc

O

RO

HO OBn OBn OBn OAll

+

AcO

OAc

i,ii

RO

BnO

O

O

OR

OBn OBn OBn OAll

O

O OBn OBn OBn OR

iii-iv

R1O

R = Ac R=H

OBn

R = CH2CH=CH2; R1 = H R = CH=CHCH3; R1 = H

v-vii

BnO

O

O

OBn OBn OBn OH

O Et3 BnO

vi-vii

O

O

P O

N3

O

O

OR

O

P

X

O

O

O

O

O +Na–O

OR

OR

viii-xi

RO RO

P O OH

OBn OBn OBn OH

O Et3NH+ –O

NH+ –O

OR OR OR O O P O– Na+

O

O

OR

OR OR OR OH

X = N3; R = Bn X = NH2; R = H

O

HO HO

O

O

O

+Na–O

H N

N

P

O

O

O

OH

OH OH OH O O P

O– Na+

O

O

OH

7

O O

S O

O

O

N H

Protein 20-25

i) BF3Et2O, CH2Cl2. ii) CH3ONa, CH3OH. iii) BnCl, BuSnO, NaH, Bu4NI. iv) tBuOK, DMSO, 100oC. v) PCl3, imidazole, CH3CN. vi) N3(CH2)2O(CH2)2OH, I2. vii) AcOH-H2O, 80oC. viii) PivCl, Py. ix) Py-H2O. x) H2, Pd-C, EtOH-H2O-EtOAc-AcOH, 1.5 atm. xi) cation exchange resin on Sephadex SP-C25.

FIGURE 6.27. Synthetic carbohydrate conjugate vaccine Quimi-Hib.

OH OH OH OH

References OH OH OH OH OH OH O O O HO O O O NHAc OHO H3 C O OH Globo H HO OH OH

OHOH

HO

AcHN OH O O OH HO

H 3C O

OH O OH

CO2 H O

HO

STn

OH OH Tn O HO AcHN O

O

OH O

O

HO AcHN

OH HNAc

301

H N

O

O N H

O O OH OH OH OH OH OH OH O O O O HO O O O O O NHAc OH H3 C OH O AcHN OH HO OH OH O O O Lewisy OH OH OH OH

H N

O N H

H N O

H N

Linker

S O Carrier protein

TF

FIGURE 6.28. Various tumor antigenic agents coupled to a linker developed as potential synthetic vaccine.

Another alternative therapeutic strategy for inducing immune response through the use of synthetic carbohydrate vaccines has been proposed by Danishefsky et al., involving the attachment of different tumor antigenic agents (Globo H, STn, Tn, Lewis y ), coupled to a linker and this to a protein carrier (Figure 6.28).83

References 1. J.F. Robyt, Essentials of Carbohydrate Chemistry, Springer-Verlag, New York, Inc. 1998, 279. 2. A. Morell, R.A. Irvine, I. Sternliev, I.H. Scheinberg, and G. Ashwell, G. J. Biol. Chem. 1968, 243, 155. 3. H.D. Fischer, A. Gonzalez-Noriega, W.S. Sly, and D.J. Morre, J. Biol. Chem. 1980, 255, 9608. 4. P. Stahl, P.H. Schlesinger, E. Sigardson, J. Rodman, Y.C. Lee, Cell 1980, 19, 207. 5. P.M. Rudd, T. Elliot, P. Cresswell, I.A. Wilson, and R.A. Dwek, Science 2001, 291, 370. 6. I.N. Reitter, R.E. Means, and R.C. Desrosiers, Nature Med. 1998, 4, 679. 7. G.F. Springer, Science 1984, 224, 1198. 8. J. Samuel, A.A. Noujaim, G.D. MacLean, M.R. Sureshand, and B.M. Longenecker, Cancer Res. 1990, 50, 4801. 9. M. Fukada, Biochim. Biophys. Acta, 1985, 780, 119. 10. R. Kornfeld, and S. Kornfeld, Annu. Rev. Biochem. 1985, 54, 631. 11. Y.C. Lee, FASEB J. 1992, 6, 3193–3200. 12. R. Dwek, Chem. Rev. 1996, 683. 13. H. Lis and N. Sharon, Chem. Rev. 1998, 98, 637. 14. W. Gaastra and A.-M. Svennerholm, Trends. Microbiol. 1996, 4, 444. 15. I.J. Goldstein, H.C. Winter, R.D. Poretz, In Glycoproteins, Elsevier 1997, 403–474. 16. W.I. Weis and K. Drickamer, Annu. Rev. Biochem 1996, 65, 441.

302

6. Glycoconjugates

17. R. Ravishankar, N. Ravindran, A. Suguna, A. Surolia and M. Vijayan, Curr. Science, 1997, 72, 855. 18. N. Sharon, Trends Biochem. Sci. 1993, 18, 221. 19. W.I. Weis, and K. Drickamer, Annu. Rev. Biochem. 1996, 65, 441. 20. N. Sharon, and H. Lis, Essays Biochem. 1995, 30, 59. 21. J.H. Naismith and R.A. Field, J. Biol. Chem. 1996, 271, 972. 22. K. Drickamer, J. Biol. Chem. 1988, 263, 9575-9560. 23. F. Fukumori, N. Takeuchi, T. Hagiwara, H. Ohbayashi, T. Endo, N. Kochibe, Y. Nagata, and A. Kobata, J. Biochem. 1990, 107, 190. 24. L.A. Lasky, Annu. Rev. Biochem. 1995, 64, 113. 25. S. Hemmerich, H. Lefﬂer, and S.D. Rosen J. Biol. Chem. 1995, 270, 12035. 26. H. Kunz, Angew. Chem. Int. Ed. Engl. 1987, 26, 294. 27. H. Garg and R.W. Jeanloz, Adv. Carbohydr. Chem. Biochem. 1985, 43, 135. 28. H. Kunz, Pure & Appl. Chem. 1993, 65, 1223. 29. H. Kunz and C. Unverzagt, Angew. Chem. Int. Ed. Engl. 1984, 23, 436. 30. J. M¨arz and H. Kunz, Synlett 1992, 589. 31. C.H. Wong, M. Schuster, P. Wang, and P. Sears, J. Am. Chem. Soc. 1993, 115, 5893. 32. M. Mizuno, K. Haneda, R. Iguchi, I. Muramoto, T. Kawakami, S. Aimoto, K. Yamamoto, and T. Inazu, J. Am. Chem. Soc. 1999, 121, 284. 33. P. Sears and C.-H. Wong, Science 2001, 291, 2344. 34. P.P. Deshpande, H.M. Kim, A. Zatorski. T.K. Park, G. Raguphathi, P.O. Livingston, D. Live and S.J. Danishefsky, J. Am. Chem. Soc. 1998, 120, 1600. 35. C.R. Bertozzi, D.G. Cook, W.R. Kobertz, F. Gonzalez-Scarano, M.D. Bednarski, J. Am. Chem. Soc., 1992, 114, 10639. 36. L.M. Obei, C.M. Linardic, L.A. Karolak, and Y.A. Hannun, Science 1993, 259, 1769. 37. J. Xue, N. Shao, and Z. Guo, J. Org. Chem. 2003, 68, 4020-4029. 38. U. Kempin, L. Henning, D. Knoll, P. Welzel, D. M¨uller, and J. Markus, van Heijenoort, Tetrahedron, 1997, 53, 17669. 39. S. Loya, V. Reshef, E. Mizrachi, C., Silbertein, Y. Rachamim, S. Carmeli and A. Hizi, J. Nat. Prod. 1998, 61, 891. 40. A. Persidis, Nature Biotechnology 1997, 15, 479. 41. T. Buskas, Y. Li and G.-J. Boons, Chem. Eur. J. 2004, 10, 3517. 42. J.Ø Duus, P.M.St. Hilaire M .Meldal, and K. Bock, Pure. Appl. Chem. 1999, 71, 755. 43. (a) B. G. Davis, Chem. Rev. 2002, 102, 579. (b) C.R. Bertozzi and L.L. Kiessling, Science 2001, 23, 2357. 44. Y.C. Lee, C.P. Stowell, and M.J. Krantz, Biochemistry 1976, 15, 3956. 45. G.R. Gray, Arch. Biochem. Biophys. 1974, 163, 426. 46. C.R. McBroom, C.H. Samanen, and I.J. Goldstein, Methods Enzymol. 1972, 28, 212. 47. D.H. Buss and I.J. Goldstein, J. Chem. Soc. C 1968, 1457. 48. C. Qu´etard, S. Bourgerie, N. Normand-Sdiqui, R. Mayer, G. Strecker, P. Midoux, A.C. Roche, and M. Monsigny, Bioconjugate Chem. 1998, 9, 268. 49. R.U. Lemieux, D.R. Bindle, and D. A. Baker, J. Am. Chem. Soc. 1975, 97, 4076. 50. W.O. Baek and M.A. Vijayalaksmi, Biochim. Biophys. Acta 1997, 1336, 394. 51. K.-Y. Jiang, O. Pitiot, M. Anissimova, H. Adenier,and M.A. Vijayalakshmi, Biochim, Biophys. Acta. 1999, 1433, 198. 52. V.P. Kamath, P. Diedrich, and O. Hindsgaul, Glycoconjugate J. 1996, 13, 315. 53. G.A. Lemieux and C.R. Bertozzi, TIBTECH 1998, 16, 506. 54. S.E. Cervigni, P. Dumy, and M. Mutter, Angew. Chem. Int. Ed. Engl. 1996, 35, 1230. 55. Y. Zhao, S.B.H. Kent, and B.T. Chait, Proc. Natl. Acad. Sci. USA, 1997, 94, 1629.

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7 Hydrolysis of Glycosides

The glycosidic bond might be degraded by chemical and/or enzymatic agents. Comparative studies revealed that chemical hydrolysis is nonspeciﬁc and on the other hand the enzymatic is regio and stereospeciﬁc. The glycosides are chemically susceptible to acid conditions, and only in some cases to basic conditions. In general, the acid sensitivity is attributed to the sugar moiety and the basic nonstability to the aglycon nature.

7.1 Acidic Hydrolysis When a glycoside is subjected to acid conditions, a process called acetolysis takes place. This phenomenon is more clearly seen on O-glycosides, where even weak acid conditions can be sufﬁcient for O-glycoside breakage. Some simple glycosides such as β methyl-2,3,4,6-tetra-O-methyl-D-glucopyranose are hydrolyzed under diluted HCl conditions to yield a hydroxy-2,3,4,6-tetra-Omethyl-D-glucopyranose. Likewise β ethyl-glucopyranose is hydrolyzed to a mixture of anomers (Figure 7.1). In general, S-glycosides are more resistant than their counterparts O-glycosides to an acidic medium; however, the former can be hydrolyzed under the conditions described in Figure 7.2. Disaccharides can be readily hydrolyzed under weak acidic conditions, producing their constitutive monomers in equivalent quantities (Table 7.1). Depending on the strength of the hydrolytic conditions, polysaccharides undergo fragmentation, producing oligosaccharides, disaccharides, and monomers. The degradation degree relies on acid concentration, branching, and solubility. Thus, cellulose, being the most abundant natural polisaccharide in nature, requires high acidic concentrations in order to be fully degraded to glucose. On the contrary, some other polysaccharides at lower concentrations produce dimers and monomers (Table 7.2). Partial hydrolysis is important in certain cases in which disaccharides are not either affordable materials or easily obtained ones through synthetic means. Such 304

7.1. Acidic Hydrolysis OMe O MeO MeO

OMe

HO

OMe O

i

MeO MeO

OMe OH O

HO

OMe OH

OH O

i

OH OEt

HO

HO

OH O

+

OH OH

HO

HO

OH

i) HCl dil.

FIGURE 7.1. Acid hydrolysis of simple O-glucosides.

OAc O

SPh

OAc O

i

AcO

OH

AcO AcO

305

OAc

AcO

OAc

i) NBS/acetone-H2O.

FIGURE 7.2. Hydrolysis of thioglycosides.

TABLE 7.1. Acid hydrolysis of disaccharides. Disaccharide

Hydrolysis product

(+)-sucrose

D-(+)-glucose D-(−)-fructose

(+)-lactose

D-(+)-glucose D-(+)-galactose

(+)-cellobiose

D-(+)-glucose D-(+)-glucose

TABLE 7.2. Acid hydrolysis of polysaccharides. Polysaccharide

Partial hydrolysis

Total hydrolysis

cellulose laminarin curdlan quitine manan pululan

1,4-cellobiose 1,3-laminaribiose 1,3-laminaribiose 1,4-N-acetyl glucosamine 1,4-mannobiose 1,4-maltotriose

D-glucose D-glucose D-glucose 2-amino-2-deoxy-D-glucose D-glucose D-glucose

OH

306

7. Hydrolysis of Glycosides

is the case of 1,3-laminaribiose synthetically obtained in poor yields (9.5%),1 but readily available from polysaccharide curdlan.2

7.2 Basic Hydrolysis Some glycosides have been shown to be partially sensitive against basic conditions, besides their naturally high acid sensitivity. It is been experimentally found that three classes of O-glycosides might be subject to basic hydrolysis:3 1. phenolic glycosides 2. enolic glycosides 3. β-substituted alcohol glycosides

7.2.1 Phenolic Glycosides A typical example of phenolic glycoside decomposition under basic conditions is observed in the treatment of salicin with barium hydroxide giving as result a cyclic acetal and the release of the aglycon (Figure 7.3).

7.2.2 Enolic Glycosides Within this type of glycosides, the three varieties to be considered are (1) 4hydroxycoumarins, (2) purine and pirimidin glycosides,and (3) simple enols (Figure 7.4).

7.2.3 β-Susbstituted Alcohol Glycosides Glycoside picrocine is hydrolyzed in diluted potassium hydroxide solution, through a mechanism that involves an intermediate carbanion formation to give a conjugated unsaturated product and glucose as breakage product (Figure 7.5). Contrary to acid hydrolysis of disaccharides where degradation products are their constitutive units, in most of the cases for basic conditions, nonsugar derivatives are produced as result (Table 7.3).

OH O OH

O

OCH3 O

OCH3

O i

HO

OH

+

OH

OH OH

OH

i) Ba(OH)2.

FIGURE 7.3. Basic hydrolysis of phenolic glycosides.

7.2. Basic Hydrolysis H3C

OH

OH

O OH

N

O

O

NH

O

OH O

OH

N

OH

OH

a

OH

CH3

OH

O

N

b

O

O

O

O

OH OH

c

OH

FIGURE 7.4. Basic hydrolysis of enolic glycosides.

OH

OH

O

O

O

OH OH

OH

CHO OH

OH

O

OH

CHO OH

OH O

OH

OH

+ CHO

OH OH

FIGURE 7.5. Basic hydrolysis of β-substituted alcohol glycosides.

TABLE 7.3. Degradation products of disaccharides under basic conditions. Disaccharide cellobiose gentobiose lactose maltose

Hydrolysis conditions (KOH)

Temperature (◦ C)

Product

1.5 N 2N 0.2 N 0.15 N

50 50 100 25

lactic acid lactic acid D-galactose fenilhydrazone of D-mannose

307

308

7. Hydrolysis of Glycosides

7.3 Enzymatic Hydrolysis β-Glycosides are the natural substrates for hydrolytic enzymes known as βglycosidases. So far, at the biochemical level, the rule of most of glycosidases is not totally well understood. However, some of them have been related to feeding, detoxiﬁcation processes, or even as a defense mechanism against herbivorous pathogens through releasing of tiocyanates, cianides, and phytohormones. It is been established that there is an speciﬁc glycosidases for each aldopyranose, being the sugar composition responsible for the recognition pattern. Some of the best-studied hydrolyses are the β-glycosidases and among them β-glucosidases, β-glucuronidases, β-cellulase, β-glucanasas, β-quitinases, all of them with important biological and economical implications.4

7.3.1 β-Glucosidases There is strong evidence indicating that their action is mainly directed toward the defense mechanism and growth regulation. For instance, cyanogenic glycosides are hydrolyzed, for the releasing of cyanide ions as a defense mechanism against animals. In humans the equivalent of β-glucosidase is called glucocerebrosidase (with low genomic homology to the plant counterpart) and catalyzes the degradation of glucosylceramide inside lysosome. The lack or deﬁciency of this enzyme produces the Gaucher disease characterized by accumulation of esphyngosylglucosides and glucosylceramides.

7.3.2 β-Glucanasas, β-Quitinases The natural substrates for these oligosaccharide hydrolytic enzymes are laminarin and quitine, respectively, being present in fungi, yeast, and insects. Some of the processes related to the activity of these enzymes are seed degradation, cellular elongation control, growth regulation, pollen growth regulation, digestion, and fertilization. Moreover, within the context of the defense mechanisms, these enzymes can be able to digest the fungi cellular wall and also to release oligosaccharides that induce the production of antimycotic substances called phytolexines.

7.3.3 β-Cellulase Cellulose is the most abundant natural polisaccharide on earth. Cellulitic enzymes, particularily cellobiohydrolases CBHI, CBHII, EGI and EG II found in fungi Trichoderma reesei, have been thoroughly studied for determing the threedimensional structure, the genomic sequence, receptors, and substrate speciﬁcity.

7.3.4 β-Glucuronidase In animals this enzyme is responsible for the detoxiﬁcation processes, coupling mainly aromatic compounds and eliminating them as glucuronides. In plants there is not detectable β-glucuronidase activity; however, the development of the

7.3. Enzymatic Hydrolysis

309

CBH I and CBH II

= 1,4-glucopyranosyl

= 1,4-galactopyranosyl

= hydrolytic site

= reduction terminal

FIGURE 7.6. Enzymatic speciﬁcity on low–molecular-weight substrates.

GUS gene fusion containing E. coli β-glucuronidase has been widely used as a gene marker.5 Transgenic plants containing exogenic information fused to the βglucuronidase gene marker can be conveniently monitored by using ﬂuorogenic o histochemical glucuronides.

7.3.5 Glycosidase Enzymatic Activity Detection Detection can be achieved not only qualitatively, but also quantitatively; for doing so, high- and low-molecular-weight substrates have been designed. Claeyssens6 demonstrated hydrolytic speciﬁcity of cellulases CBH I and CBH II through the use of synthetic fruorogenic substrates containing the highly ﬂuoroescent coumarin umbelliferone or p-nitrophenol, in the form of O-glucosides. The cleavage of the glycoside releases the chromophore, which can be easily measured in a ﬂuorometer or spectrophotometer. The synthetic design of mono-, di-, tri-, and tetrasaccharides attached to the mentioned chromophores have been of great advantage to determine the speciﬁcity during enzymatic cleavage (Figure 7.6).

7.3.6 Regarding β-1,4-Glucanases (EG) The utilization of polysaccharides covalently attached to dyes has been reported. The complex Ostatin Brilliant Redhydroxyethylcellulose (OBR-HEC) is applied as a speciﬁc substrate for EG, Remazol Brilliant Blue-xylan (RBB-X) the speciﬁc substrate for β-1,4-xylanases. Likewise, β-1,3-glucanases are detected by using an electrophoresis technique on polyacrilamide gels utilizing laminarin as substrate. The generated fragments are reacted further with azoic stain 2,3,5-triphenyltetrazolium to produce a color

310

7. Hydrolysis of Glycosides CH3

RO

OR O

RO

O

O

O

N O

H2N R = glucose, galactose, glucucronic acid, N-acetylglucosamine.

FIGURE 7.7. Fluorescent O-glycosides.

complex.7 Despite their high sensitivity, this method cannot distinguish between endo and exo glucanase.

7.3.7 Fluorescent O-Glycosides As mentioned before, ﬂuorogenic aglycons are very useful molecules to monitor enzymatic activity. In principle, the ﬂuorescent compound does not exhibit ﬂuorescence in the glycoside form and exerts its ﬂuorescence when released as a result of the enzymatic activity (Figure 7.7). Some of the ﬂuorescent compound widely used for enzymatic detections are umbelliferone, ﬂuoresceine, and resoruﬁn, having been coupled to most of the biologically important sugars as O-glycosides. The generated ﬂuorescence is quantiﬁed in ﬂuorometers constituted basically by a radiation source and two monocromatic mirrors (f1 and f2). The ﬁrst one selects the light for producing ﬂuorescence activation, and the second transmits selectively ﬂuorescence emission. A detector will measure the intensity of the ﬂuorescence generated (Figure 7.8).

source

sample f1

f2

detector

FIGURE 7.8. Basic diagram of ﬂuorometer.

OR

7.3. Enzymatic Hydrolysis

311

FIGURE 7.9. Absorption glycosides. O2N

OR

R = glucose, galactose, glucuronic acid, N-acetylglucosamine.

7.3.8 O-Glycosides Measured by Absorption Quantiﬁcation of enzymatic activity following absorption detection is based on the use of synthetic p-nitrophenol in the form of O-glycosides as substrate (Figure 7.9). The release of the aglycon from the sugar moiety produces slight yellow color measured as absorbance.

7.3.9 Histochemical O-Glycosides Generally, a histochemical substrate is to be considered as a good candidate, should be such that in the form of O-glycosides it is water-soluble and when the enzyme hydrolyzes the glycosidic bond releases the aglycon, which precipitates immediately. A compound that closely fulﬁlls these requirements is 5-bromo-4-chloro-Nacetyl-3-indoxyl, which has been attached to most of the biologically important monosaccharides. Although this is commercially available, it is highly sensitive, producing an easily detectable blue precipitate. It shows some diffusion before the monomers undergo dimerization in the presence of oxygen, to produce the blue indigo precipitate (Figure 7.10). O Cl

Cl

O

OH Ac N

Br

Br

i

N Ac

N Ac

Br HO leuco form

Cl

O Ac N

Br ii

N Ac

Br O

Cl

indigo blue precipitate i) glycosidase. ii) O2.

FIGURE 7.10. 5-Bromo-4-chloro-indoxyl aldopyranose hydrolysis.

Cl

312

7. Hydrolysis of Glycosides FIGURE 7.11. Phenylazonaphtol glucuronides as histochemical substrates.

CH3

CH3 N N CO2H OO OH OH OH

Alternatively, phenylazonaphtols O-glycosides (Figure 7.11) known as Sudan glucuronide has been tested as histochemical substrate for enzymatic detection of gene marker β-glucuronidase in transgenic plants.8,9 The water-soluble Sudan glucuronide releases the fenilazo naphtol stain after enzymatic hydrolysis, which can be seen in the sites of enzymatic activity as red crystals. The mechanism and stereochemistry of enzymatic hydrolysis may occur with either inversion or retention of the conﬁguration at the anomeric center. The ﬁrst type of hydrolysis is carried out by the called inverting glycosidases, and the second by retaining glycosidases, being the vast majority of β-glucosidases of the latter type. This has been proved through NMR studies, by measuring the chemical shift and magnitude of the coupling constant of the anomeric carbon. The most accepted Glu O

O

H

O

O

OH

O

H

inversion

OR O O

O

NH2

O

Glu

O

OR

O

O H

Glu

O H

O

O

retention

OH

FIGURE 7.12. Schematic representation of retention and inversion hydrolysis mechanism.

References

313

mechanism involves protonation of substrate, carboxylate participation attached to enzyme, intermediate formation glycoside-enzyme, and displacement as shown in Figure 7.12.4

References 1. 2. 3. 4. 5. 6.

P. B¨achli,and E.G. Percival, J. Chem. Soc. 1952, 1243. L.-X. Wang, N. Sakairi, and H. Kuzuhara, J. Carbohydr. Chem., 1991, 10, 349. C.E. Ballou, Adv. Carbohydr. Chem., 1954, 9, 59. A. Esen, β-Glucuronidase, Biochemistry and Molecular Biology, ACS, 1993, 7. R.A. Jefferson, S.M. Burges, and D. Hirsh, Proc. Natl. Acad. Sci. USA, 1986, 83, 8447. M. Clayssens, H. Van Tildeburgh, P. Tomme, T., Wood, and S. McRae, Biochem. J., 1989, 261, 819. 7. S.Q. Pan, X.S. Ye, and J. Kuc, Anal. Biochem., 1989, 136, 182. 8. N. Terryn, M. Brito-Arias, G. Engler, C. Tire, R. Villarroel, M. van Montagu, and D. Inze, Plant Cell, 1993, 5, 1761. 9. E. Van der Eycken, N. Terryn, J.L. Goemans, G. Carlens, W. Nerinckx, M. Claeyssens, J. Van der Eycken, M. Van Montagu, M. Brito-Arias, and G. Engler, Plant Cell Report 2000, 19, 966.

8 Nuclear Magnetic Resonance of Glycosides

8.1 NMR of O-Glycosides Nuclear magnetic resonance (1 H, 13 C NMR), X-ray diffraction, and mass spectrometry are considered among the most important analytical methods for structural elucidation. Characterization by means of 1 H, 13 C NMR, mono- and bidimensional spectroscopy is a powerful tool for structural assignment of simple and complex glycosides. Pioneering studies1−4 on simple monosaccharides were essential for understanding through the chemical shifts and coupling constants the conformational behavior of sugars. Some basic considerations derived from the referred studies mentioned above that apply to simple saccharides are Pyranoside rings of the D-series generally prefer to assume conformation 4 C1 and those of the L-series the conformation 1 C4 . The anomeric proton usually resonate at lower ﬁeld than methine protons, whereas methylene protons resonate at somewhat higher ﬁelds. In D-pyranoses with 4 C1 conformation, the α-anomer resonance is downﬁeld compared to the β-anomer, and the value of the coupling constant between H-1 and H-2 at three bond distance 3 J1−2 determine if the anomeric proton is equatorial or axial, and therefore if the glycoside is α or β. Usually for axial-axial interactions, the observed values are 8-10 Hz and for axial-equatorial or equatorial-equatorial 23 Hz. Thus for β-glucose, 3 J1,2 = 8 Hz, 3 J2,3 = 3 J3,4 = 3 J4,5 = 10 Hz, H-1 appears as doublet, and H-2, H-3, H-4 appear as 10 Hz triplets, and H-5 as double double doublet as it is coupled to the two H6s. The α-galactose presents 3 J1,2 = 3 Hz, 3 J2,3 = 10 Hz, 3 J3,4 = 4 Hz, 3 J4,5 < 1 Hz. H-1 appears as 3 Hz doublet, H-2 and H-3 as double of doublets, H-4 as doublet, and H-5 as triplet for coupling with two H6s. The different possible arrangements are for better understanding represented in Newman proyection (Figure 8.1). Equatorial protons are positioned at lower ﬁeld than chemically equivalent axial protons except in those cases were there is a carbonyl group adjacent to H-equatorial, or when there is a synaxial interaction with H-axial, in which a deshielding effect is observed.1c

314

8.1. NMR of O-Glycosides

O1 O2

H2

H1

O5

O3

C3

O2

J = 8 Hz

H2

H3

C4

O3

C1

H2

eg. Glc H2/H3;Glc H3/H4

β-Gal H1/H2

Glc H4/H5;Gal H2/H3

H3

C4

H1

C1

O2

H2

O1

O5 C3

J = 4 Hz

J = 3 Hz

eg. Man H2/H3

eg. α-Glc H1/H2

J = 10 Hz

β-Glc H1/H2

O2

315

α-Gal H1/H2

Gal H3/H4

Man H3/H4;Man H4/H5

C6 H4

O4

H5

O5

O1

C3

H2

J = 1 Hz

O2

H1

O5

O1

C3

H2

J = 1 Hz

eg. α-Gal H4/H5

eg. β-Man H1/H2

O2

H1

O5 C3

J = 1.7 Hz eg. α-Man H1/H2

FIGURE 8.1. Newman projections showing the arrengements of hydrogens in 4 C1 and 4 C1 chair conformations and the expected coupling constants.

The magnitude of coupling constant 3 JH-H besides torsion angle dependence may be affected by other factors such as substituent electronegativity, bond length, and bond distance. Solvent effects on 3 J(HH) appear to be relatively minor, except in cases where solvent-induced conformational changes occur.5 The 13 C chemical shift may also reveals along with de 1 H NMR the anomeric conﬁguration, but the one bond 13 C-1 H coupling constants can be remarkably useful to determine the anomeric conﬁguration in pyranoses. For instance, the 1 JCH for the α-anomer is 170 Hz and for the β-anomer 160 Hz, being for the L-isomer the reverse.6 The chemical shift values of the ring protons are dependent of the groups attached to the hydroxyl groups. For instance, a characteristic shift of ring-proton resonances to lower ﬁeld occurs when the hydroxyl group is esteriﬁed with acetyl, sulfate, or phosphate where normally downﬁeld shifts of ∼0.2-0.5 ppm are observed. If the protecting group is acetate, for nonaromatic solvents C-6 resonates at lower ﬁeld, followed by C-2, C-4, and at highest ﬁeld the 3-acetoxyl signal.4 The proton magnetic resonance of 4,6-O-benzylidene pyranosides have been measured and the values of the coupling constants J1,2 , J1,3 , J2,3 , and J3,4 support the assignment of the chair conformation to the pyranoid ring.2 The coupling constants 3 JH-H values on saturated systems can be predicted by applying the Karplus equation,7 which correlates the dihedral angle θ values with the magnitude of the coupling constant 3 JH-H . 3

JH-H = A + B cosθ + C cos2θ

where θ is the dihedral angle between H1-C1-C2-H2, A = 4.22, B = −0.5, and ˚ C = 4.5 Hz for C-C bond distance 1.543 A.

316

8. Nuclear Magnetic Resonance of Glycosides Ha Hb Ha

3

JH-H

(Hz)

9

FIGURE 8.2. Relationship between coupling constant and torsion angle.

Hb

8 7 6 5 4

Ha Hb

3 2 1 0

30

60

90 120 150 180 torsion angle (θ)

Coupling constants for vicinal protons at three bond distances are two or three times bigger when they are eclipsed or antieclipsed (0◦ or 180◦ ) to each other than when they are synclinal or gauche (60◦ ) (Figure 8.2). Karplus analysis is more accurate when comparative studies are performed between structurally similar compounds. For the study of conformational differences between structurally similar molecules the Karplus equation adopts the form of 3

JH-H = K cos2θ

where K is dependent on H1-C1-C2-H2 fragment, when θ is having values between 50 and 70◦ , or 110 and 130◦ , slight variations are observed, while for values close to 0, 90 and 180◦ , no observable changes are detected. The effect of the relative orientation and electronegativity of substituents on the magnitude of 3 J(aa), 3 J(ae), and 3 J(ee) has been predicted by a simple set of additivity constants. The step followed in the derivation of the additivity constants considers that antiperiplanar substituents exert a negative and gauche substituents a positive effect on J. The resulting data were ﬁtted equation 3 J = 3 J0 + J (x), where 3 J0 represents the reference value. Some of the additivity constants J (x) for a given substituent are given in Table 8.1.5 More recently, a computer program known as ALTONA was developed for the calculation of dihedral angles from 1 H NMR. This program calculates plots of H-C-C-H diedral angles from proton-proton NMR vicinal coupling constants using an empirically generalized Karplus-type equation, which takes into account the

8.1. NMR of O-Glycosides

317

TABLE 8.1. Additivity constants J (x) for a substituent X. X H,C I,S Br N N3 Cl O F

J (ae)(x) or J (ee)(x) X anti

X gauche

J (aa)(x) X gauche

0.0 −0.3 −0.9 −1.1 −1.4 −1.2 −1.8 −2.5

0.0 +0.1 +0.3 +0.3 +0.4 +0.4 +0.5 +0.7

0.0 −0.3 −0.7 −0.6 −1.1 −1.0 −1.4 −2.0

electronegativity and the orientation of the substituents attached to the considered fragment.8 The NMR spectra of fully acetylated 1-thioaldopyranosides having the conﬁgurations β-D-xylo, α-L-arabino, β-D-ribo, β-D-gluco, and β-D-galacto were determined in different solvents, observing that the H-1 signal in these derivatives appears ∼0.35 ppm to higher ﬁeld than its position in the 1-oxygenated analogs.9 Also, detailed studies of 1 H NMR spectra of a series of hexopyranosyl halides have been accomplished. The ﬁrst-order assignments revealed several stereospeciﬁc dependencies, mainly upon the orientation of the halogen substituent with respect to the pyranose ring and the relative orientation of other substituents attached to the ring.3b,10,11 1 H and 13 C chemical shifts and J-coupling patterns for common D-aldohexoses, D-aldopentoses, and some methyl monosaccharides are described in Tables 8.2 and 8.3.12,13 A wide number and variety of O- and to a lesser extent C-glycosides islolated from natural sources have been reported and their NMR analysis described. The chemical shifts and coupling constants of some of them are described just as representative examples in Table 8.4. Nuclear Overhauser effects (NOE) is a dipole-dipole relaxation experiment and has been one of the most useful experiments for the structural assignments of glycosides on the basis of shielding and deshielding effects.19 Glycosylation sites can be identiﬁed by comparison of 1 H NMR spectral data of the peracetylated and the nonprotected sugar, since free OH groups causes signiﬁcant downﬁeld shift (in the range of 1 to 0.5 ppm). The approach known as “structural-reporter-group” has been introduced to identify individual sugars or sequences of residues and can be used to identify structural motifs or speciﬁc sugars and linkage compositions found in relevant databases.13 For complex molecules the interpretation is often problematic, especially due the presence of internal motion. Some of the difﬁculties encountered for NMR structural assignment for oligosaccharides are20 The limited number of C,H dipolar couplings measured across a single bond The distribution of C,H bond vectors is not isotropic due to the geometry of the pyranose ring.

318

8. Nuclear Magnetic Resonance of Glycosides

TABLE 8.2. 1 H chemical shifts and couplings (3 JH-H ) of D-aldohexoses and aldopentoses measured at 400 MHz in D2 O. Compound

H-1

H-2

H-3

H-4

H-5

H-6a

H-6b

α-glucose

5.09 3.6 4.51 7.8 5.16 3.8 4.48 8.0 5.05 1.8 4.77 1.5

3.41 9.5 3.13 9.5 3.72 10.0 3.41 10.0 3.79 3.8 3.85 3.8

3.61 9.5 3.37 9.5 3.77 3.8 3.56 3.8 3.72 10.0 3.53 10.0

3.29 9.5 3.30 9.5 3.90 1.0 3.84 1.0 3.52 9.8 3.44 9.8

3.72

3.72 2.8 3.75 2.8 3.70 6.4 3.70 3.8 3.74 2.8 3.74 2.8

3.63 5.7, 12.8 3.60 5.7, 12.8 3.62 6.4 3.62 7.8 3.63 6.8, 12.2 3.60 6.8, 12.2

β-glucose α-galactose β-galactose α-mannose β-mannose 1H

3.35 4.00 3.61 3.70 3.25

chemical shifts and couplings (3 JH-H ) of D-aldopentoses measured at 400 MHz in D2 O.

Compound

H-1

H-2

H-3

H-4

H-5a

H-5b

α-xylose

5.09 3.6 4.47 7.8 4.40 7.8 5.12 3.6 4.75 2.1 4.81 6.5 4.89 4.9 4.74 1.1

3.42 9.0 3.14 9.2 3.40 9.8 3.70 9.3 3.71 3.0 3.41 3.3 3.69 3.6 3.81 2.7

3.48 9.0 3.33 9.0 3.55 3.6 3.77 9.8 3.83 3.0 3.98 3.2 3.78 7.8 3.53 8.5

3.52

3.58 7.5 3.82 5.6 3.78 1.8 3.54 2.5 3.82 5.3 3.72 4.4 3.71 3.8 3.84 5.1

3.57 7.5 3.22 10.5, 11.4 3.57 1.3, 13.0 3.91 1.7, 13.5 3.50 2.6, 12.4 3.57 8.8, 11.4 3.58 7.2, 12.1 3.15 9.1, 11.7

β-xylose α-arabinose β-arabinose α-ribose β-ribose α-lyxose β-lyxose

3.51 3.83 3.89 3.77 3.77 3.73 3.73

Due to the ﬂexibility of the glycosidic bond that connects the different sugars moieties, different alignment tensors can be observed. More recently the use of a novel procedure known as “residual dipolar coupling” has been introduced by Tian & Prestegard as an alternative approach for studing the conformational and the motional properties of oligosaccharides.21 The approach is based on the solution for each ring of an order matrix that combines different types of couplings, 1 DCH , 2 DCH , and DHH . Dipolar couplings arise from through space spin-spin interactions and are dependent of both internuclear distance (r) and an angle between the magnetic ﬁeld and the internuclear vector (θ) as described by the equation (3 cos2 θ − 1) Dij = ξij (1/r3 ) 2

8.1. NMR of O-Glycosides TABLE 8.3.

13

319

C chemical shifts of some aldoses

Compound

C-1

C-2

C-3

C-4

C-5

C-6

α-glucose β-glucose α-galactose β-galactose α-mannose β-mannose α-arabinose β-arabinose α-ribose β-ribose

92.9 96.7 93.2 97.3 95.0 94.6 101.9 96.0 97.1 101.7

72.5 75.1 69.4 72.9 71.7 72.3 82.3 77.1 71.7 76.0

73.8 76.7 70.2 73.8 71.3 74.1 76.5 75.1 70.8 71.2

70.6 70.6 70.3 69.7 68.0 67.8 83.8 82.2 83.8 83.3

72.3 76.8 71.4 76.0 73.4 77.2 62.0 62.0 62.1 63.3

61.6 61.7 62.2 62.0 62.1 62.1

where ξ ij is a constant that depends on the properties of nuclei i and j. Direct measurements of dipolar interactions can be achieved by dissolving molecules in oriented media such as crystals composed of bicelles or phage. Despite the fact that molecular tumbling remains fast in these media, the sampling of orientations are no longer isotropic, and consequently the dipolar couplings do not average to zero and splittings are observed between the dipolar coupled spin pairs. The knowledge of the molecular geometry of a fragment and the measurement of ﬁve or more interdependent residual couplings from the fragment allows the determination of the Saupe order matrix elements (Sij) from a set of linear equations relating dipolar couplings to the known geometry factors and the unknown order tensor elements. Dresid α Si j cos θi cos θ j ij

where θij are the angles between the internuclear vectors. Determination of the Saupe order matrices for individual rigid fragments of a molecule allows both structural characterization and assestments of internal motions between fragments.10 NMR studies carried out by De Bruyn,22 using as models series of disaccharides, provided valuable information about conformational behavior from the chemical shifts and the torsion angles present around the glycosidic bond (Figure 8.3). Also it has been reported that the 13 C chemical shifts for the glycoside and the aglycone carbon can be directly correlated with one of the torsion angles psi (ψ) deﬁned by the bonds C(1)-O(1)-C(4)-H(4).19 The sign of θ and ψ has been previously calculated through the method known as hard sphere exoanomeric effect23 which predicted the relative stability of the different conformers around the torsion angles, considering the bond length, bond angle, and atomic size. It has been observed that for a number of disaccharides there is a variation of the chemical shifts as a function of ψ, compared with the values of their corresponding monosaccharides (Table 8.5).

320

8. Nuclear Magnetic Resonance of Glycosides

TABLE 8.4. 1 H chemical shifts and couplings (3 JH-H ) of natural O- and C-glycosides Natural glycoside O

O

HO

H-3

H-4

H-5

4.63 d(8.0)

3.17 dd (8.0, 9.0)

3.36 t (9.0)

3.26 t (9.0)

3.30 m

5.04 d (7.5)

3.50 dd (9.0, 7.5)

3.44 m

3.38 t (9.0)

3.44 m

3.71 dd (5.5, 12.3), 3.91 dd (2.0, 12.3)

15

4.32 d (8.0)

3.59 dd (8.0, 10.0)

3.54 br dd (3.0, 10.0)

3.63 br d (3.0)

3.58 dq (1.0, 6.0)

1.21 d

16

4.88 d (10.0)

5.35 t (10.0)

3.49 m

3.49 m

3.33 m

3.58 dd (5.5, 12.5), 3.78 m

17 Zou et al

3.47 dd (1.9, 9.2)

3.66, t (9.2)

3.76 dd (2.8, 9.2)

5.21 br s

3.37 d (13.1), 3.88 dd (1.8, 13.1)

O 6

6'

14

O

O

HO 4 HO

Ref

H-2

O HO

H-6, H-6’ 3.66 dd (6.0, 12.0); 3.89 dd (2.0, 12.0)

H-1

5 2

3

OH 1

Glc OH

HO

HO

6

6'

O

O

O

HO 4 HO

5 2

3

OH 1

Glc O

HO 1

2 HO 3

O

OH 5

6

Fuc

OH

OH

O

O

6'

O

6

HO HO

4

OH

1

5

O

2

4 3

HO

O

Glc O

OH

OH

CH3 5

1

O 3

4

O

Ph O

HO

OH 2

18 Diaz et al

5.10 5.41 5.38 5.36 5.41 5.41 4.63 4.73 4.63 4.51 — —

3.56 3.55 3.56 3.57 3.59 3.58 3.37 3.37 3.37 3.32 — —

H2

3.80 3.80 3.77 3.76 3.68 3.69 3.52 3.54 3.54 3.52 — —

H3

3.47 3.47 3.47 3.45 3.42 3.42 3.42 3.42 3.42 3.42 — —

H4 3.86 3.86 4.02 4.02 3.72 3.74 3.46 3.49 3.49 3.50 — —

H5 3.85 3.78 3.82 3.82 3.74 3.77 3.75 3.73 3.73 3.75 — —

H6a 3.78 3.78 3.82 3.82 3.74 3.77 3.75 3.73 3.73 3.75 — —

H6b 5.45 4.81 5.24 4.67 5.23 4.66 5.45 5.23 4.74 4.67 5.23 4.67

H1 3.64 3.39 3.63 3.36 3.58 3.28 3.65 3.73 3.44 3.29 3.50 3.27

H2

1 = kojibiose; 2 = nigerose; 3 = maltose; 4 = soforose; 5 = laminaribiose; 6 = cellobiose; 7 = glucose

α-1 β-1 α-2 β-2 α-3 β-3 α-4 α-5 β-5 β-6 α-7 β-7

H1

TABLE 8.5. Chemical shifts of disaccharides and anomers of glucopyranose. 3.83 3.59 3.86 3.64 3.97 3.77 3.87 3.92 3.74 3.60 3.71 3.45

H3 3.47 — 3.67 3.64 3.64 3.62 3.47 3.52 3.49 3.65 3.36 3.35

H4 4.03 — — 3.48 3.93 3.60 3.87 3.88 3.49 3.58 3.82 3.82

H5

3.85 3.90 — — 3.82 3.92 3.84 3.85 3.90 3.97 — —

H6a

3.76 3.56 — — 3.82 3.77 3.77 3.79 3.74 3.82 — —

H6b

8.1. NMR of O-Glycosides 321

322

8. Nuclear Magnetic Resonance of Glycosides FIGURE 8.3. Torsion angles around the glycosidic bond. O O

O

ψ

θ

The development of Karplus relationship for three-bond C-O-C-C spin-coupling constants by Bose et al.24 suggests that 3 JCOCC obeys a Karplus relationship similar to that observed for 3 JHH ,3 JHC , and other vicinal spin-coupling constants. However, the precise form of this relationship that is the shape and amplitude of the Karplus curve is unknown. Also in this work, 3 JCOCH values have been measured to asses the phi (φ) and the psi (ψ) torsion angles (Figure 8.4) and Karplus relationships have been reported for this vicinal coupling.25 Comparative conformational studies using a combination of NMR spectroscopy and molecular mecanics of lactose disaccharide (βGal[1-4]Glc) and its C-analog showed that for the former the population in solution is about 90% syn and 10% anti, while for the latter the conformation is more ﬂexible in the forms 55%, 40%, and 5% syn, anti, gauche-gauche, respectively.26 1 H NMR spectra of oligosaccharides follows in many cases complex patterns due to extensive overlap within the region δ 3.0-4.2. However, the use of pyridine-d5 improves the signal dispersion, increasing the resolution especially in overcrowded regions. The localization of anomeric protons is a valuable tool for recognizing the number of monosaccharide residues. A number of one- and two-dimensional methods provide thorough information to assert the complete assignment unambigiously. One-dimensional NMR analysis provides useful information about the chemical shifts and scalar couplings of well-resolved signals such as anomeric protons (δ 4.4-5.6) and methyl groups for 6-deoxy monosaccharides (fucose, quinovose, ramnose) at (δ 1.1-1.3). The effect on the proton chemical shift of glycosylation is a typical deshielding of the proton across the glycosidic bond and the two neighboring positions of the aglycone. This behavior is due to repulsion between hydrogens and to the effect of the lone pair of the oxygen to the hydrogens.27

CH2OH

CH2OH

O φ C1 C2

HO HO

JC1,C4´,

3

OH

JC2,C4´,

3

ψ

H1 H4

couplings sensitive to 2

O

C5´ C4´ C3`

O

OH

HO

φ

JC4´,H1

O

couplings sensitive to 2

JC1,C3´,

3

JC1,C5´,

3

JC1,H4´

ψ

FIGURE 8.4. Couplings sensitive to φ and ψ torsion angles.

8.1. NMR of O-Glycosides

323

Conformational analysis on more complex glycosides is based mainly on the inter-residue 1 H-1 H Nuclear Overhauser effects (NOE).28 and also 13 C-1 H longrange coupling constants across the glycosidic linkage for studying the preferred conformation of oligosaccharides in solution. Selective irradiation of the anomeric proton reveals inter-residual contacts with aglycone protons. In this way 1→2, 1→3, 1→4, and 1→6 combinations as well as −α and −β linkages may be determined.29 Long-range 1 H-1 H couplings involving four bonds between anomeric and aglycone protons (4 JHCOCH ) are usually very small that could be detected but not measured.30 Two-dimensional NMR is a reliable method for determining inter-ring connectivity. Through space dipolar interactions between the anomeric and the trans glycosidic proton can be detected in the form of NOE signals and represent the basis for linkage and sequence analysis.31 Also, the interglycosidic connectivities are established on the basis of long-range (3 JCH ) by HMBC studies.27 The usefulness of this method has been later demonstrated in a number of structural elucidations.32 Bidimensional homonuclear techniques such as the TOCSY experiment have been useful for the NMR characterization of the naturally occurring complex glycosides such as glycoresin tricolorin E,33 allowing the total assignment of the sugar region, including the anomeric protons for each of the 4 monosaccharides established (Figure 8.5). Likewise, the complete 1 H and 13 C assignments of a synthetic octasaccharide fragment of the O-speciﬁc polysaccharide of Shigella dysenteriae type 1 by using 2D TOCSY at 600 MHz was described. In the contour plot it is possible to observe the connectivity between the sugar units and the detailed assignment of the protons.34 Moreover, a 2D selective TOCSY-DQFCOSY experiment for identiﬁcation of individual sugar components in oligosaccharides is described, assuming that unambiguous sequential assignment of the proton signals for individual components is reached.35 High-resolution 1 H NMR spectroscopy has been applied in the structural analysis of glycoproteins. The initial efforts to assign all the anomeric and nonanomeric protons were done by using spin decoupling and nuclear Overhauser spectroscopy.36 Nuclear magnetic resonance of carbohydrate related to glycoconjugates has been analyzed. One of the ﬁrst high-resolution studies was reported back in 1973 on intact glycolipids in a 220-MHz magnet.37 Subsequent studies on underivatized and permethylated glycosphingolipids in dimethylsulfoxide-d6 and chloroform, respectively, allowed one to assign all the anomeric protons and a number of nonanomeric proton resonances.38 Early studies on high-resolution NMR. spectra of glycans chain in D2 O allowed one to assign the anomeric and nonanomeric protons as well as the coupling constants of sugar residues found in glycoproteins.36,39 More recently, the complete resolution of acetyl protected sialic acid glycopeptides was achieved by using NOESY and DQF-COSY technique.6 For the NMR-analysis of carbohydrate-protein complexes the transfer nuclear Overhauser effect (trNOE) experiment seems to be a promising alternative.40

324

8. Nuclear Magnetic Resonance of Glycosides

FIGURE 8.5. Expanded region of TOCSY spectrum for characterization of tricolorin E.

Recent advances on conformational analysis of oligosaccharides allows to determine the inter-residue interactions based on the dihedral angles φ and ψ along the interglycosidic linkage.41 In this connection, recent conformational advances on E-selectin-sialyl Lewisx complex have led to the determination of the bioactive conformation of the silayl Lewisx tetrasaccharide.42

8.2. N-Glycosides

325

NMR spectroscopy of glycoproteins has been achieved by using a combination of homo- and heteronuclear experiments at natural abundance.43 Increased reﬁnement is possible when a 15 N-labeled sample was used and the mobility of the glycan chain could be assessed by the measurements of 13 C line widths obtained from the high-resolution HSQC spectra.41

8.2 N-Glycosides The conformational analysis of N-glycosides has been extensively studied on the basis of chemical shifts and coupling constant determinations mainly around the C-N linkage. Torsion angles symbolized as χ for furanosides rings are also dependent on the Karplus equation, and similarly plays an important rule for the conformational analysis of 5-member rings.44 For purines the angle χ is formed between O4 -C1-N9-C4 atoms, and O4 -C1-N1-C2 for pyrimidines. When torsion angles O4 -C1 N9-C4 for purines, and O4 -C1-N1-C2 for pyrimidines are eclipsed, then χ = 0◦ . Positive angles of χ occurs for rotation clockwise for N9C4 for purines and N1-C2 for pyrimidines. The conformation syn in nucleosides corresponds to the angle χ = 0 ± 90◦ , and anti to 180 ± 90◦ (Figure 8.6). Regarding furanoside rings, there are different nonplanar conformations possibly assumed in terms of ﬁve endocyclic torsion angles symbolized as v0 , v1 , v2 , v3 , v4 , corresponding to the bonds O4 -C1 , C1 -C2 , C2 -C3 , C3 -C4 , and C4 -O4 . The two most common conformations founded are the envelope (E), referring to 4 atoms on the plane, and twist (T) for 3 atoms on the plane. The puckering of the furanoside rings of nucleosides is explained by Sorenssen et al.45 Unmodiﬁed nucleosides are present as an equilibrium between the C-3 -endo conformation, located around P = 18◦ , and the C-2 -endo conformation centered around P = 162◦ (Figure 8.7). Besides the torsion angle described for the N-glycosidic bond, there are for the case of oligosaccharides, additional torsion angles symbolized as ω,ω ,φ,φ ,ψ,

O

O

HO

N O

OH OH anti

N

HN

NH O

O HO

N O

OH OH syn

NH2

NH2

HO

N O

N N

N

N

N

N HO

O

OH OH anti

FIGURE 8.6. Syn-anti conformations for purines and pyrirmidines.

OH OH syn

326

8. Nuclear Magnetic Resonance of Glycosides OH

B O

3 2T 2E

3

E

0o

OH

342

o

18o

North

OH

4E

O B

B

oE

O

270o

90o

198o 3E

South 180

o

E

1E

162o

o 2

E

2

3T OH

B O

FIGURE 8.7. Pseudorotacional cycle of the furanoside ring in nucleosides.

and ψ corresponding to the bonds P-O5, P-O3 , O5 -C5 , O3 -C3 , C5 -C4 , C4 O3 , respectively (Figure 8.8). The vicinal coupling constants at 3 bond distance are dependent of the dihedral angle θ, and the relationship determined by the Karplus equation. 3

JHH = A cos 2θ − B cos θ + C

where A, B, and C are constants and their values are given in Table 8.6.

ω

P O

φ ψ

O

O P

N

χ

OH

FIGURE 8.8. Torsion angles for oligosaccharides.

8.2. N-Glycosides

327

TABLE 8.6. Karplus A, B, C constant values for nucleotide molecular fragments. torsion angle HO-CH-CH-OH H-C-C-H H-C-O-H

H-C-O-P

sugar ring ψ φ φ φ φ

J (Hz) J1 2 ,

J2 3 ,

J4 5 , J4 5 J2 OH2 J3 OH3 J5 OH5 J3 P J5 , J5

J3 4

A

B

C

10.2 9.7 10.4

0.8 1.8 1.5

0 0 0.2

18.1

4.8

0

TABLE 8.7. 3 J2 3 (Hz) values for furanoside ring in nucleosides. Nucleoside β-D-ribonucleosides pyrimidine (anti) pyrimidine (syn) purine (anti) purine (syn) β-D-deoxyribonucleosides pyrimidine (anti) pyrimidine (syn) purine (anti-syn) C-nucleoside pyrimidic C-nucleoside puric

3 J +3 1 2 J3 4

3J 23

(Hz)

(Hz)

9.9 (± 0.2) 10.3 (± 0.2) 9.7 (± 0.3) 9.6 (± 0.3)

5.3 (± 0.2) 6.2 (± 0.2) 5.2 (± 0.1) 5.5 (± 0.2)

10.6 (± 0.2) 11.0 (± 0.1) 6.3 (± 0.1) 10.6 (± 0.3) 10.1 (± 0.3)

6.7 (± 0.2) 8.0 (± 0.1) 6.3 (± 0.1) 5.2 (± 0.2) 5.0 (± 0.2)

The exchange of –OH for -OPO3 does not affect sensibly the Karplus relationship; therefore, the values are valid for both nucleosides or oligonucleosides. However, as mentioned, 3 J there is a dependence of other factors such as bond length, bond angle, electronegativity, and substituent orientation. Some of the values reported for ribose and deoxyribose are described in Table 8.7. The analysis of the C-Nucleosides β-pseudouridine (β–ψ) and α-pseurouridine (α–ψ) in aqueous solution have been described and the observed coupling constants given in Table 8.8.1b TABLE 8.8. Coupling constant (Hz) for β- and α-pseudouridine at 30◦ Coupling constant J61 J1 2 J2 3 J3 4 J4 5 B J4 5 C J5 B5 C

β–ψ 0.8 5.0 5.0 5.2 3.2 4.6 −12.7

α–ψ 1.3 3.3 4.2 7.9 2.4 5.7 −12.7

328

8. Nuclear Magnetic Resonance of Glycosides

References 1. (a) R.U. Lemieux and A.R. Morgan, Can. J. Chem., 43, 2199 (1965). (b) G. Kotowycz and R.U. Lemieux, Chem. Rev., 73, 669 (1973). (c) R.U. Lemieux and J.D. Stevens, Can. J. Chem., 43, 2059 (1965). 2. B. Coxon, Tetrahedron, 21, 3481 (1965). 3. (a) L.D. Hall, Adv. Carbohydr. Chem., 19, 51 (1964). (b) L.D. Hall, J.F. Manville, and N.S. Bhacca, Can. J. Chem., 47, 1 (1969). 4. D. Horton, and J.H. Lauterbach, Carbohydr. Res., 43, 9 (1975). 5. C. Altona, and C.A.G. Haasnoot, Organic Magnetic Resonance, 13, 417 (1980). 6. E. Breitmaier, Structure Elucidation by NMR I Organic Chemistry 3th Ed. John Wiley & Sons pp 46 (2002). 7. M. Karplus, Chem. Phys., 11, 30 (1959). 8. C.M. Cerda-Garc´ıa-Rojas, L. G. Zepeda, and P. Joseph-Nathan, Tetrahedron Computer Methodology, 3, 113, (1990). 9. C.V. Holland, D. Horton, M.J. Miller, and N.S. Bhacca, J. Org. Chem., 32, 3077 (1967). 10. G. Hajdukovic, M.L. Martin, P. Sina¨y, and J.R. Pougny, Organic Magnetic Resonance 7, 366 (1975). 11. D. Horton and W. N. Turner, J. Org. Chem. 30, 3387 (1965). 12. K. Bock and H. Thøgerson, Annual Reports on NMR Spectroscopy Ed by G.A Webb, Academic Press 13, 37 (1982). 13. J. Ø Duus, C.H. Gotffredsen, and K. Bock, Chem. Rev., 100, 4589 (2000). 14. A. Delazar, M. Byres, S. Gibbons, Y. Kumarasamy, M. Modarresi, L. Nahar, M. Shoeb, and S. D. Sarker, J. Nat. Prod. 67, 1584 (2004). 15. G. Bohr, C. Gerh¨auser, J. Knauft, J. Zapp, and H. Becker, J. Nat. Prod. 68, 1545 (2005). 16. S.O. Lee, S. Z. Choi, S. U. Choi, K. C. Lee, Y. W. Chin, J. Kim, Y. C. Kim, and K.R. Lee, J. Nat. Prod. 68, 1471 (2005). 17. J-H Zou, J.S. Yang, and L. Zhou, J. Nat. Prod. 67, 664 (2004). 18. F.Diaz, H-B Chai, Q.Mi, B-N.Su, J.S.Vigo, J.G. Graham, F.Cabieses, N.R. Farnsworth, G.A. Cordell, J.M. Pezzuto, S.M. Swanson, and A. D.Kinghorn, J. Nat. Prod. 67, 352 (2004). 19. P. Manitto, D. Monti, and G. Speranza, J. Chem. Soc. Perkin Trans. 1, 1297 (1990). 20. H. Neubauer, J. Meiler, W. Peti, and C. Griesinger, Helv. Chim. Acta, 84, 243 (2001). 21. F. Tian, H.M. Al-Hashimi, J.L. Craighead, and J.H. Prestegard, J. Am. Chem. Soc., 123, 485 (2001). 22. A. De Bruyn, J. Carbohydr. Chem., 10, 159 (1991). 23. R.U. Lemieux and S. Koto, Tetrahedron, 30, 1933 (1974). 24. B. Bose, S. Zhao, R. Stenutz, F. Cloran, P.B. Bondo, G. Bondo, B. Hertz, I. Carmichael, and A.S. Serianni, J. Am. Chem. Soc., 120, 11158 (1998). 25. K. Bock, A. Brignole., and B.W. Sigurskjold, J. Chem. Soc. Perkin Trans. II, 1711 (1996). 26. A. Imberty, Current Opinion in Structural Biology, 7, 617 (1997). 27. P.K. Agrawal and A.K. Pathak, Phytochemical Analysis, 7, 113 (1996). 28. I. Tvaroska, M. Hricovini, and E. Petrakova, Carbohydr. Res., 189, 359 (1989). 29. (a) Y.S. Bae, J.F.W. Burger, J.P. Steynberg, D. Ferreira, and R.W. Heminway, Phytochemistry, 30. (a) G. Batta and A. Liptak, J. Am. Chem. Soc., 106, 248 (1984). (b) G. Masiiot, C. Lavaud, C. Delaude, G.V Binst, S.P.F. Miller, and H.M. Fales, Phytochemistry, 29, 3291 (1990).

References

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31. J.H. Prestegard, T.A.W. Koerner, P.C. Demou, and R.K. Yu. J. Am. Chem. Soc., 104, 4993 (1982). 32. (a) N.M. Duc, R. Kasai, K. Ohtani, A. Ito, N.T. Nham, K. Yamasaki, and O. Tanaka, Chem. Pharm. Bull., 42, 634 (1994). (b) T. Nakamura, T. Takedo, and Y. Ogihara, Chem. Pharm. Bull., 42, 1111 (1994). (c) B. Razanamahefa, C. Demetzos, A.-L Skaltsounis, M. Andriantisiferana, and F. Tillequin, Heterocycles, 38, 357 (1994). (d) T. Nakanishi, K. Tanaka, H. Murata, M. Somekawa, and A. Inada, Chem. Pharm. Bull, 41, 183 (1994). (e) S.T. Thulborg, S.B Christensen, C. Cornett, C.E. Olsen, and E. Lemmich, Phytochemistry, 36, 753 (1994). 33. (a) M. Bah and R. Pereda-Miranda, Tetrahedron, 41, 13063 (1996). (b) R. PeredaMiranda and M. Bah, Current Topics in Medicinal Chemistry, 3, 1 (2003). 34. B. Coxon, N. Sari, G. Batta, and G. Pozsgay, Carbohydr. Res., 324, 53 (2000). 35. H. Sato, and Y. Kajihara, J. Carbohydr. Chem., 22, 339 (2003). 36. F.G.J. Vliegenthart, L. Dorland, and H. van Halbeek, Adv. Carbohydr. Chem. Biochem., 41, 209 (1983). 37. M. Martin-Lomas and D. Chapman, Chem. Phys, Lipids, 10, 152 (1973). 38. (a) J. Dabrowski, P. Handﬂand, and H. Egge, Biochemistry, 19, 5652 (1980). (b) K.E. Falk, K.-A. Karlsson, and B.E. Samuelson, Arch. Biochem. Biophys., 192, 164 (1979). 39. L.S. Wolfe, R.G. Senior, and N.M.K. Ng Yin Kin, J. Biol. Chem., 249, 1838 (1974). 40. (a) F. Ni, Prog. Nucl. Magn. Reson Spectros., 26, 517 (1994). (b) F. Casset, T. Peters, M. Etzler. E. Korchagina, S. Nifantev, Perez, and A. Imberty, Eur. J. Biochem., 239, 710 (1996). 41. (a) T. Peters, and B.M. Pinto, Current Opinion in Structural Biology, 6, 710 (1996). (b) T. Weimar, S.L. Harris, .J.B. Pitnar, K. Bock, and B.M. Pinto, Biochemistry, 34, 13672 (1995). 42. K. Schefﬂer, B. Ernst, A. Katopodis, J.L. Magnani, W.T. Wang, R. Weisemann, and T. Peters, Angew. Chem. Int. Ed., 34, 1841 (1995). 43. (a) D.F. Wyss, and J.S. Choi, and G. Wagner, Biochemistry, 34, 1622 (1995). (b) D.F. Wyss, J.S. Choi, J. Li, M.H. Knoppers, K.J. Willis, A.R.N. Arulandaman, A. Smolyar, E.L. Reinherz, and G. Wagner, Science, 269, 1273 (1995). (c) R. Liang, A.H. Androtti, and D. Kahne, J. Am. Chem. Soc., 117, 10395 (1995). 44. D. Davies, Progress in NMR Spectroscopy, 12, 140 (1978). 45. M.H. Sorenssen, C. Nielsen, and P. Nielsen, J. Org. Chem., 66, 4878 (2001).

9 X-Ray Diffraction of Glycosides

X-ray crystallography is a powerful tool for obtaining molecular information regarding bond lengths, bond angles, hydrogen bond interactions, and torsion angles, which are necessary elements for understanding the conformation of glycosides. Improved diffractometers, faster computational processors, and newer mathematical programs have made possible the structural resolution of simple and complex substances of glycosidic nature particularly those with noncentrosymmetric space groups. Early studies on simple glycosides allowed to conﬁrm that the sugar residue is pyranoid (and not acyclic), assuming two possible chair conformations (4 C1 and 1 1 4 C ), usually orienting the substituent to the equatorial position. In hydrogen bond interactions on pyranoids some facts that almost occur invariably are (a) the ring-oxigen atom is always a hydrogen bond acceptor, (b) each hydroxyl group is associated with two hydrogen bonds, one as donor and one as acceptor, (c) in disaccharides there might be intramolecular hydrogen bonding between two residues, (d) the hydrogen bond O-O distance has values around 2.68 ˚ to 3.04 A. Crystallographic observations on the anomeric effect demonstrated that the bond shortening and preferred gauche conformation of the glycosidic bonds in pyranoses was a consequence of an electronic distribution in the hemiacetal and acetal moiety of these molecules.2 On the other hand, the primarily alcohols can be present in three staggered orientations, deﬁned as gg, gt, and tg, refering to torsion angles O5-C5-C6-O6 and the second to C4-C5-C6-O6 [g ± 60◦ , t 180◦ ]. An alternative nomenclature refers O5-C5-C6-O6 as +g = gt, −g = gg, t = tg. The general standard molecular dimensions for pyranosides are described in Table 9.1, being the C-C bond length in the range of 1.523–1.526, C-C-C angles 110.4–110.5◦ , and usually shorter glycosidic bond 1.398 for axial and 1.385 for equatorial disposition (Table 9.1).3 The distortion degree from the ideal chair conformation has been studied by Cremer and Pople,4 who, by following a mathematical approximation, were able to propose three puckering parameters described as spherical polar set Q (total puckering amplitude), and the angles θ and φ, describing the distortion suffered 330

9.1. X-Ray Diffraction of O-Glycosides

331

TABLE 9.1. Standard molecular dimensions for 4 C1 chair conformations in pyranosides. Bond ˚ lengths (A)

Bond ˚ lengths (A)

C-C ring C-C exo C-O exo

1.526 1.516 1.420

1.523 1.514 1.426

C5-O5 axial C1-O5 axial C1-O1 axial C5-O5 eq. C1-O5 eq.

1.434 1.419 1.398 1.426 1.428

1.436 1.419 1.415 1.436 1.429

Bond type

Angle type

Bond angle (◦ )

Bond angle (◦ )

C-C-C ring C-C-C exo C-C-O ring C-C-O exo C5-O5-C1 O5-C1-O1 C5-O5-C1 O5-C1-O1

110.4 112.5 110.0 109.7 114.0 112.1 112.0 108.0

110.5 112.7 110.0 109.6 114.0 111.6 112.0 107.3

4 atoms ring

Torsion angles (◦ )

C-C-C-C C-C-C-O C-C-O-C C-C-C-C C-C-C-O

53 56 60 53 57

C pole

θ HB

HC

Q φ

B

equator

TB

FIGURE 9.1. One octant of the sphere on which the conformations of six-membered rings can be mapped for a constant Q.

by six-member rings from the ideal chair conformation. The chair corresponds to θ = 0◦ , φ = 0◦ ; boat for θ = 90◦ , φ = 0◦ ; and twist boat for θ = 90◦ , φ = 90◦ (Figure 9.1). The pyranoside ring varies slightly and in terms of Cremer and Pople ˚ with θ within 5◦ of puckering parameters, the range of values is Q = 0.55–0.58 A ◦ 3 0 or 180 .

9.1 X-Ray Diffraction of O-Glycosides One of the pioneering studies about sugar X-ray analysis was presented by Levy and Brown5 reporting the structure of sucrose and sucrose NaBr.H2 O. Through these studies it was observed that although they were energetically equivalent, the chair conformation was different, due to slight hydrogen bridge interactions on the furanoside moiety (Figure 9.2).

332

9. X-Ray Diffraction of Glycosides

FIGURE 9.2. X-ray diffraction of sucrose and sucrose NaBr.H2 O.

Another disaccharide characterized by X-ray crystallography was octa-Oacetyl-β-D-cellobiose, which presents space group P21 ,21 ,21 , with both pyranoside residues in 4 C1 chair conformation slightly more distorted in comparison with cellobiose. Moreover, the torsion angles determined were for O5-C1-C4 −77◦ , and for C1-O1-C4-C5 104◦ (Figure 9.3). The sign value indicates according with the Klyne & Prelog notation to the right if positive and to the left if negative.6

FIGURE 9.3. Chair conformation for octa-O-acetyl-β-D-cellobiose.

9.1. X-Ray Diffraction of O-Glycosides

333

FIGURE 9.4. Thermal ellipsoid drawing and packing diagram showing the hydrogen bonding along [001] of phenyl methyl 2,3,4,-tri-O-acetyl-b-D-fucopyranoside.

The crystal structure of benzyl 2,3,4-tri-O-acetyl-β-D-fucopyranoside is described,7 presenting a monoclinic system, space group P21 , with bond distances ˚ C-C 1.513 A, ˚ and shorter C-O 1.380 A ˚ for equatorially anomeric C-O 1.423 A, bond. The angle disposition for the endocyclic bond C1-O5-C5 is of 112.4 (3)◦ , being this value typical for chair conformation 4 C1 in pyranoside with substituents positioned at equatorial positions. The perspective view of the molecule shows equatorial disposition for all substituents except position 4 that remains axial (Figure 9.4).

334

9. X-Ray Diffraction of Glycosides

FIGURE 9.5. Perspective Ortep view of phenylmethyl glucosyl fucopyranosyl derivative showing the distortion degree between 5- and 6-member fused rings on chair conformation.

Disaccharide phenylmethyl-O-(2,3-di-O-acetyl-4,6-O-benzylidene-β-D-glucopyranosyl)-(1→2)-3,4-O-isopropylidene-β-D-fucopyranoside shows for fucopyranoside moiety a distorted chair due the ﬁve-member ring acetonide at O3 and O4 positions, with Cremer and Pople puckering parameters of Q = 0.556 (3), θ = 159.9 (3)◦ , and φ = 220.8 (8)◦ . In contrast for the glucopyranosyl moiety with a six-member ring benzylidene ring attached at positions O4 and O6, the chair conformation is less distorted with Cremer and Pople puckering parameters of Q = 0.597 (3), θ = 170.5 (3)◦ , and φ = 156.0 (16)◦ (Figure 9.5).8 The solid-state crystal structure of glycoresin tricolorin A was solved by using an intense syncroton radiation to collect data. The cystals belongs as usual to the P21 having cell dimensions a = 14.025(1), b = 33.337(1), c = 25.512(1) ˚ β = 91.07(1)◦ . The energy maps were calculated as a function of two glycoA, sidic linkage torsion angles deﬁned as φ = (O5-C1-O1-Cx) and ψ = (C1O1-Cx-C(x+1), indicating a higher level of conformational freedom along the ψ-axis. The size of the crystal unit cell demonstrate the presence of four independent tricolorin A molecules per asymmetric unit and the reﬁned structure showed the presence of 18 water molecules forming a channel along the hydrophilic region (Figure 9.6).9 Other selected pyranosides analyzed by X-ray diffraction and their parameters determined are shown in Table 9.2.

9.1. X-Ray Diffraction of O-Glycosides

335

FIGURE 9.6. ORTEP representation of tricolorin A and graphical representation of the unit cell.

336

9. X-Ray Diffraction of Glycosides

TABLE 9.2. X-ray diffraction parameters of some selected pyranosides. Structure

Symmetry cell

OPiv

orthorhombic

Symmetry space P 21 21 21

Conformation

monoclinic

P 21

monoclinic

P21

orthorhombic

P 21 21 21

monoclinic

C2

orthorhombic

P 21 21 21

monoclinic

C2

4C

monoclinic

P21

4C

Ref

4

C1

10

4

C1

11

chair for α-anomer and boat β-anomer

12

OH O HO PivO OMe OCOCH2Ph O N H PhH2COCO

O

PhH2COCO

OMe

O

Ph

O

O PhO2S

HN OMe Ph

Ph N N

OAc

4

C1

13

1 4C

14

4

C1

15

1 4

C1

16

1 4

C1

17

S

O

AcO AcO

S OAc

Me

O N MeOOC

O N OAc O

MeO

NH

OAc

OAc OH

OH O

O OH OH

OH OH Me

O OH

O

O OH HO

OH OMe

OH OH

OH HO O OH

OH

. 5 H2O

O HO

O OMe OH

MeOH

9.2. X-Ray Diffraction of Nucleosides

337

9.2 X-Ray Diffraction of Nucleosides A number of N -glycosides and C-glycosides has been solved by X-ray analysis, presenting as common features space group P21 21 21 orP21 , the furanoside ring in the twist conformation, and symmetric system monoclinic or orthorhombic. For instance, the hypermodiﬁed nucleoside queuosine presents a space group ˚ and symmetric P21 21 21 , cell dimensions a = 26.895, b = 7.0707, c = 23.883 A, system orthorhombic (Figure 9.7). The three-dimensional structure determined by X-ray has been also helpful to understand the recognition process at the tRNA level. Thus, based on this information it was possible to determine that the bulky group cyclopentenediol due to the trans disposition assumed is not involved in any interaction codon-anticodon, therefore suggesting that another type of interaction was taken place.18 The unusual conformation of α-D anomer of 5-aza-7-deaza-2 -deoxyguanosine has been reported by Seela et al.19 In this work it is described that the title compound adopts a high-anticonformation with the C1 -C2 and N9-C8 bonds nearly eclipsed with torsion angle C1 -C2 -N9-C8 = 30.3 (4)◦ . It can be also observed that for 2 deoxy-α-D-ribonucleosides the C2’ endo sugar puckering with either a half chair or envelope conformation is preferred (Figure 9.8). The solid-state conformation of constrained carbocyclic nucleosides (N)methano-carba-AZT and N-(S)-methano-carba-AZT was determined by X-ray

FIGURE 9.7. Perspective view of hypermodiﬁed nucleoside queuosine.

338

9. X-Ray Diffraction of Glycosides

FIGURE 9.8. Perspective view of α-D anomer of 5-aza-7-deaza-2 -deoxyguanosine.

diffraction. As expected with the prediction, their thermal ellipsoid presented a rigid pseudoboat conformation for the bicycle [3.1.0] hexane system, which makes them assume in nearly perfect 2 E and 3 E envelope conformations in the pseudorotational cycle (Figure 9.9).20 Other selected N-nucleosides that have been analyzed by X-ray diffraction and their parameters determined are shown in Table 9.3.

FIGURE 9.9. X-ray structure of (N)methano-carba-AZT.

TABLE 9.3. N-Glycosides and their X-ray diffraction parameters.

340

9. X-Ray Diffraction of Glycosides

TABLE 9.3. (Continued)

References 1. (a) S. Furberg, and C.S. Petersen, Acta Chem. Scan. 1962, 16, 1539. (b) Reeves, R.E. J. Am. Chem. Soc. 1950, 72, 1499. 2. G.A. Jeffrey, J.A. Pople, J.S. Binkley, and S. Vishveshwara, J. Am. Chem. Soc. 1978, 100, 373. 3. G.A. Jeffrey, Acta Cryst. 1990, B46, 89. 4. D. Cremer and J.A. Pople, J. Am. Chem. Soc. 1975, 97, 1354. 5. G.M. Brown and H.A. Levy, Science, 1963, 141, 921. 6. F. Leung, H.D. Chanzy, S. P´erez,and H., Marchessault, Can J. Chem. 1976, 54, 1365. 7. M.A. Brito-Arias, E.V.Garc´ıa-Baez, E. Dur´an-P´aramo, and S. Rojas-Lima, J. Chem. Crystallogr., 2002, 32, 237. 8. M.A. Brito-Arias, E. Duran-Paramo, I. Mata, and E. Molins, Acta Cryst., 2002, C58, o537. 9. A. Rencurosi, E.P. Mitchell, G. Cioci, S. P´eres, R. Pereda-Miranda, and A. Imberty, Angew. Chem. Int. Ed. 2004, 4, 2. 10. I. Matijasic, G. Pavlovic, and R. Trojko Jr, Acta Cryst., C59, o184 (2003). 11. O. Renaudet, P. Dumy and C. Philouze, Acta Cryst., C57, 309 (2001). 12. C.G. Suresh, B. Ravindran, K.N. Rao, and T. Pathak, Acta Cryst., C56, 1030 (2000). 13. Z.-Z. Qiu, X.-P. Hui, P.-F. Xu, Acta Cryst, C61, O475 (2005). 14. N. Low, C. Garcia, M. Melguizo, J. Cobo, M. Nogueras, A. S´anchez, M.D. L´opez, and M.E. Light, Acta Cryst., C57, 222, (2001).

References

341

15. Z.-H. Cheng, T. Wu, S.W.A. Bligh, A. Bashall, and B.-Y. Yu, J. Nat. Prod. 2004, 67, 1761. 16. L. Eriksson, R. Stenutz, and G. Widmalm, Acta Cryst., C56, 702 (2000) 17. R. Stenutz, M. Shang, and A. S. Serianni, Acta Cryst., C55, 1719 (1999). 18. S. Yokohama, T. Miyazawa, Y., Litaka, Z. Yamaizumi, H. Kasai, and S. Nishimura, Nature, 1979, 107, 282. 19. F. Seela, H. Rosemeyer, A. Melenewski, E.-M. Heithoff, H. Eickmeier, and H. Reuter, Acta Cryst., C58, O142 (2002). 20. V.E. Marquez, A. Ezzitouni, P. Russ, M.A. Siddiqui, H. Ford, Jr., R.J. Feldman, H. Mitsuya, C. Goerge, J.J. Barchi, Jr., J. Am. Chem. Soc. 1998, 120, 2780. 21. F. Seela, P. Chittepu, J. He, and H. Eickmeier, Acta Cryst., C60, O884 (2004). 22. F. Seela, Y. Zhang, K. Xu, and H. Eickmeier, Acta Cryst., C61, O60 (2005). 23. F. Seela, K. Xu, and H. Eickmeier, Acta Cryst., C61, O408 (2005). 24. W. Lin, K. Xu, H. Eickmeier, and F. Seela, Acta Cryst., C61, O195 (2005). 25. F. Seela, K.I. Shaikh, and H. Eickmeier, Acta Cryst., C61, O151 (2005). 26. F. Seela, V.R. Sirivolu, J. He, and H. Eickmeier, Acta Cryst., C61, O67 (2005). 27. W. Lin, F. Seela, H. Eickmeier, and H. Reuter, Acta Cryst., C60, O566 (2004). 28. F. Seela, K.I. Shaikh, and H. Eickmeier, Acta Cryst., C60, O489 (2004). 29. J. W. Bats, J. Parsch, and J. W. Engels, Acta Cryst., C56, 201 (2000). 30. F. Seela, A.M. Jawalekar, and H. Eickmeier, Acta Cryst., C60, O387 (2004).

10 Mass Spectrometry of Glycosides

High-resolution mass spectrometry has become another valuable tool for characterization of simple and complex glycosides. The method is based on the collision of a high-energy electron against a sample under study producing as result a cation radical fragment known as the molecular ion, which should match with the molecular weight of the molecule. The mass spectrum also registers a number of fragments being the most intense the base peak assigned a relative intensity of 100. Mass spectrometry can be applied as high and low ionization experiments, being for the former the most suitable for glycosides electron impact and for the latter fast atom bombardment (FAB) and electrospray ionization the routine experiments for characterization of glycosides. In terms of sensitivity of the measurement this instrumental method requires small amount of sample, even in the order of nanogram quantities. The fragmentation patterns of acetyl-protected pentoses and hexoses was studied and their main m/z fragment established. For instance, for methyl-β-Dxylopyranoside triacetate the main fragment follows the two alternative routes shown in Figure 10.1.1 High ionization experiments such as electron impact has been found to be a suitable approach for the determination of the molecular weight through their corresponding molecular ion of protected glycosides such as peracetylated Oglycosides of low molecular weight. For instance, by using electron impact it was possible to determine the molecular weight, and the common fragmentation patterns, of m/e 331 and 169 (100) of the phenylazonaphtol-β-D-glucopyranoside pentaacetate (Figure 10.2).2 However, for most of nonprotected glycosides, high ionization does not provide reliable information and commonly decomposition is observed due to thermal unstability. The introduction of shoft ionization techniques such as fast atom bombardment (FAB) and electrospray ionization has produced great progress for the structural characterization of simple and complex glycosides. This important analytical procedure is especially useful for determining the molecular weight through detection of the molecular ion, as well as sugar sequence. The choice of the matrix and the solubility of the sample are essential aspects to consider for obtaining

342

10. Mass Spectrometry of Glycosides O OCH3 OAc

O

-CH3O

-CH3COOH

OAc

OAc

m/e: 199 A1 - 60

OAc OAc

343

OAc

A1= M-31 (m/e: 259)

MW 290 -CH3COOH

-CH2CO

m/e: 139

m/e: 97

A2

A3

O OCH3 OAc

-CH2CO

AcO

OAc

m/e 128

-CH2CO

B2 OAc

MW 290

m/e 86 B2-42

OAc

m/e 170 B1

FIGURE 10.1. Fragmentation pattern of methyl-β-D-xylopyranoside.

the best resolution. Glycerol is the matrix most commonly used and it is the best choice for underivatized carbohydrates and glycopeptides. Some other matrices used alternatively for hydrophobic samples are thioglycerol, tetraethyleneglycol, and triethanolamine.3 The use of derivatives also plays an important rule and may improve the spectral interpretation and the sensitivity. The most commonly used derivatives are the per-O-acetyl and the per-O-methyl. Usually for the former the fragmentation pathways are less speciﬁc and furnish more information, although the spectrum is more difﬁcult to interpret. For the assignment of the molecular ion it is important to recognize the pseudomolecular ions produced during a FAB experiment, which can be positive-ion and negative-ion mode. In the positive-ion mode the usually present signals are [M+H]+ , [M+NH4 ]+ , [M+Na]+ , and [M+K]+ , and for the negative [M−H]− , and for those molecules that cannot lose a proton [M+Cl]− , or [M+SCN]− . Some of the most common fragmentation pathway produced by polysaccharides and glycoconjugates are represented in Figure 10.3:4 Likewise, application of different ionization techniques in the study of natural glycosides has been performed and derived. From this it has been possible to assign the main potential fragmentation sites in O- and C-glycosides (Figure 10.4).5 Negative ion FAB-MS in triethanolamine of synthetically prepared glycoresin composed by fucose, glucose, and quinovose attached to jalapinolic acid (Figure 10.5) shows [M−H]− peak (m/z 1216) in agreement with the expected molecular weight.6

344

10. Mass Spectrometry of Glycosides

FIGURE 10.2. Mass spectrum of Phenylazonaphtol glucoside.

Mass spectrometry has been also applied successfully for glycoprotein structural determination of primary structure. The ﬁrst glycoprotein primary structure was determined through electron impact and chemical ionization;7 however, soft ionization methods of fast atom bombardment (FAB), electrospray (ES), or matrixassisted laser desorption ionization (MALDI) are conducting most of the glycoprotein structural determinations. FAB is particularly useful for analyzing the permethyl derivatives of oligosaccharides released from glycoproteins by chemical or enzymatic methods. When the atom or ion beam collides with the matrix, a substantial number of sample molecules are ionized producing positively charged species called quasimolecular ions [M+H]+ and [M+Na]+ .8 In order to optimize fragment ion information of glycoproteins, three approaches are currently being used: inducing fragmentation by collisional activation,

10. Mass Spectrometry of Glycosides

345

Pathway A

CH2OH

CH2OH

O

+

O

O

O

O HO

CH2OH

OH

O HO

+

H

+

O

OH

HO

OH

Pathway B

CH2OH

CH2OH

O

+ or –

O

+ or –

CH2OH

O

O

O

O

+ or –

H

HO

O

+ or –

H

H HO

HO

OH

OH

HO

OH

Pathway C

CH2OH

CH2OH

O O

O O

H OH

OH

+ or –

CH2OH

O O

HO

+ or –

+ or –

H

O

OH

OH HO

+ or –

H

OH

Pathway D CH2OH

CH2OH

O

+ or –

+ or –

CH2OH

O O

O

O

O H

HO

OH

+ or –

+ or –

H OH

O

H O

O

OH HO

OH

FIGURE 10.3. The most common fragmentation pathways.

H

346

10. Mass Spectrometry of Glycosides

Pathway E

CH2OH

CH2OH

O

+ or –

+ or –

CH2OH

O

O O

O H HO

+ or –

O

OH

H

+ or –

O OH

H

O

OH

OH HO

OH

FIGURE 10.3. (continued)

monitoring natural ionization-induced fragmentation, and selecting derivatives that enhance and direct fragmentation. During collisional activation of collected fractions from an enzymatic digest, the ﬁrst step is to identify in the MS mode the fractions containing sugar fragmentions. Then switching to the MS/MS mode of a doubly or triply charged ion a composite spectrum containing fragmentation of saccharide and peptide is obtained. Since glycosidic bonds are weaker than peptide bonds, the basic oligosaccharide sequence is determined.9 The natural fragmentation approach relies on the fragmentation created by internal energy transfer to the ion during the ionization process, and now is becoming most limited in use than the previous one.10

Gly

O

Aglycone Gly

C

Aglycone

S

A + frag S A

A A + Gly'

S A + Gly S'

Gly'

O

Gly

O

S + S'

Gly'

Aglycone A

O

Gly

O

Aglycone

S' A + Gly

S, S' = free sugar unit

A

A = aglycone Gly, Gly' = inked glycosidic unit

FIGURE 10.4. The main potential fragmentation sites in O- and C-glycosides.

10. Mass Spectrometry of Glycosides

347

FIGURE 10.5. FAB-MS negative mode of synthetically prepared protected glycoresin.

Derivatization methods are likewise divided into tagging of reducing ends and protection of most of all of the functional groups. The ﬁrst type facilitates chromatographic puriﬁcations and enhances the formation of reducing end fragment ions. The second type involves primarily the permethylation, which form abundant fragment ions arising from cleavage on the reducing side of each HexNAc residue. The permethylation of Tamm-Horsfall glycoprotein was effected and the FAB mass spectrum obtained, showing molecular ions for core 2-type structures carrying up to three sialyl Lex moieties (Figure 10.6).11

348

10. Mass Spectrometry of Glycosides

FIGURE 10.6. Partial FAB mass spectrum of permethylated O-oligosaccharides from glycoprotein uromodulin.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

K. Bieman, J. Am. Chem. Soc. 85, 2289 (1963). M. Brito-Arias, unpublished results. K. Harada, M. Suzuki, and H. Kambara, Org. Mass. Spectrom, 1982, 17, 386. M. Dell, Advances in Carbohydrate Chemistry and Biochemistry, 1987, 45, 19. J.-L. Wolfender, M. Maillard, A. Marston, and K., Hostettman, Pytochemical Analysis, 1992, 3, 193. M.A. Brito-Arias, R. Pereda-Miranda, and C.H. Heathcock, J. Org. Chem., 2004. Morris, et al., J. Biol. Chem. 1978, 253, 5155. A. Dell and H.R. Morris, Science, 2001, 291, 2351. Teng-umnuay et al., J. Biol. Chem. 1998, 273, 18242. A. Dell, Adv. Carbohydr. Chem. Biochem. 1987, 45, 19. R.L. Easton, M.S. Patankar, G.F. Clark, H.R. Morris, and A. Dell, J. Biol. Chem. 2000, 275, 21928.

Index

A Abacavir, 179, 220 Acarbose, 35 Aceptor, 95 Acetate, 46, 145 Acetobromoglucose, 39, 40 Acetonide, 49 Acyclovir, 223, 224 Adenosine, 157 Adenosine triphosphate, 138 Aldohexoses, 318 Aldopentoses, 318 Aldolases, 22 Aldol condensation, 22 Aloin, 247 Altromycin B, 247 Aminosugars, 29 Anomeric effect, 38 Antigens Globo H, 112 KH-1, 292 Lewis, 292 MBr1, 292 Antisense, 172 Antivirals, 179 Aristeromycin, 210, 219 Armed-Disarmed approach, 95, 96 Arthrobacilin A, 109 Aurodox, 247 AZT, 191

B Benzoyl, 47, 145 Benzyl, 47, 146 Benzylidene, 50 Biosynthesis, 11 Bleomycin, 74

Boronate, 50 Brigl epoxide nmr spectrum, 101 preparation, 100

C Cadeguomycin, 155 Calcitonine, 283 Calicheamicin, 82 Capuramycin, 138, 195 Carbapyranosides, 34 Carbonate, 50 Carbovir, 220 Carminic acid, 247 Castanospermine, 28 Cellobiose, 332 Cellulase, 308 Chair conformation 1, 3, 4 Chromophores, 310 Concanavalin, 279 Cross-metathesis, 260 Cucumerin, 247 Cyclodextrin, 124 Cytidine, 161

D Deazapurines, 155 Deoxynojirimycin, 27 Disaccharides, 319 Disarmed sugars, 95, 96 Donor, 39, 95

E Electrospray, 344 Eleutherobin, 88

349

350

Index

Epirubicine, 77 Exoglycals, 265

F Fast atom bombardment, 344 Fischer projections 3–7 Fluoresceine, 310 Formycin, 2002, 207 Fucose, 72, 272, 333 Furfural, 10

G Galactose, 16, 272 Ganglioside, 81, 115 Gentobiose, 75 Glycal nmr spectrum, 99 preparation, 98 Glucanase, 308 Glucosamine N-acetyl, 272 Glucuronidase, 308 Glycans, 274 Glyceraldehyde, 6 Glycosidase, 308 Glycosides alpha, 104, 110 cis, 73 reactivity, 35 trans, 73 Glycosyltransferases, 113–115 Guanosine, 157

H Heck type reactions, 166, 167 Herbicidin, 247 Hikizimycin, 138

I Iminosugars, 25 Imidate formation, 82 Imidate reaction, 81 nmr spectrum, 82 Indole O-glycoside, 311

J Jalapinolic acid, 83

K Kanamacin A, 77 Karplus, 315, 316 Kifunensine, 28 Kiliani-Fischer synthesis, 9

L Lactosamine, 117 Laminaribiose, 74 Lasalocid A, 247, 255 Leaving group, 35 Lectins, 277 Lipase, 142 Lodenosine, 193 Lubocavir, 219 Luganol, 108

M Malayamycin A, 207, 209 Mannose, 272 Mannostatin, 28 Methoxybenzyl, 48 Monosaccharides deﬁnition, 1 distribution, 2 reactions, 3–11

N Nectricine, 28 Neplanocin, 210, 221 Nicotinic acid adenine dinucleotide, 138 Nikkomycin B, 138 p-Nitrophenol, 311 Nojirimycin, 29 Nuclear Overhauser effect, 317, 323 Nucleosides, 138, 155, 179, 327

O Octosyl acid A, 138 Oxidations, 3–5

P Palladium type reactions, 166, 167, 187 Palytoxin, 247 Phenylazonaphtol glycoside, 312, 344 Phosphonate, 172 Phosphoramidite, 170

Index Pseudouridine, 202, 205, 327 Pyramicine, 202 Pyrazomycin, 202 Pivaloyl, 47, 145 Protecting groups, 43 Puckering parameters, 330

Q Queuine, 195 Queuosine, 138, 337 Quitinase, 308

R Raﬁnose, 77 Rearrangements enediol, 9 Pummerer, 241 Ramberg-B¨acklund, 21, 262 sigmatropic, 110 Residual dipolar coupling, 318 Resoruﬁn, 310 Rhodiooctanoside, 117 Ruff degradation, 10

S Samarium, 260 Sangivamicine, 154 Saponarin, 247 Scoparin, 247 Selectins, 84, 281 Showdomycine, 202, 206 Sialic acids, 12, 272 Sialyl Lewis, 104 Silyl protecting groups, 146, 147, 160 Sinefungin, 138 Spongistatin, 247 Sucrose, 331

Sugar amino acids, 18 Sulfotransferases, 117 Swainsonine, 28

T Tebbe reagent, 265 Thiazofurine, 202, 205 Thioglycosides, 89 Thiomonosaccharides, 34 Thionucleosides, 228 Thuringiensin, 138 Transglycosylation, 117 Tricolorin, 83, 324, 334 Toluyl, 145 Toyocamicine, 154 Trans rule, 157 Triciribine, 200 Trityl, 47, 146 Tubercidine, 154 Tunicamycin V, 138, 198

U Umbelliferone, 70, 310 Umpolung, 257

V Vineomicinone B2, 256

W Wang resin, 121 Wittig-type reaction, 265 Wye base, 138

X Xylose, 272, 343

351