Advances in Carbohydrate Chemistry and Biochemistry, Volume 44

Advances in Carbohydrate Chemistry and Biochemistry

Volume 44

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Advances in Carbohvdrate Chemistrv and Biochemistry rl

d

Editors R. STUART TIPSON

DEREK HORTON

Board of Advisors GUYG. S. DUTTON BENCT LINDBERC HANS PAULSEN NATHANSHARON ROYL. WHISTLER

LAURENSANDERSON STEPHENJ. ANCYAL HANS H. BAER CLINTONE. BALLOU JOHN S. BRIMACOMBE

Volume 44 1986

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

Orlando San Diego New York Austin Boston London Sydney Tokyo Toronto

COPYRIGHT @ 1986 BY ACADEMIC PRESS. INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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9 8 7 6 5 4 3 2 1

CONTENTS PREFACE.

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Fred Shafizadeh. 1924-1983 GARYD . MCCINNIS Text

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

Vibrational Spectra of Carbohydrates MOHAMEDMATHLOUTHIA N D

JACK

L . KOENIG

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . I1. Background . . . . . . . . . . . . . . . . . . . . . . . . . .

111. Computational Calculation of Vibrational Frequencies. and Band Assignments . . . . . . . . . . . . . . . . . . . . . . . . . IV . Fourier-transform. Infrared Spectroscopy . . . . . . . . . . . V . Laser-Raman Spectroscopy . . . . . . . . . . . . . . . . . VI . Current Problems . . . . . . . . . . . . . . . . . . . . . . .

7 10

. . . .

31 56 67 85

Monosaccharide Isothiocyanates and Thiocyanates: Synthesis. Chemistry. and Preparative Applications ZBICNIEW J . WITCZAK

1. Introduction

. . . . . . . . . . . . . . . . . . . . . . . . .

I1. Monosaccharide Isothiocyanates . . . . . . . . . . . . . . . . .

111. Monosaccharide Thiocyanates . . . . . . . . . . . . . . . . . IV . Spectroscopic Properties of Monosaccharide Isothiocyanates . . . . V . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . .

. .

91 93 123 139 140

Enzymic Analysis of Polysaccharide Structure

BARRYV . MCCLEARY A N D NORMANK . MATHESON 1. I1. 111. IV . V. VI . VII . VIII . IX . X.

147 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Polysaccharides Having a (1+4)-P-D-Clucan Backbone . . . . . . . 150 Polysaccharides Having a P-D-Xylan Backbone. . . . . . . . . . . 158 Polysaccharides Based on a (1+4)-P-~-Mannan Backbone . . . . . . 164 182 Pectic Polysaccharides . . . . . . . . . . . . . . . . . . . . . Agarose and Related Polysaccharides . . . . . . . . . . . . . . . 186 191 Alginic Acid . . . . . . . . . . . . . . . . . . . . . . . . . Bacterial Peptidoglycan, Chitin, and Chitosan . . . . . . . . . . . 195 198 Glycosaminoglycans . . . . . . . . . . . . . . . . . . . . . . 217 Bacterial Polysaccharides . . . . . . . . . . . . . . . . . . . . V

CONTENTS

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XI. XI1. XIII . XIV .

Glycoconjugates . . . . . . . . . . . . . . . . . . . . . . . . Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . a-D-Glucans . . . . . . . . . . . . . . . . . . . . . . . . . P-D-Glucans . . . . . . . . . . . . . . . . . . . . . . . . .

231 247 252 266

Biosynthesis of Bacterial Polysaccharide Chains Composed of Repeating Units VLADIMIRN . SHIBAEV

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . I1. Glycosyl Esters of Nucleotides and Polyprenyl Glycosyl Phosphates in Polysaccharide Biosynthesis . . . . . . . . . . . . . . . . . . . I11. Biosynthesis of Monosaccharide Components. and Their Activation for Polymeric-Chain Formation . . . . . . . . . . . . . . . . . . . IV . Inter-monomeric Linkages in Bacterial Polysaccharides . . . . . . . V . Assembly of Polymeric Chains . . . . . . . . . . . . . . . . . VI . Enzymic Synthesis of Bacterial Polysaccharides from Modified Precursors . . . . . . . . . . . . . . . . . . . . . . . . . .

277 279 286 305 309 335

Lipid-linked Sugars as Intermediates in the Biosynthesis of Complex Carbohydrates in Plants

RAFAEL PONTLEZICA.GUSTAVO R . DALEO.AND PRAKASH M . DEY I. 11. I11. IV . V.

Introduction . . . . . Lipid-linked Sugars . . Complex Carbohydrates Functional Aspects . . Concluding Remarks . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

341 347 358 378 384

Glycolipids of Marine Invertebrates

NICOLAIK . KOCHETKOVA N D GALINA P. SMIRNOVA I. I1. 111. IV . V. VI . VII .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Occurrence of Glycosphingolipids. Their Isolation. and Purificatian . . Composition of Glycosphingolipids. . . . . . . . . . . . . . . . Determination of the Structure of Glycosphingolipids . . . . . . . Glycolipids of Various Groups of Marine Invertebrates . . . . . . . Biological Role of the Sialoglycolipids of Echinoderms . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . .

INDEX . SUBJECT INDEX .

AUTHOR

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387 391 396 398 409 435 436 439 471

PREFACE In this volume, M. Mathlouthi (Dijon) and J. L. Koenig (Cleveland) discuss the vibrational spectra of carbohydrates in an article that updates and vastly expands those by W. B. Neely in Volume 12 and by H. Spedding in Volume 19 of this series. Important advances in both infrared and Raman spectroscopy have stemmed from discovery of the fast Fourier-transform algorithm, the introduction of efficient minicomputers, the development of Fourier-transform spectrophotometers, and the use of lasers for Raman spectroscopy. Although vibrational spectroscopy has been overshadowed for many years by n.m.r. spectroscopy as a tool for studying molecular structure and interactions, the new developments now readily permit normal coordinate analysis of molecules of the complexity presented by carbohydrates, and the technique is of particular importance for studying hydrogen-bonding interactions of carbohydrates. In an article that collates information not extensively treated before, Z. J. Witczak (West Lafayette) describes the synthesis, chemistry, and preparative applications of monosaccharide thiocyanates and isothiocyanates; the thiocyanate anion is an ambident nucleophile of great synthetic versatility in approaches to nucleoside analogs and to thio and deoxy sugars. B. V. McCleary (Rydalmere) and N. K. Matheson (Sydney) present a broad discussion of the analysis of polysaccharide structure by use of specific degradative enzymes and bring up to date the treatment of the subject as devoted to D-glucans by J. J. Marshall in Volume 30. The biosynthesis of bacterial polysaccharide chains composed of repeating units is treated by V. N. Shibaev (Moscow), who coordinates our knowledge of the manner in which nucleoside and polyprenyl glycosyl diphosphates serve to generate polysaccharides of great structural diversity. A complementary discussion, by R. Pont Lezica and G. R. Daleo (Mar del Plata) and P. M. Dey (Egham), treats the role of lipid-linked sugars as intermediates in the biosynthesis of complex carbohydrates in plants. The final article, by N. K. Kochetkov and G. P. Smirnova (Moscow), on glycolipids of marine invertebrates complements that by E. Lederer in Volume 16 on those of acid-fast bacteria, by Y.-T. Li and S.-C. Li on the biosynthesis and catabolism of glycosphingolipids (Volume 40), and by R. T. Schwarz and R. Datema on the lipid pathway of protein glycosylation and its inhibitors (Volume 40). Finally, an obituary of Fred Shafizadeh is provided by his former student, G. D. McGinnis.

R. STUARTTIPSON DEREKHORTON

Kensington, Maryland Columbus, Ohio July, 1986

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1924-1983

FRED SHAFIZADEH*

1924-1983

Fred Shafizadeh was born on January 26, 1924, and named Fraidoun, in Teheran, Persia, and died as Fred, on October 1, 1983, of a heart attack in Missoula, Montana. He is survived by his wife, Doreen; his daughter, Alexandra S. Startin; and his grandson, Taylor Startin. His premature death, at age 59, removed from the active mainstream of carbohydrate chemistry a major contributor. Fred was a unique individual, best described as a first-rank innovator, an enthusiastic teacher and scientist, and a strong believer in individual rights and responsibilities. A jovial man, he was 5ft 10” tall, somewhat portly, weighing 155 lbs, and had brown eyes and originally brown hair. Dr. Shafizadeh obtained his early education in Persia, receiving a B.S. degree in Chemical Engineering from the Technical College in Teheran in 1946, and then a Ph.D. in Organic Chemistry from Birmingham University, England, in 1950. In Birmingham, he adopted a long and unusual course, which included 2 years of undergraduate, 2 years of graduate, and 2 years of post-doctoral studies. During that period he established himself as a first-class carbohydrate chemist, with several publications on deoxy sugars, and considerable experience on DNA and the biochemistry of cancer to his credit. To broaden the scope of his interests, he spent another year as a postdoctoral fellow in the Physics Department of Pennsylvania State University, working on the X-ray analysis of biological compounds. Equipped with an exceptionally broad and multidisciplinary education and experience, he proceeded to The Ohio State University, Columbus, Ohio, to work with Professor M. L. Wolfrom. His initial job was to investigate the ignition of cellulose nitrate, a project left over from World War 11. In Fred’s hands, this project was turned into an isotopic investigation of the biosynthesis and degradation of cellulose. At this time, there was very little known about the preparation of specifically labeled sugars, let alone the biosynthesis of *The kind assistance of Drs. Donald F. Root, Keith Osterheld, Allan Bradbury, and Murray Laver, and Professor A. B. Foster is greatly appreciated.

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Copyright @ 1986 by Academic F’ress, Inc. All rights of reproduction in any form reserved.

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GARY D. McGINNIS

specifically labeled cellulose, or suitable methods for determining the isotopic distribution within the labeled polysaccharides. Furthermore, the modern methods for the isolation and radiochemical analysis of numerous degradation products of cellulose were not known. The preparation of labeled cellulose, involved, among other things, experiments with cultures of Acetobacter xylinurn and the growing and treatment of cotton plants north of the Mason-Dixon line, both of which exceeded the traditional expertise of a carbohydrate chemist. Despite all these problems, Fred’s achievements went far beyond his original expectations, and resulted in several publications on the incorporation of D-glucose from the metabolic pool into cellulose, determination of the distribution of the I4C label in labeled cellulose, the mechanism of the thermal decomposition of cellulose nitrate, and even the biosynthesis and fragmentation of cotton-seed oil. These are now considered to be classical achievements, but, at that time, they had to be presented to and argued with Professor Wolfrom in order to gain his acceptance. This in itself was not an easy task, especially when the more precise, isotopic data that Fred had obtained contradicted some of the previously published results. Professor Wolfrom’s exacting manners and standards provided a challenge, rather than a hurdle, for Fred, whose thorough handling of this project resulted in the incidental discovery of L-iduronic acid, a by-product of the synthesis of D - ~ ~ u c o s ~ -which ~ - ~ ~since C , has been found to be a component of heparin and chondroitin sulfate. Also, incidental to the problems of isolating, as phenylhydrazones, the fragmentation products of 14 C-labeled cellulose nitrate, it was found that reduction of the hydrazone provides a practical method for the synthesis of amino sugars. Wolfrom, being an astute and exacting research director, did not lose any time in getting Fred to develop the leads that he had found for the synthesis of biologically significant amino sugars, and provided him with some graduate-student help in order to proceed in this direction. In this way, some of the rare and hitherto unobtainable amino pentoses and amino hexoses were synthesized. This original procedure has since been modified, and used for the synthesis of a variety of amino sugars. Fred was not content with the idea of synthesizing new compounds, and, by combining the knowledge on DNA and modified sugars that he had acquired in Birmingham, he developed a major program at Ohio State for the synthesis of modified nucleosides for testing in cancer chemotherapy. After organizing the aforementioned program, which employed a number of graduate students and postdoctoral fellows, Fred decided to accept a job with the Weyerhaeuser Company in Seattle, Washington. At Weyerhaeuser, Fred was assigned to one of the most difficult problems of the wood-products industry, namely, development of a practical and economic

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method for the dimensional stabilization of wood. Here, again, Fred adopted a basic approach to the problem. The processes that he developed in a short while were tested and patented. The results sufficiently impressed the management of that resource-oriented company that they created a new department for Pioneering Research, and promoted Fred to manage it. A new laboratory, close to the University of Washington in Seattle, was leased and remodeled, and Fred’s staff and responsibility were expanded to embrace a catalog of the hitherto-unsolved problems of the wood-products industry, including waste utilization, lignin utilization, new and better flame-proofing methods, new modifications of cellulose, and new methods of combining plastics with wood products. Several patents were issued to Weyerhaeuser as a result of Fred’s program, including the dimensional stabilization of wood (U.S. Pat. 3,284,231), levoglucosan (US. Pats. 3,305,542 and 3,414,560), and levulinic acid from hexoses of wood. Noteworthy among the areas of research that he directed were fire-retardant treatments of wood, wood preservation, sustained-release herbicide and nutrient formulations, and the polymer coating of wood products (U.S. Pat. 3,616,028). Some of the data that he had obtained on the combustion and pyrolysis of cellulose were presented at national meetings, and he was a participant in the United Nations F A 0 meeting on wood saccharification, October, 1960, in Tokyo, Japan. He became a naturalized American citizen in June, 1970. In 1966, the University of Montana chose Fred Shafizadeh to become Professor of Chemistry and Forestry, and the director of its newly created Wood Chemistry Laboratory. When Fred moved to Montana, there were minimal amounts of space, money, and equipment available for his program. Fred had two choices-he could be content with the facilities, and live the comfortable life of a professor in a small State University, or he could try to build a strong program by attracting external support. Fred decided on the latter course. The present national and international stature of that Laboratory is truly a memorial to the talents, the energy, and the dedication that he devoted to developing it. Under his leadership, important contributions were made to our understanding of a variety of topics, including the chemistry of plant constituents, the chemical taxonomy of plants, the mechanism of combustion of wood and cellulosic materials, the control of the combustion process in wood and paper, the chemistry of biomass gasification, and the chemical utilization of wood and cellulosic wastes. His unusual ability to design fundamental studies of problems of practical importance contributed greatly to the significance of his contributions. Fred’s major contributions were in the area of combustion and pyrolysis of cellulosic materials. At Montana, he developed a research program for unravelling the complex, consecutive and concurrent reactions involved in

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the pyrolysis, combustion, and flame-proofing of cellulosic materials. The interaction of natural fuels and energy, resulting in the formation of volatile, combustible materials and the spreading of flaming combustion, was investigated through analysis of the thermal properties and the pyrolytic reactions of the various components, including cellulose and hemicelluloses. The thermal degradation of these compounds was, in turn, investigated by using a variety of model compounds, in order to ascertain the mechanism of cleavage of the glycosidic bond and the decomposition of the sugar units at different temperatures. The methods of thermal analysis developed in this program threw a new light on an area of carbohydrate chemistry that previously was completely in the dark. The results, published in various journals, are of such a basic and broad nature that their significance transcends the chemistry of cellulosic fires, and covers many fundamental aspects of carbohydrate chemistry, such as the physical transitions and molecular motions, anomerization, polymerization, transglycosylation, dehydration, fission, and carbonization of the carbohydrate compounds. The thermal-analysis methods were also used to determine, not only the heat of combustion, but also the rate of heat release and the seasonal variation of combustibility, matters of practical significance for the protection and conservation of forest resources. In the area of waste utilization, Fred’s program on the heat content, gasification, and carbonization of forest fuel is now recognized as a major step in our understanding of forest fires. The acid-catalyzed pyrolysis of cellulosic waste to afford 1,6-anhydro-3,4-dideoxy-p-~-glycero-hex-3enopyranos-2-ulose (“levoglucosenone”) pointed the way to another method of chemical conversion of cellulosic wastes similar to “cat-cracking” in the petrochemical industry. On the sagebrush program, isolation of the minute amounts of the extractable sesquiterpene lactones, and structural determination thereof, mainly through interpretation of the n.m.r., mass, and i.r. spectra of various derivatives, were achievements of the first magnitude for any organic chemist. In this program, Fred had again gone beyond the traditional scientific barriers by correlating these compounds and their properties with the taxonomy, physiology, and ecology of the Artemisia species, showing the penetration and depth of his inquiries, and his understanding and appreciation of the biological problems involved. Fred’s courage, and his capability to delve into multidisciplinary problems involving a range of subjects from physical chemistry to cellular biology, are clearly reflected in his penetrating analysis and discussion in an article on the morphology and biogenesis of cellulose and plant cell-walls. This article unfolded more than a century of multidisciplinary developments in a critical and coherent manner that constituted a hallmark in cellulose

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5

chemistry. It started with a consideration of the composition and ultrastructure of the fibers, and ended with a discussion of the role of various cell-organelles in producing them. Other reviews and contributions by Fred, on pyrolysis and combustion of cellulosic material and on cleavage of the oxygen ring, showed the same qualities of timeliness and scholarship. Fred Shafizadeh published over 160 research papers and review articles, was co-editor of two books, and was the inventor or co-inventor for six patents. He was frequently invited to speak at national and international meetings, and, in June, 1975, he visited laboratories in Moscow, Leningrad, Riga, and Tashkent, U.S.S.R., under an exchange program of the National Academy of Sciences of the United States of America and the Academy of Sciences of the U.S.S.R. In 1972, the University of Birmingham, England, awarded Professor Shafizadeh the D.Sc. degree in recognition of his important contributions on carbohydrates and sesquiterpene lactones. He was a member of the American Chemical Society, and of its Carbohydrate Division (Chairman, 1972-1973) and its Cellulose, Paper, and Textile Division (Chairman, 1971-1972). He was also a member of The Chemical Society (London), The Society of the Sigma Xi, the Combustion Institute, the Torrey Botanical Club, the Technical Association of the Pulp and Paper Industry, and the Montana Academy of Sciences. He served on the editorial boards of the Journal of Analytical and Applied pVrolysis and the Journal of Wood Chemistry and Technology. He chaired a number of symposia and conferences, including the July, 1983, Gordon Conference on Analytical Pyrolysis. Through election by the University of Montana faculty, he served on the Faculty Senate and on the Executive Committee of the Senate. In 1980, he was awarded the University of Montana’s first Distinguished Research Award. Some of the contributions of Fred Shafizadeh the scientist have just been detailed but that description represents only a part of Fred Shafizadeh the man, and fails to recognize what a complex and colorful man he was. He came from Persia (now Iran), a country of people having a 3,000-year-old cultural tradition, in which devotion to thought, to education, and to freedom were then central. That cultural tradition continued to be important to Fred, even after he had acquired his pragmatic, scientific education in our western culture. Despite his international stature in research, it was important to Fred Shafizadeh to be involved in teaching. In fact, he insisted each year on teaching a freshman-chemistry course. It was imperative to him to teach the meaning of inquiry, and to convey the spirit and thrill of discovery. His absolute devotion to academic excellence came as much from Persian as from western culture. At the advanced level, his graduate students and postdoctoral fellows left his laboratory remarkably able to move into respon-

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sible positions. Fred always, to an unusual degree, delegated responsibilities to his students and research staff. On leaving his laboratory, these investigators were ready to proceed with research independently, from the conception of a problem to reporting the results obtained. The successes of these people were a source of great pride to Fred. As hardworking and demanding as he was at the University, Fred was at home a quiet, relaxed, and devoted family-man. Fred’s house-parties were superbly hosted by him and his wife, Doreen, and were enjoyed by us all. Although Fred did not himself partake of alcoholic .beverages, he and Doreen would sometimes join the rest of the lab. workers at the Friday-night get-together at a local bar. Any who worked for Fred in his Wood Chemistry Laboratory will invariably say how fortunate they were to have had the experience. Fred’s lab., like the man himself, was one of a kind. Whether it was the inevitable odor of a pyrolyzed carbohydrate, the scenic backdrop of the Rattlesnake Mountains from the lab. window, or just the sight of an exuberant Fred discussing the latest data, the Wood Chemistry Laboratory under Fred Shafizadeh will remain a fond memory for all of us who ever worked there. Fred was a backgammon player, a fisherman at Flat Head Lake, a collector of Oriental rugs, and our good and respected friend. His presence will be missed, but his memory and his contribution will live on through his many friends and students. GARYD. MCGINNIS Forest Products Utilization Laboratory Mississippi State University Mississippi State, Mississippi 39762

ADVANCES IN CARBOHYDRATE CHEMISTRY A N D BIOCHEMISTRY. VOL. 44

VIBRATIONAL SPECTRA OF CARBOHYDRATES

BY MOHAMEDMATHLOUTHI* A N D JACK L. KOENIG Deparimeni of Macromolecular Science, Case Wesiern Reserve University, Cleveland, Ohio 441 06

I. INTRODUCTION

Since the article by Spedding‘ on infrared spectroscopy and carbohydrate chemistry was published in this Series in 1964, important advances in both infrared and Raman spectroscopy have been achieved. The discovery* of the fast Fourier transform (f.F.t.) algorithm in 1965 revitalized the field of infrared spectroscopy. The use of the f.F.t., and the introduction of efficient minicomputers, permitted the development of a new generation of infrared instruments called Fourier-transform infrared (F.t.4.r.) spectrophotometers. The development of F.t.4.r. spectroscopy resulted in the setting up of the software necessary to undertake signal averaging, and perform the mathematical manipulation of the spectral data in order to extract the maximum of information from the ~ p e c t r a . ~ The intense absorption of water over most of the infrared spectrum restricts the regions where aqueous solutions of carbohydrates can be usefully studied. Absorbance subtraction makes it possible to eliminate water absorbance and magnify the remaining spectral features to the limit of the signal-to-noise ratio. Many other data-processing techniques, such as the ratio method: the least-squares refinement,5 and factor analysis: should be of benefit in the study of carbohydrate mixtures. Although carbohydrate chemists and biochemists are familiar with the use of conventional infrared spectroscopy for structural and conformational

* Present address: Institut Universitaire de Technologie, Dtpartement “Biologie Appliqute”, B.P. 510, 21014 Dijon Ctdex, France. (1) (2) (3) (4) (5) (6)

H. Spedding, Adv. Carbohydr. Chem., 19 (1964) 23-49. J. W. Cooley and J. W. Tukey, Math. Comput., 19 (1965) 297. J. L. Koenig, Ace. Chem. Res., 14 (1981) 171-178. J. L. Koenig, L. D’Esposito, and M. K. Antoon, Appl. Spectrosc., 31 (1977) 292-295. M. K. Antoon, J. H. Koenig, and J. L. Koenig, Appl. Specrrosc., 31 (1977) 518-524. M. K. Antoon, L. D’Esposito, and J. L. Koenig, Appl. Spectrosc., 33 (1979) 351-357.

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Copyright @ 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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MOHAMED MATHLOUTHI AND JACK L. KOENIG

studies of mono- and poly-saccharides, few are acquainted with Raman spectroscopy, which provides the “other half” of the vibrational spectra. Since the pioneering investigations of the Birmingham school7-” and the extensive work at the National Bureau of Standards”-I5 in the field of infrared spectroscopy of carbohydrates, no systematic work has been undertaken on the Raman spectra of sugars and their derivatives. The infrared results have been reviewed by T i ~ s o n ,but, ~ ~ as . ~noted ~ by Tu,” no review article has been written on the Raman spectra of carbohydrates. Historically, very few Raman spectra of sugars were when the Raman technique used mercury-arc sources and required timeconsuming, photographic detection with low signal-to-noise ratio. However, in the past few years, the use of modern laser sources has permitted the recording of high-quality Raman spectra in minutes. With the advent of the laser, Raman spectroscopy has experienced a rebirth, and the number of articles on laser Raman spectra of carbohydrates and their derivatives is growing very fast. Our objective in this article is not to introduce the theory of Fourier transform-infrared or laser-Raman spectroscopy; this has already been done for F.t.4.r. in such books as those of Griffiths,” Ferraro and Bade:’ and (7) S. A. Barker, E. J. Bourne, M. Stacey, and D. H. Whiffen, J. Chem. Soc., (1954) 171-176. (8) S. A. Barker, E. J. Bourne, R. Stephens, and D. H. Whiffen, J. Chem. SOC.,(1954) 3468-3473. (9) S . A. Barker, E. J. Bourne, R. Stephens, and D. H. Whiffen, J. Chem. Soc., (1954) 4211-4215. (10) S. A. Barker and R. Stephens, J. Chem. Soc., (1954) 4550-4555. (11) S. A. Barker, E. J. Bourne, J. M. Pinkard, and D. H. Whiffen, Chem. Ind. (London), (1958) 658-659. (12) H. S . Isbell, F. A. Smith, E. C. Creitz, H. L. Frush, J. D. Moyer, and J. E. Stewart, 1. Res. Narl. Bur. Stand., Sect. A, 59 (1957) 41-78. (13) R. S. Tipson, H. S. Isbell, and J. E. Stewart, J. Res. Natl. Bur. Stand., Sect. A, 62 (1959) 257-282. (14) R. S. Tipson and H. S . Isbell, J. Res. Natl. Bur. Stand., Sect. A, 64 (1960) 230-263. (15) R. S. Tipson and H. S. Isbell, J. Res. Narl. Bur. Stand., Sect. A, 66 (1962) 31-58. (16) R. S. Tipson, NatL Bur. Stand. (U.S.) Monogr., 110 (1968) 1-21. (17) R. S. Tipson and F. S. Parker, in W. Pigman and D. Horton (Eds.), The Carbohydrates, Vol. IB, Academic Press, New York, 1980, pp. 1394-1436. (18) A. T. Tu, Raman Spectroscopy in Biology: Principles and Applications, Wiley, New York, 1982, pp. 234-255. (19) F. H. Spedding and R. F. Stamm, J. Chem. Phys., 10 (1942) 176-183. (20) R. Kishore and M. Padmanabhan, Roc. Indian Acad. Sci., Sect. A, 33 (1951) 360-363. (21) P. R. Griffiths, Chemical Infrared Fourier Transform Spectroscopy, Wiley, New York, 1975. (22) J. R. Ferraro and L. J. Basile, Fourier Transform Infrared Spectroscopy, Vols. 1 and 2, Academic Press, New York, 1978.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

9

Painter, Coleman and K ~ e n i gand , ~ ~the general references on laser-Raman spectroscopy are n u m e r ~ u s . We ~ ~ intend - ~ ~ only to describe the techniques, and to comment on the results obtained with these vibrational spectroscopic methods. Besides the computerization of these methods, the use of fast digital computers has radically changed the approach and interpretation of spectra. Whereas, before the advent of the computer, it was a slow, demanding, tedious task2' to make a normal coordinate analysis (n.c.a.) of a small molecule, it now takes only minutes to carry out the normal coordinate analysis of large molecules. The normal coordinate analysis of a-D-glucose2' was achieved for the first time in 1972, and it is now possible to analyze the large molecules of polysaccharides and make parametric refinements by comparison with the experimental As carbohydrates are very sensitive to modifications of the environment, especially when they are in solution, it is of interest to focus attention on molecular interactions in aqueous solution. Vibrational spectroscopy has been ~ h o w n ~to' -be ~ ~one of the techniques most adapted to the study of hydrogen bonding, which is the indicator of molecular interaction in aqueous solutions of sugars. The study of such techniques as FA.-i.r., computerized laser-Raman, or n.c.a., however great their degree of sophistication, should have practical utility for carbohydrate chemists and biochemists. That is why, amid the current problems elucidated by the interpretation of the vibrational spectra of carbohydrates and their derivatives, a section has been reserved for discussion of structure-properties relationships.

(23) P. C. Painter, M. M. Coleman, and J. L. Koenig, The Theory of VibrationalSpectroscopy and its Application to Polymeric Materials, Wiley, New York, 1982. (24) T. R. Gilson and P. J. Hendra, Laser Raman Spectroscopy, Wiley, New York, 1970. (25) J. A. Koningstein, Zntoduction to the Theory of the Raman Eflect, Reidel, Dordrecht, 1972. (26) M. C. Tobin, Laser Raman Spectroscopy, Wiley, New York, 1971. (27) E. B. Wilson, Jr., J. C. Decius, and P. C. Cross, Molecular Vibrations, McGraw-Hill, New York, 1955. (28) P. D. Vasko, J. Blackwell, and J. L. Koenig, Carbohydr. Res., 23 (1972) 407-416. (29) J. J. Cael, K. H. Gardner, J. L. Koenig, and J. Blackwell, J. Cbem. Pbys., 62 (1975) 1145-1153. (30) J. J. Cael, J. L. Koenig, and J. Blackwell, Biopolyrners, 14 (1975) 1885-1903. (31) A. S. N. Murthy and C. N. R. Rao, Appl. Spectrosc. Rev., 2 (1968) 69-191. (32) F. S. Parker, Applications of Infrared Spectroscopy in Biochemistry, Biology and Medicine, Hilger, London, 1971. (33) J. Umemura, G. I. Birnbaum, D. R. Bundle, W. F. Murphy, H. J. Bernstein, and H. H. Mantsch, Can. J. Chem., 57 (1979) 2640-2645.

10

MOHAMED MATHLOUTHI A N D JACK L. KOENIG

11. BACKGROUND

Although progress in experimental techniques and data processing has allowed vibrational spectroscopy to undergo rapid evolution, very little new, fundamental theory has emerged. The vibrations associated with a molecule may be described as bond stretching, bending or angle deformation, and torsional vibrational modes. The frequencies of the various types of vibration are determined by the mechanical motion of the molecule, and depend on the force constants of the bonds between atoms and the masses of the vibrating atoms. The intensities of the infrared absorptions and of the inelastic scattered light (Raman) are determined by such electrical factors as dipole moments and polarizabilities. At the time of the pioneering studies on the infrared spectra of carbohydrates by the Birmingham ~ c h o o l , ~calculations -~’ of the vibrational frequencies had been performed only for simple molecules of fewer than ten atom^.^'*^^*^' However, many tables of group frequencies, based on empirical or semi-empirical correlations between spectra and molecular structure, are a ~ a i l a b l e . ~ * ~ ~ ~ - ~ ~ The widespread use of infrared spectroscopy at that time was probably due to the observation that many chemical groups absorb in a very narrow range of frequency. Furthermore, within this frequency range, the observed frequency may be correlated to specific chemical structures. For example, aldehydes can be differentiated from ketones by the characteristic stretching frequency of the carbonyl group near 1700 cm-’, and the spectral pattern may be likened to a “molecular fingerprint.” However, the application of group vibrational frequencies to the molecular structural problems posed by carbohydrates is only valid when the group concerned is a terminal one and the force constants of the bonds, and the masses of the atoms in the group, differ from those in the rest of the molecule. The approximation of assuming that a molecular vibration is localized in a particular group of atoms is not valid, especially when it deals with the internal skeletal vibrations of the molecule.38This is probably the reason why the classical results are generally localized in the 1200700 cm-l range of frequencies, which corresponds to the vibrations of the groups of atoms peripheral to the pyranoid or furanoid rings of the sugar (34) G. Herzberg, Infrared and Raman Spectra of Polyatomic Molecules, Van Nostrand, New York, 1945. (35) N. B. Colthup, L. H. Daly, and S. E. Wilberly, Introduction to Infrared and Raman Spectroscopy, Academic Press, New York, 1964. (36) L. J. Bellamy, Aduances in Infrared Group Frequencies, Methuen, London, 1968. (37) M. Avram and G. Mattescu, Infrared Spectroscopy, Wiley, New York, 1972. (38) L. H. Little, Infrared Spectra of Absorbed Species, Academic Press, New York, 1966.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

11

molecules. Indeed, this range of frequencies was namedI6 the “fingerprint” or anomeric region39 of the infrared spectra of carbohydrates. The structural analysis of carbohydrates, which is the major interest in the interpretation of their vibrational spectra, necessitates making a synthesis of the information given by differenttechniques. It may be seen from Scheme 1 that the determination of a structure lies at the crossroads of different kinds of information.

Calculations: n.c.a. (to minimize disagreement with experimental results)

Spectroscopic data: ix., Raman, n.m.r., light-scattering

SCHEME 1.-Determination of a Structure.

It is not possible to present the computer calculation of frequencies and the description of the newer ‘techniques (F.t.4.r. and laser-Raman) without developing some background. Structural information that is “carved in stone” is given by the crystallographic data. The bond distances and valence angles used in the calculations are given by the X-ray or neutron diffraction results, and, very often, the geometry of the monosaccharides in the crystalline state is taken as the basis of analysis of their behavior in the polymer or in solution. The substantial amount of spectroscopic information given by the classical, noncomputerized methods (especially i.r. spectra) is not to be neglected, and should be analyzed in the light of subsequent results. 1. Structure Factors in Carbohydrates A polyatomic molecule, such as a sugar, may be regarded as a system of masses joined by bonds having spring-like properties. The vibration of each of the masses (atoms) can be resolved into components parallel to the x, y, and z axis of a Cartesian system of coordinates. This means that each atom has three degrees of freedom, and a system of N nuclei has 3 N (39)

V. M. Tul’chinsky, S. E. Zurabyan, S . A. Asankozhoev, G. A. Kogan, and A. Ya. Khorlin, Carbohydr. Res., 51 (1976) 1-8.

12

MOHAMED MATHLOUTHI AND JACK L. KOENIG

degrees of freedom. For nonlinear molecules, 6 degrees of freedom correspond to translations and rotations of the molecule, and this leaves 3 N - 6 vibrations. The number of vibrational degrees of freedom (3 N -6) is equal to the number of fundamental vibrational frequencies or normal modes of vibration (66 for a hexose). Knowledge of the symmetry elements of a molecule helps in defining the symmetry operations that can be performed. Each symmetry operation results in an interchange of atoms, without changing the configuration of the molecule.23A group of symmetry operations leaves one point unchanged, namely, the center of gravity of the molecule, and such a group is called a point group. It is important to know the classes of symmetry operations in a particular point group if it is desired to determine the number of normal modes of vibration. Most carbohydrates have no symmetry element other than the identity E (or, in some texts, I). This operation, where the molecule remains in the same position, although possessed by every molecule, is useful in the mathematical treatment of the normal coordinate analysis. For such molecules, all of the vibrations are active in both the infrared and Raman spectra. Usually, certain of the vibrations give very weak bands or lines, others overlap, and some are difficult to measure, as they occur at very low wavenumber values.40 Because the vibrations cannot always be observed, a model of the molecule is needed, in order to describe the normal modes. In this model, the nuclei are considered to be point masses, and the forces between them, springs that obey Hooke’s law. Furthermore, the harmonic approximation is applied, in which any motion of the molecule is resolved in a sum of displacements parallel to the Cartesian coordinates, and these are called fundamental, normal modes of vibration. If the bond between two atoms having masses M1 and M2 obeys Hooke’s law, with a stiffness f of the spring, the frequency of vibration u is given by

where M,is the reduced mass

This approximation shows that the vibrational frequency is inversely proportional to the mass, and directly proportional to the force constant. The force constants are defined in terms of internal coordinates of the molecules; they correspond to the forces resisting stretching and bending (40) E. F. H. Brittain, W. 0. George, and C. H. J. Wells, Introduction to Moleculur Spectros-

copy, Theory and Experiment, Academic Press, New York, 1970.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

13

of, and torsions around, the bonds between atoms. The vibrational analysis of a sugar molecule requires accurate knowledge of the atomic coordinates, and a defined set of force constants. In the case of polysaccharides, the problem is reduced by symmetry arguments to determination of the vibrations of the repeating unit.23 Fortunately, data concerning the atomic coordinates are available for a large number of carbohydrates. Many of the sugars and their derivatives that are available in crystalline form have been analyzed by X-ray diffraction. Neutron diffraction data, and refinement calculations of the structures, are relatively scarce. The crystallographic results have been regularly reviewed in this Series:' However, the potential constants are generally compiled from results given in the literature on molecules having the same groups of atoms as carbohydrates, such as aliphatic ethers," or carboxylic but then the problem of transferability of data from one molecule to another arises.23 a. Structure and Atomic Coordinates.-The free monosaccharides exist in the lactol ring-form. In the crystalline form, they generally favor the pyranose over the furanose. Among the possible conformations of an aldopyranose ] is generally found to be the most table.^' the "C,(D)[or ' C 4 ( ~ )conformer When the crystalline sugar is dissolved in water, an equilibrium is established between the lactol ring-forms and the aldehydo or keto acyclic form. On relactolization, the sugar enters into a dynamic equilibrium involving anomerization [axial ( a ) disposition of the OH group attached to the anomeric carbon atom C-1 of an aldose ( (Y anomer of a D sugar) or equatorial (e) ( p anomer of a D sugar)] and ring ( 5 - or 6-membered) i s o m e r i ~ a t i o n . ~ ~ Other modifications of sugar molecules may take place, such as an aldoseketone interconversion, or the isomerization of a glycosylamine to an amino sugar. Anhydro sugars may also be formed. When such a variety of forms is dealt with, it is difficult to find good agreement with experiments if the normal-coordinates calculations are based on standard bond-lengths and valence angles (that is, C-0,143 pm; C-H, 109 pm; 0-H,96 pm; valence angles, tetrahedral). Because of the presence of many oxygen atoms having (41) G. A. Jeffrey and M. Sundaralingam, Ado. Carbohydr. Chem. Biochem., 30 (1974) 445-466; 31 (1975) 347-371; 32 (1976) 353-384; 34 (1977) 345-378; 37 (1980) 373-436; 38 (1981) 417-529; 43 (1985) 203-421. (42) J. H. Schatschneider and R. G . Snyder, Spectrochim. Acfa, 19 (1963) 117-168. (43) R. G. Snyder and J. H. Schatschneider, Spectrochim. Acta, 21 (1965) 169-195. (44) R. G. Snyder and G . Zerbi, Spectrochim. Acfa, Part A, 23 (1967) 391-437. (45) W. V. Brooks and C. M. Haas, J. Chem. Phys., 7 1 (1967) 650-655. (46) Y. Mikawa, J. W. Basch, and R. J. Jakobsen, J. Mol. Specfrosc., 24 (1967) 314. (47) R. E. Reeves, Ado. Carbohydr. Chem., 6 (1951) 107-134. (48) R. U. Lemieux, in P. de Mayo (Ed.), Molecular Rearrangements, Wiley-Interscience, New York, 1964.

MOHAMED MATHLOUTHI AND JACK L. KOENIG

14

different orientations ( a or e), the conformational analysis of carbohydrates is concerned with dipole-dipole interactions that strongly affect the i.r. absorption. Rotation around a linkage between two sugars may take place. This influences the shape of the disaccharide, and affects the conformation that the polysaccharide will adopt. When a carbohydrate polymer is obtained in crystalline form, characterization of its shape is possible by using X-ray diffraction. However, it is not at all certain that this “X-ray conformation” will be that of the active form in the biological environment. Nevertheless, it constitutes a basis for formulating hypotheses concerning the shape in a biological environment. Another structure problem arises as to whether or not it is reasonable to extend the solid-form structure-results to aqueous solution. This is probably possible for polysaccharides, because it is generally found that they are ordered in a highly hydrated en~ironment:~but mono- and di-saccharides in aqueous solutions are much more flexible. It has been shown’’ that, although the problem presents such complexities as the difficulty of including stereospecific, potential-energy functions in the conformational analysis of rotamers capable of forming intramolecular, hydrogen bonds, the extrapolation of crystal structures to carbohydrate conformations in solution will apply to furanoses and to non-hydrogen-bonding solutions. The results concerning the conformational families49(for example, helices and chains) of synthetic polymers are generally transferable to carbohydrate polymers. However, the most important step in the determination of polysaccharide shapes, and vibration analysis of carbohydrates, remains the knowledge of structure factors in mono- and di-saccharides. The analysis of crystallographic results shows the important role played by the anomeric center in the structure of sugars. In Table 15’-59 are reported the C-C and C-0 bond-lengths in some hexopyranoses in both anomeric forms, and in some disaccharides. (49) (50) (51) (52) (53) (54) (55) (56) (57)

D. A. Rees, Polysucchuride Shapes, Chapman & Hall, London, 1977. G . A. Jeffrey, Adu. Chem. Ser., 32 (1973) 177-196. G. M. Brown and H. A. Levy, Science, 147 (1965) 1038-1039. B. Sheldrick, Acra Crysrullogr., Secr. B, 32 (1976) 1016-1020. R. C. G. Killean, W. G. Femer, and D. W. Young, Acru Crysrullogr., 15 (1962) 911-912. H. M. Berman and S. H. Kim, Acru Ctysrullogr., Sect. B, 21 (1968) 897-904. S. C. C. Chu and G. A. Jeffrey, Acru Crysrullogr., Secr. B, 21 (1968) 830-838. B. Lindberg, P. J. Garegg, and G. G. Shwann, Acru Chem. Scund, 27 (1973) 380-381. D. C. Fries, S. T. Rao, and M. Sundaralingam, Acru Crystullogr., Secr. B, 27 (1971) 994-1005.

(58) S. C. C. Chu and G. A. Jeffrey, Acru Crysrallogr., Secr. B, 20 (1967) 1038-1049. (59) G. M. Brown and H. A. Levy, Science, 141 (1963) 921-923.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

15

TABLEI C-C and C - 0 Bond Lengths

Sugars

Average C-C length (Pm)

a Anomer D-Glucose D-Galactose D-Glucose, monohydrate Methyl a-D-glucopyranoside

152.4 152.6 (1.5) 153.2 151.9 (2.5)

D-Glucose D-Galactose Methyl p-D-galactoside Disaccharide a-lactose monohydrate

Average C-0 length, excluding

c-1-0- 1

c-1-0-1

(Pm)

(pm)

References

142.6 142.9 (1.2) 143.3 142.4 (1.5)

139.0 140.0 (1.2) 138.0 141.1 (0.4)

51 52 53 54

152,O (0.2) 152.2 (0.4) 151.6 (0.6)

142.5 (0.2) 143.1 (0.4) 142.9 (0.5)

138.3 (0.4) 139.6 (0.4) 137.5 (0.8)

55 52 56

152.4 (0.5)

143.5 (5)

138.8 (0.4)

57

fi Anomer

(bridge) Methyl p-rnaltopyranoside Sucrose

152.0 (0.8) 152.5 (1.4)

142.7 (0.8) 141.8 (1)

137.5 (0.8) 142.7

58 59

The differences between CY and p anomers on the one hand, and the shortening of the C-0 bond6' attached to the anomeric carbon atom on the other, are, among other structure factors, to be taken into account when calculations of normal coordinates are made. Furthermore, the exocyclic C-C bonds are shorter than the average bond. It is only when neutron diffraction analysis6' is achieved and the atomic coordinates are refined by using anisotropic extinction corrections, as in the case of a-D-glucose,6' that reliable data are obtained that could be taken as a good basis for calculations.

b. Hydrogen Bonding.-Another factor that influences the structure of carbohydrates is hydrogen bonding. The position of the hydrogen atoms is determined with precision only when neutron diffraction is applied in crystal-structure analysis. The number of carbohydrates analyzed by neutron diffraction is relatively small!* However, a compilation of hydrogen-bond data in pyranose monosaccharides, methyl glycosides, and disaccharides (60) H. M. Berman, S. S. C. Chu, and G. A. Jeffrey, Science, 157 (1967) 1576-1577. (61) G . M. Brown and H. A. Levy, Acta Crystallop., Sect. B, 35 (1979) 656-659. (62) G . A. Jeffrey and S. Takagi, Ace. Chem. Res., (1978) 264-270.

16

MOHAMED MATHLOUTHI A N D JACK L. KOENIG

has been presented: and, when it is desired to minimize the disagreement between calculations and experiment, it should be helpful to enter this kind of information in the calculated model. The importance of hydrogen bonding and knowledge of the glycosidic bond have been shown to be essential in the understanding of polysaccharide conformation^^^ The conformations of furanosides were with a special interest in the role of furanosyl groups in the structure of nucleosides and nucleotides. Another approach to the structure of carbohydrates is the application of molecular-mechanics calculations. This method may yield predicted geometries in good agreement with the crystal-structure analysis? but, as for p-maltose in solution, may be at variance with the crystal data.65 The calculated 4 and JI torsional angles around the glycosidic linkage of the disaccharide are dependent on intra- and inter-molecular interactions. The solvation energy must be taken into account in order to predict the conformational behavior of a polysaccharide in solution.66 Moreover, some experimental, spectroscopic r e s ~ l t s ~ ’may * ~ * be interpreted as a demonstration of the flexibility of a disaccharide such as sucrose in water. It is to be emphasized that, in the absence of elements of symmetry, as is the case for carbohydrates, determination of the molecular structure should be based on both the experimental, vibrational spectra and the calculated frequencies. In order to minimize the differences between experimental and calculated results, the structure factors utilized in the calculation should take into account the previous conformational studies. The peculiarities of carbohydrate structures, such as anomeric and exo-anomeric effects, are revealed by bond shortening and torsion-angle modifications. These modifications are accompanied by a change in the vibrational-energy level, and hence, by the corresponding information in their infrared or Raman spectra.

2. Classical Infrared and Raman Results Instead of giving a compilation of the group-frequencies characteristic ’,’~ of carbohydrates, which has already been done for i.r. r e s ~ l t s , ’ ~ * ~we intend to comment on the data for each of the characteristic regions of the spectrum. Although i.r. spectroscopy has been extensively applied in carM. Sundaralingam, J. Am. Chem. Soc., 87 (1965) 599-606. G. A. Jeffrey and R. Taylor, J. Compur. Chem., 1 (1980) 99-109. I. TvaroSka, Biopolyrners, 21 (1982) 1887-1897. I. TvaroSka and T. Kozar, J. Am. Chem. Soc., 192 (1980) 6929-6936. M. Mathlouthi, C. Luu, A. M. Meffroy-Biget, and D. V. Luu, Curbohydr. Res., 81 (1980) 213-223. (68) M. Mathlouthi, Curbohydr. Res., 91 (1981) 113-123. (63) (64) (65) (66) (67)

VIBRATIONAL SPECTRA OF CARBOHYDRATES

17

bohydrate chemistry during the past 30 years, the investigations were generally limited to a region of frequencies, and the results have sometimes been controversial. This is probably due to the fact that the purpose of the studies was firstly analytical. It consisted in determining the identity of, or distinguishing between, different carbohydrate samples;69that is the reason why the “fingejprint” region was the region most used.I6 It is also probable that the state of the technique did not permit recording of well resolved spectra above 1000 cm-’ (below 10 pm) and below 667 cm-’ (above 15 pm). Another approach consists in correlating the frequencies to the most likely vibrations, and including among the reasons for assignments the energy arguments that are essential in assessing a vibrational spectrum That is what was done by Sivchik and Zhbankov”; after calculation and interpretation of the vibrational spectrum of the cellobiose molecule, they distinguished frequency regions that could be extended to all of the vibrational spectra of carbohydrates. These regions are generally adopted” for their significance in the structural analysis of sugars. ( a ) Region of 36002800cm-’: where the stretching vibrations of CH and OH contribute to 100% of the potential-energy distribution (p.e.d.). (b) Region of 15001200 cm-’ : which may be called “the local symmetry” region, because it is mainly constituted of the deformational vibrations of groups having a local symmetry, such as HCH, and the vibrations of the CH,OH group. (c) Region of1200-950 cm : the C - 0 stretching region. For their calculations, Sivchik and Zhbankov” associated C - 0 and C-C contributions, but they noticed that the contribution from C - 0 to the p.e.d. appreciably exceeds that from C-C. ( d ) Region of 950-700 cm-’ : the side-groups deformational-region (COH, CCH, OCH), which includes the important “fingerprint” or anomeric bands between 930 and 840 cm-I, and an appreciable contribution from the stretching of C-C. (e) Region offrequencies below 700cm-’: the skeletal region, which could be split in two, namely, the 700-500-cm-’ range, called39 the “crystalline region,” where the exocyclic deformations (CCO) are observed, and below 500 cm-’, for the endocyclic (CCO, CCC) deformations. It is even possible to separate a low-frequencies region, below 200 cm-’, where the molecular interactions (hydrogen bonding, intercrystalline forces) are revealed. The classical infrared and Raman results will be reviewed by reference to the aforementioned regions of frequencies.

-’

(69) L. P. Kuhn, J. Am. Chem. Soc., 74 (1952) 2492. (70) V. V. Sivchik and R. G . Zhbankov, Zh. Prikl. Spekrrosk, 97 (1977) 853-859. (71) G . A. Kogan, V. M. Tul’chinsky, M. L. Shulman, S. E. Zurabyan, and A. Ya. Khorlin, Carbohydr. Rex, 26 (1973) 191-200.

MOHAMED MATHLOUTHI AND JACK L. KOENIG

18

a. 3600-2800-cm-' region.-The early studies, such as those of the Birmingham school7-" or Verstraeten7' do not refer to this region. The investigations at the National Bureau of Standards were extensive (for example, 56 sugar acetatesI2 and 28 cyclic acetals of sugar^'^). They did not neglect the 3600-2800-cm-' region, where the 3595 cm-I band (2.78 pm) was assigned to free 0 - H stretching in penta- 0-acetyl-aldehyde-D-galactose aldehydro1 and that at 3485 cm-' (2.87 pm) to hydrogen-bonded 0-H. On the other hand, no precise information concerning the C-H stretching was found, and the data connected with the acetals are somewhat inconsistent with preceding assignments, as the free 0 - H stretching band was localized at 3472 to 3279 cm-' (2.88 to 3.05 Fm). The C-H stretching band (28802840 cm-') of 21 methyl aldopyran~sides'~ seem to be characteristic of the glycosidic methoxyl group, regardless of the configuration, or of substitution at C-5. The position and the shape of the band for 0 - H stretching is generally used in studying the hydrogen bonding in carbohydrate solutions. Likewise, orientation studies have been carried out in this region of frequencies. Hydrogen bonding and orientation, as well as mutarotation, investigations are reported in Section II,3.

b. 1500-1200 cm-'.-This region is one of the richest in structural information as it deals with symmetrical deformation of CH2 and the numerous C-OH deformations encountered in carbohydrates. However, it was only moderately discussed. It was noticed' that this region is crowded (more than 17 bands), and that assignment of the observed bands by classical, group-frequencies correlations is difficult. G ~ u l d e recorded n~~ the infrared spectra of aqueous solutions of sugars in the 1500-1000-cm-' range. Frequencies related to CHI (scissoring, wagging, and twisting) and COH vibrations have been studied by using the deuteration technique.74s75 It is known that primary hydroxyl groups are the most reactive hydroxyl groups in the monosaccharides. That is probably why it has been found76 that this region is strongly affected by the interaction of D-mannitol, Dglucose, and D-ribose with boric acid when the pH is raised to 10. These reactions yielded an extensive loss of the intensity of OH and CH deformation bands, which was inter~reted'~ as due to the complexing of OH (in CH20H) by borates, or to self-association. The influence of the C H 2 0 H group on the structurally sensitive regions from 900 to 700 cm-' and on the L. M. J. Verstraeten, Anal. Chem., 36 (1964) 1040-1044. J. D. S. Goulden, Specrrochim. Acra, 9 (1959) 657-671. S. A. Barker, R. H. Moore, M. Stacey, and D. H. Whiffen, Nature, 186 (1960) 307-308. J. L. Koenig, in T. M. Theophanides (Ed.), Infrared and Roman Spectroscopy of Biological Molecules, Reidel, Dordrecht, 1979, pp. 125- 137. (76) H. B. Davis and C. J. B. Mott, Trans. Faraday SOC.,76 (1980) 1991-2002.

(72) (73) (74) (75)

VIBRATIONAL SPECTRA OF CARBOHYDRATES a+

H-6’

40-6

FIG. I.-Possible

19 I

H-6’

Dispositions of the CH,OH Group?’

1500- 1200-cm-’ range of frequencies was a n a l y ~ e d ’for ~ a-D-glucose, a - ~ mannose, and a -D-galactose. The compared previous experimental results78 to their calculations, and deduced the conformationally most likely orientations for CH20H, which are g- and t for a-D-glucose and a-D-mannose, respectively, and g+ and g- for a-D-galactose (see Fig. 1).

C - 0 region may be extended beyond 1200 cm-’. c. 1200-950 cm-’.-The The C-0 stretching bands have been observed between 1272 and 1205 cm-’ in sugar acetals12. The acetyl groups of 56 sugar acetates were shown to absorb at 1250 cm-’ (8 pm) and 1220 cm-’ (8.2 pm). Furanose derivatives of pentoses have been o b ~ e r v e d to ’ ~ give a short band at 1250 cm-’ which may distinguish them from pyranoid derivatives. Strong bands for C-0 stretching were reportedI6 to occur at 1250 to 1170 cm-’ for aliphatic esters, and in two regions (1300 to 1250 and 1150 to 1100 cm-’) for aromatic esters. The 1200-1060-cm-’ region has not been sufficiently discussed.’ What renders the assignment uncertain is the coupling of C - 0 and C-C vibrations, and the weak differences between endo- and exo-cyclic C-0 contributions, which overlap, as well the configurational positions of each of the C-0 groups attached to the ring. d. 950-750 cm-’.-This region of frequencies generally called the “fingerprint” or the “anomeric” region3’ is the most discussed. The investigations of the Birmingham school7-’’ were concluded by a classification of the observed absorption bands into 3 types of bands, with different varieties for type 2 (types 2a, 2b, and 2c). It was possible, by using the characteristic types of bands, to identify a and p anomers in monosaccharides and higher saccharides7The p anomers of D-glucose and derivatives invariably showed a type 1 band at 915*5cm-’, type 2a at 874*6cm-’, and type 3 at (77) V. V. Sivchik and R. G. Zhbankov, Zh. Prikl. Spektrosk, 32 (1980) 1056-1059. (78) B. Schneider and J. Vodnansky, Collecr. Czech. Chem. Commun., 28 (1963) 2081-2083.

20

MOHAMED MATHLOUTHI AND JACK L. KOENIG

767 f 8 cm-', whereas a-D-glucopyranose derivatives gave an absorption at 921 f 4 cm-' (type l), 890f5 cm-' (type 2b), and 774f9 cm-' (type 3). By comparing a large number of carbohydrates (37 compounds) having in common a glucopyranoside tetraacetate group, it was possible79to assign characteristic bands of a and p anomers. During the deformation mode of the anomeric C-H, an axial hydrogen on C-1 (LI-D-G~C)comes closer to that on C-5, leading to an increase of Van der Waals forces, and hence, to an increase of freq~ency.~'"In the identification of glucans, it was possible to distinguish the a-(1 + 4) linkage (930zt4 cm-', type 1; and 758 f 2 cm-', type 3) from the (1 + 6) linkage (9171t2 cm-', 768f 1 cm-I). It was suggested* that the type 2c absorption at 871 f 7 cm-' might be useful in distinguishing between D-galactopyranose, and, at 876 f 9 cm-' Dmannopyranose derivatives, from D-glucopyranose and glucans, which do not display this band in their spectra. The type 2c peak was assigned to an equatorial C-H deformation? The ring methylene groups in 2- and 3-deoxy derivatives of gluco-, manno-, and galacto-pyranoses give rise to a CHI rocking mode at 867 f 2 cm-', whereas this vibration occurs at 853 f 6 cm-' for quercitols.' The same region (960-730 cm-') was used in ring-isomerism ~ t u d i e s . In ' ~ ~this ~ ~case, four bands were noted: type A at 924cm-', B at 879 cm-', C at 858 cm-', and D at 799 cm-', but, although types A and D were shown" by aldopyranoses, and B and C by furanoid compounds, this result cannot be extended to products other than those which were studied." This is probably due to the fact that these bands may be confused with types 1,2, and 3 bands. Assignments were proposed,72with some certainty, to the furanose ring at 850 f 6 cm-', and to the 2-hexuloses at 817 f 7 cm-' and 8745 9 cm-'. The conformational stability of the pyranoid ring having at least one axial hydroxyl group was correlated71 with the absorbance at 781 * 5 cm-'. However, the interference of C - 0 and C-C stretching, and the overlapping and combinations of different modes, make it somewhat hazardous to assign configurations in this region, other than the anomeric, which has been observed for many samples by different authors. The similarity between carbohydrates and polyols was observed in the anomeric region.79-79b The spectral ranges 855-820, 885-860, and 920-885 cm-' were found to be characteristic of ea, ae, and aa structural elements (where a (79) S. H. Doss and W. M. Miiller, Aust. J. Chem, 24 (1971) 2711-2715. (79a) A discussion of the work of the Birmingham school has been and it was especially noted that the type 2a band at 842 cm-', normally used in diagnosing of anomers, is absent from the infrared spectra of a-D-xylopyranose (and its derivatives) and a-L-arabinopyranose. Such an absence. or weakness, of an absorption may be resolved e~perimentally'~~ by the use of time-dependent, Fourier-transform spectra, or justified by normal-coordinate analysis. (79b) D. M. Back and P. L. Polavarapu, Carbohydr. Res., 121 (1983) 308-311.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

21

TABLEI1

observed Frequencies in the 950-700-cm-' Range and the Orientationsso of H-1 Orientations sugars

cr-D-Glucose p anomer a-D-Mannose /3 anomer a-D-GalaCtOSe p anomer a

H-1

H-2

H-3

e

a a

a

e a

e e

e a

a

a a a a

a

a

H-4 H-5 (I

a

a a

a

a e

a a

e

a

a

v (cm-'1" 914 s, 837 s, 774 s 909 m, 896 vs, 856 w 907 s, 872 s, 824 s, 798 s 896 m, 861 m, 854 m, 770 s 888 vs, 833 vs, 792 vs, 764 vs 897 s, 881 s, 776 s

Key: m, medium; s, strong; vs, very strong; w, weak.

and e respectively represent axial and equatorial C-H groups). The correlation between CH orientation and the frequencies observed is shown in Table I1 for the anomers of D-glucose, D-mannose, and D-galactose. e. Below 700 cm-'.-Most of the classical i.r. investigations were restricted to the region below 15 p,m (above 667 cm-'). The spectra of 28 cyclic acetals of sugars were recorded13between 15 and 40 p,m (667 to 250 cm-I). It was observed13 that the crystalline materials show more absorption bands than the spectra of the same compounds in solution. Sub~equently?~ it was noted that the 700-500 cm-' region permits differentiation of crystalline monoand oligo-saccharides from amorphous, solid samples. It was also observed" that the aspect of the spectrum changes when the potassium halide pellet is hydrated. The sensitivity of the spectrum to the presence of moisture is due to the i.r. absorption of water, or to the libration movement of water revealed by the Raman effect.82 The nonplanar bending absorptions of On studying hydroxyl groups in the 700-500-cm-' range was pointed i.r. spectra from 725 to 680cm-' of a large number of cyclic acetals of hexuloses, Patil and B o ~ esuggested ~ ~ that the absorption observed at 683-680cm-' could be due to the ring-breathing mode. The range of frequencies below 700 cm-' was used in low-temperature investigation^.^' Skeletal and hydrogen-bonding vibrations were below 500 cm-I. The correlation83abetween group frequencies and the observed (80) V. P. Komar, R G. Zhbankov, and A. M. Prima, Zh. Strukt. Khim., 8 (1967) 252-257. (81) S. A. Barker, E. J. Bourne, W. B. Neely, and D. H. Whiffen, Chem. Ind. (London), (1954) 1418. (82) G . E. Walrafen, J. Chem Phys., 47 (1976) 114-126. (83) J. R. Patil and J. L. Bose, Carbohydr. Res., 7 (1968) 405-409. (83a) D. E. Dorman and J. D. Roberts, J. Am. Chem SOC.,92 (1971) 1355.

22

MOHAMED MATHLOUTHI AND JACK L. KOENIG

frequencies is difficult to establish in this region, because of the interactions of vibrations and the high sensitivity of skeletal bending and twisting vibrations to small changes in the structure of the molecule. The application of classical Raman spectroscopy, using the mercury radiation at A 253.6 nm as the excitation source, permitted recording2' of more than 20 peaks for sucrose below 500 cm-'. The observed frequencies below 100 cm-' were interpreted as due to inter-ring oscillations, which was also the conclusion reached from a far-infrared studya4 of glucose and sucrose. 3. Spectral Results by Non-computer Methods

Analysis of the classical i.r. and Raman results permitted classification of the observed bands into characteristic ranges of frequencies. One merit of the pioneering infrared investigations (Birmingham school and N.B.S.) was that they dealt with a very large number of samples. The adoption in our classification of the energy arguments is an attempt to reconcile the early empirical assignments with subsequent potential-energy contributions to the vibrational frequencies. Before approaching the computer calculations of frequencies, the qualitative and quantitative use of the classical results will be reviewed. The noncomputer, vibrational spectra have found important application in the analysis and identification of sugars in the food industries. On a more fundamental level, an understanding of the structure of carbohydrates helps in deciphering their mechanisms of reaction. Accordingly, numerous investigations were devoted to the study of configurationand conformation. Studies at sub-ambient temperatures, which have been found to yield spectra of better quality, were often applied. Hydrogen bonding was actively investigated both in solid samples and aqueous solutions. a. Analysis and Identification.-1.r. and Raman spectroscopy have been of major interest" in the analysis and identification of food carbohydrates. G ~ u l d e nwas ~ ~one of the first to apply i.r. spectroscopy to a semi-quantitative analysis of glucose-galactose mixtures in water. The rapid quantitative determination of lactose concentrations6 in milk was achieved with an accuracy of *1.5%. It was suggested73that measurement of the i.r. transmission at 1050 cm-' provides a possible method for the continuous monitoring of lactose concentration during the evaporation of whey. The solid(84) M. Hineno and H. Yoshinaga, Nippon Kagaku Zasshi, 43 (1970) 3308-3309. (85) A. Eskamani, in E. G. Brame and J. G. Grasselli (Eds.), Infrared and Raman Specfroscopy, Part B, Dekker, New York, 1977, pp. 629-634. ( 8 6 ) J. D. S. Goulden, 1. Dairy Res., 26 (1959) 151-159.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

23

state spectra give rise to difficulties of interpretation, owing to the amorphous or crystalline nature of the sample.87 Such difficulties may be eliminated by applying the far-infrared absorption procedure, using the Nujol oil-mull technique, and barium carbonate as the internal standard, as for the evaluation of the crystallinity of a-lactose.88 Application of this method to solid wheys, or dehydrated dairy products, in the region from 660 to 570cm-I (-15 to 17.5 pm) permitted the determination of lactose crystallinity with a standard error of 1.51% and a maximum error of -2%. The i.r. spectrophotometric method was adopted as the official method for lactose measurement in milk after a collaborative study” of 5 laboratories in comparing the i.r. method to the standard method and finding good agreement between them. 1.r. spectroscopy has also been used in the analysis of wines in the range of wavelengths of 0.8 to 2.4 pm. An investigationg0 of 26 samples of two wines led to the conclusion that the infrared technique is very suitable for rapid instrumental determination of the composition of wine in regard to alcohol, sugar, and acid content. It was possibleg1to predict concentrations of food carbohydrates in dry mixtures by near-infrared, reflectance spectroscopy. Conventional i.r. and laser-Raman spectra of malto-oligosaccharides were recorded?* Distinct absorption bands were observed in the glucose and maltose spectra in the regions of 1320-1220 and 960-730 cm-’, but no important differences were shown by the higher polymers.92 The pectic substances in food and pharmaceuticals have been analyzed” by i.r. spectroscopy. In particular, the degree of esterification, which is an indicator of gel formation, was for pectins of different origins by monitoring the intensity ratio of the bands v ( C 0 ; ) at 1608 cm-’ and v(C=O, ester) at 1745 cm-’. The polysaccharides used as thickeners in the food industries have been characterizedg4by their i.r. spectra. It was shown, by recording 38 spectra of such thickeners as derivatives of starch and cellulose, gums, and alginates, that it is quite easy to differentiate between these polysaccharides, and to determine the influence of their degree of substitution from their i.r. spectra.94 The sugar industry is another field of application of infrared spectroscopy. The constituents of sugar colorants, namely, caramels

(87) (88) (89) (90) (91) (92) (93) (94)

J. D. S. Goulden and J. W. White, Nature, 181 (1958) 266-267. H. Susi and J. S. Ard., J. Assoc. 08 Agric. Chem., 56 (1973) 177-180. D. Briggs, cited in Ref. 85. K. J. Kaffka and K. H. Noms, Acta Aliment. Acad. Sci. Hung., 5 (1976) 267-279. R. Giangiacomo, J. B. Mage,G. S. Birth,andG. G . Dall, L FoodSci.,46(1981) 531-534. R. Srisuthep, R. Brockman, and J. A. Hohnson, Cereal Chem., 53 (1976) 110-117. M. P. Filippov, G . A. Shkolenko, and R. Kohn, Chem. Zuesfi, 32 (1978) 218-222. R. Friese, Fresenius Z. Anal. Chem., 305 (1981) 337-346.

24

MOHAMED MATHLOUTHI AND JACK L. KOENIG

and melanoidins, were ~ t u d i e din~ the ~ . ~region ~ of 2.5 to 15 pm (4000 to 667 cm-'). Melanoidins showed characteristic peaks in the region of 35003400cm-' due to N-H stretching modes, and a weak band in the 800665 cm-' range due to the out-of-plane N-H wagging vibration of primary and secondary amides, and the caramel region seemed to lie at 16501600cm-', where bands for C = C , C=O, and a,P-diketones could be found.95 The colorants produced by alkaline degradation of carbohydrates during sugar-cane processing were identified96 from their i.r. spectra. For humic acids, model compounds consisting of phenol polymers, aminodeoxyglucoses, and chitosan-phenol polymers have been compared to natural soil-components. The i.r. spectra recorded97 in the 3600- lOOO-cm-' region indicated the characteristics of the model polymers as compared to fungal and soil humic acids. These spectra illustrate the importance and advantages of i.r. spectroscopy when model compounds are compared with natural soil, peat, and microbial, humic polymer^?^ Another i.r.-spectroscopic study of soil organic matter dealt with a fulvic acid fraction from an acidic soil called podzol?' The spectra recorded in the range of 2-16 pm permitted characterization, in the different fractions corresponding to different depths in the soil, of polysaccharides of various types, mainly pectic and uronic acids, as well as lignin residues. The extraction (and modification) of the beechwood glucuronoxylans in the prehydrolysis kraft process was monitoredg9by i.r. spectroscopy. It was found that two bands, at 1740 and 1245 cm-', are characteristic of the beechwood glucuronoxylans, and this result was confirmed by the diff erence-spectrum te~hnique.'~ These are some examples of the use of i.r. spectra in the analysis and identification of carbohydrates in foods and natural products. Very often, these spectroscopic techniques are complementary to others, such as the study of aldobiouronic acids obtained by hydrolysis of peach-gum polysaccharides by their optical rotations and their i.r. spectra.'" However, the i.r. results appear to be sufficiently reliable to be used in the detection of traces of fructose and glucose, and to determine the d.e. (dextrose equivalent) of corn syrups, as well as the quantitative carbohydrate content in different products.'" (95) S. K. D. Agarwal, P. C. Johary, and D. S. Misra, Z. Ver. Dtsch. Zucker Ind., 24 (1974) 532-535. (96) L. P. Kotelnikova and L. D. Bobrovnik, Cent. Azucor, 5 (1978) 1-6. (97) E. Bondietti, J. P. Martin, and K. Haider, Soil Sci. SOC.Am. Roc., 36 (1972) 597-602. (98) H. A. Anderson, A. R. Fraser, A. Hepburn, and J. D. Russell, J. Soil Sci., 28 (1977) 623-633. (99) S. Smiljanski and S. Stankovic, Cellul. Chem. Techno/.,8 (1974) 283-284. (100) J. Rosik, A. Kardolovi, and J. Kubala, Chem. Zvesti, 27 (1973) 551-553. (101) R. T. Sleeter, U.S.Pat. 4,102,646 (1977); Chem. Absrr., 89 (1978) 225, 7176.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

25

b. Mutarotation.-The mutarotation of several sugars was measured quantitatively."* The change in i.r. absorption at 1143 cm-' for glucose and 1163 cm-' for mannose permittedlo2 determination of mutarotation constants in 20% aqueous solutions. Another approach consisted in freezedrying the aqueous solution after mutarotation was complete. The i.r. spectra were then recorded," and compared to those of the crystalline anomers; this led to the identification of the anomers present by comparison with the solid samples. The 1012-1054- and 1054-1076-cm-' ranges enabled the mutarotation of a 20% glucose solution to be monitored, with time, by other investigator^,'^^ who found that glucose was characterized by a strong carbonyl-water interaction. The ratio of a to p anomer may be reliably Likewise, the investigation of analyzed by using i.r. spectros~opy.~~'"*~'~~~~ 37 glucopyranoside derivatives permitted79 the finding that the absorption bands characteristic of anomers remain relatively constant, regardless of the rest of the molecule. c. Conformation and Tautomem.-Although 'H- and I3C-n.m.r. spectroscopy are far more suitable for such applications, the vibrational spectra of carbohydrates may be used to give conformational and tautomeric informahave been tion. The ring isomers of 5-acetamido-5-deoxy-~-arabinose differentiated from their i.r. ~pectra."~The characterization of furanoses by the appearance of absorbance at 850 cm-', and the correlation between the stability of the pyranose ring and the absorbance at 781 cm-' were established7' for common monosaccharides. The study of oligosaccharides in the region 1000-40 cm-' permitted" elucidation of the configuration of the glycosidic linkage, and differentiation of the vibrations assigned to the pyranose or furanose rings in sucrose and raffinose. Five- and six-membered rings of cyclic acetals of hexuloses have also been differentiated from their i.r. ~pectra.'~ In a conformational study of cellulose oligosaccharides and ~ e l l u l o s e , it ' ~was ~ concluded that the significant changes that occur in the intensity and frequency of the bands near 3400 cm-' when the temperature is varied may be due to changes in intra- and inter-molecular hydrogenbonds. The effect of increasing the temperature on the ratio of intensities at 2900-1372cm-' was interpreted as a change in conformation due to greater freedom of movement of OH groups when hydrogen bonds are broken. The band at 893 cm-' was assigned to changes in conformation due to rotation about the interglycosidic bond, and that at 1429 cm-' was

(102) (103) (104) (105)

F. S. Parker, Biochim. Biophys. Acta, 42 (1960) 513-519. V. A. Afanasev and 1. F. Strel'tsova, Zh. Fiz. Khim., 39 (1965) 110-1 15. J. K. N. Jones and J. C. Turner, J. Chem. SOC.,(1962) 4699-4703. H. Hatakeyama, C. Nakasaki, and T. Yurgi, Carbohydr. Res., 48 (1976) 149-158.

26

MOHAMED MATHLOUTHI AND JACK L. KOENIG

associated with the environment of the C-6 group, for example, the formation (or breaking) of an intermolecular hydrogen bond involving 0-6. In a series of papers dealing with chain folding in polymers, Koenig and Vasko 106-ins employed spectroscopic techniques in order to elucidate the fold conformation of arnylose and amylopectin. They found'06 that i.r. spectroscopy is more sensitive to localized arrangements or conformations of a polymer chain, such as a folded region, than X-ray or electron diff raction. The 1295-cm-' band was assigned to a unique conformation within the folded amylose molecule of the V-complex crystals, and it was suggested that this conformation is a regular, tight-loop fold. The spectroscopic method permitted the conclusion that irregular, as well as regular, folds can be transformed into regular folds during annealing. Thermal treatment of V-amylose-Me,SO films was found'" to produce a high degree of regular folding, and the swelling of annealed films causes a loosening of the folded to occur conformation. The 790- and 1256-cm-' bands have been in spectra of amorphous and V-complex amylose. These bands are assigned to conformations within a noncrystalline, metastable state which, with time, are incorporated into crystalline regions of the polymer. Amylopectin complexes have also been found'"' to form folded structures. Regular folding was measured by the 1295-cm-' band in amylopectin-nonsolvent complexes. It was found that folding occurs in complexes of amylose-amylopectin mixtures."* The structure of the cellulose from the cell wall of Valonia uentricosa was studied'"' by use of infrared and Raman spectra. It was found that only one rotational orientation is present for the -CH,OH side-chains, which considerably diminishes the number of structural possibilities. d. Orientation.-Infrared dichroism in polysaccharides was applied in order to obtain information on the orientation of chemical groups in the crystalline structure. 1 1 " - 1 1 3 The absorption of infrared radiation is given by the absorbance A according to the formula

where I0 and I are the incident and transmitted intensities of the absorbing frequency, M is the transition-moment vector of the normal mode, and E (106) (107) (108) (109) (110) (111) (112) (113)

J. L. Koenig and P. D. Vasko, J. Macromol. Sci. Phys., 4(2) (1970) 347-367. J . L. Koenig and P. D. Vasko, J. Macromol. Sci. Phys., 4(2) (1970) 369-380. P. D. Vasko and J . L. Koenig, J. Macromol. Sci. Phys., 6(1) (1972) 117-127. J. Blackwell, P. D. Vasko, and J. L. Koenig, J. Appl. Phys., 41 (1970) 4375-4379. C. Y. Liang and R. H. Marchessault, J. Polym. Sci., 37 (1959) 385-395. C. Y. Liang and R. H. Marchessault, J. Pofym. Sci., 39 (1959) 269-278. R. H. Marchessault and C. Y. Liang, 1. Pofym. Sci., 43 (1960) 71-84. C. Y. Liang and R. H. Marchessault, J. Polym. Sci., 43 (1960) 85-100.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

27

is the electric-field vector of the incident beam at the absorbing frequency. When measurements are made with the electric vector parallel to or perpendicular to the direction in which a polymer chain is oriented, the dichroic ratio R can be measured. R

=AII/Al,

where All is the absorbance for linearly polarized light parallel to the chain direction, and A l is the corresponding measurement perpendicular to the chain axis. The orientational measurements in polymers made by using vibrational spectroscopy have been r e ~ i e w e d . "The ~ effect of orientation was observed in the tilting spectra of some chitin sample^."^ The orientation and the tilting effects helped in band assignments and interpretation of bands at 3106, 2962, and 1619 cm-'. Infrared dichroism was also used in the study of crystalline monosaccharides related to xylans.'16 In this investigation, the directions of the transition moments of absorption bands were determined from crystals of known structures, and compared to those observed. Oligosaccharides of xylans and cellulose, which very often crystallize with monoclinic or triclinic symmetry, do not have their dielectric ellipsoid coinciding with the crystallographic axis, which renders such an orientational study difficult to achieve. However, successive drawing out (extension) increases the molecular orientation of the polymer, and the orientation functions obtained from infrared and X-ray diffraction^"^ for regenerated cellulosic fibers with a draw ratio of 2.3: 1 were in good agreement. e. Low Temperature.-The quality of the infrared spectra of carbohydrates is generally improved by using cooled samples. Better resolution of the 0 - H absorption bands of some mono- and oligo-saccharides was obtained,"' but, even at the temperature of liquid nitrogen, there is considerable overlap of the bands. Deuteration as well as polarization techniques have been used in conjunction with low temperature in order to elucidate the structure in the 0 - H absorption region, but the information was rather poor. Most of the 0 - H vibrations have been found to be coupled. It was ~ u g g e s t e d "that ~ the band width of 0 - H stretching absorptions might be due to strong, anharmonic coupling between v(0-H) and v ( 0 - H * 0),

-

(114) B. Jasse and J . L. Koenig, J. Macromol. Sci.,Rev. Macromol. Chem., 17 (1979) 61-135. (115) R. H. Marchessault, F. G. Pearson, and C. Y. Liang, Biochim. Biophys. Acra, 45 (1960) 499- 507. (116) A. J. Michell, Aust. J. Chem., 21 (1968) 2451-2466. ( 1 17) H. Siesler, H. Krassig, F. Grass, K. Kratzl, and J. Derkosch, Angew. Makromol. Chem., 42 (1975) 159. (118) A. J. Michell, Ausr. J. Chem., 21 (1968) 1257-1266. (119) N. Sheppard, in D. Hadzi (Ed.), Hydrogen Bonding, Pergamon, London, 1959, p. 85.

28

MOHAMED MATHLOUTHI AND JACK L. KOENIG

together with Fermi resonance between u(0-H) and neighboring overtone and summation frequencies involving low-frequency fundamentals. It was not possible to demonstrate that bands arising from OH groups involved in inter- and intra-molecular hydrogen-bonds show differing sensitivities to temperature because of the lack of intramolecular bonds in the samples studied.'I8 The effect of lowering the temperature of samples was shown to result in increase of intensity, narrowing of band widths, and shifts to higher or lower frequencies for some cellulose oligosaccharides and for cellulose 11. The regions where the most noticeable changes occurred were 35003100 cm-', 1500-1350 cm-I, and 850-350 cm-'. It was concludedI2' from these changes that the increase in definition in carbohydrate spectra found on cooling occurs only for highly ordered compounds having hydroxyl groups involved in strong, intermolecular hydrogen-bonds. The technique of recording i.r. spectra of cooled samples was describedI2' as a useful one for identification, characterization, and differentiation of complex compounds of biological interest. The changes in the spectra of carbohydrates on lowering the temperature were ascribed to internal rotations that change the positions of hydrogen atoms only. The X-ray diffraction pattern, where hydrogen atoms are not well localized, does not reflect any change, and the i.r. frequency-shifts could be the sign of a temperaturedependent order-disorder transition associated with flickering of the hydrogen bonding. In a series of papers on infrared spectra of sugars at the temperature of liquid helium, Hineno and Y o ~ h i n a g a ' described ~ ~ ~ ' ~ ~increase in intensity of the absorption bands. The lowering of temperature was observedI2' to be necessary to identify, clearly, bands below 200 cm-'. The comparison of di- with mono-saccharides permitted'24 assignment of a band at 40.7 cm- ' to inter-ring interactions of cellobiose, and that at 41.0cm-' to the: same mode in lactose; the band at 47.2 cm-' of lactose and that at 45.4 cm-' of sucrose were found similar to the band at 48.5 cm-' of 0-D-glucose. The far-infrared spectra of mono-, di-, and tri-saccharides were recorded 123,125 at liquid helium temperature with removal of thermal noise. Comparison of the spectra permitted assignments of inter-ring, interaction modes. f. Hydrogen Bonding.-The width of the v(0-H) band was interpreted'26 in terms of hydrogen bonding and conformational stability for glucose, and (120) (121) (122) (123) (124) (125) (126)

A. J. Michell, Ausr. J. Chern., 23 (1970) 833-838. J. E. Katon, J. T. Miller, Jr., and F. F. Bentley, Carbohydr. Rex, 10 (1969) 505-516. M. Hineno and H. Yoshinaga, Spectrochirn. Acta, Part A, 28 (1972) 2263-2268. M. Hineno and H. Yoshinaga, Spectrochirn. Acra, Part A , 29 (1973) 301-305. M. Hineno and H. Yoshinaga, Spectrochim. Acta, Parf A , 29 (1973) 1575-1578. M. Hineno and H. Yoshinaga, Spectrochirn. Acra, Part A, 30 (1974) 441-416. 9. Casu, M. Reggiani, G. G. Gallo, and A. Vigevani, Tetrahedron, 22 (1966) 3061-3083.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

29

di-, oligo- and poly-glucoses in Me2S0, and correlated with the chemical shift of 0-2-H-0-3'-H. The study7' of hydrogen bonds in monosaccharides was carried out in the regions of 3400-3100 and 180-120cm-' at low temperature, using the deuteration technique. By comparison of the i.r. results with X-ray and neutron diffraction data, it was found that no intramolecular hydrogen-bonds exist in the crystalline monosaccharide derivatives studied. The use of solvents of diff erent proton-acceptor strength permitted'27 the gaining of some insight into the hydrogen bonding of carbohydrates in the near-infrared (n.i.r.) region. The results showed that a-D-glucose, p- D-glucose, and glycogen can be differentiated by their n.i.r. absorption maxima in Me2S0, N,N-dimethylformamide (DMF), and 19 : 1 DMF-water. The OH absorptions at 6964 and 6944 cm-' in the spectra of anhydrous and aqueous DMF were taken as the nonsolvent, hydrogenbonded species, and those at 6325 and 6231 cm-' as the solvent, hydrogenbonded species in the solutions. The variation of the temperature enabled calculations of thermodynamic parameters of nonsolvent and solvent hydrog e n - b o n d ~ .The ' ~ ~ intramolecular hydrogen-bonding was investigatedt2*by using infrared spectra of model sugars (8 monosaccharides, p-maltose, and p-cellobiose) dissolved in Me2SO-CC1,. A 5-membered saccharide chelation, classified according to the cis or trans configuration of the carbohydrates studied, was found. A systematic of the infrared spectra in the region of 3700-3300 cm-' of 30 diastereoisomers having configurations corresponding to that of cellulose or amylose permitted formulation of a hypothesis concerning the intramolecular H-bonding in cellulose, and confirmed the OH-2 * 0 - 1 chelation in a-(1+ 4)-glucans. The Raman and infrared spectra of methyl 3,6-dideoxy-p-~-ribohexopyranoside were r e ~ o r d e d ' ~at ' room temperature and lower temperatures. Correlation between the 0 . 0 distances and four bands identified at 3530, 3470, 3442, and 3216 cm-' was made. 1.r. spectroscopy has been appliedi3' to the study of inter- and intra-molecular hydrogenbonding in hexopyranoses and their derivatives. The calculated OH * 0 distances were correlated with frequency shifts measured in the v ( 0 H ) region. It appears from these different studies of hydrogen bonding that the limiting factor is to find for carbohydrates a solvent that does not interfere

-

- -

(127) G. F. Trott, E. E. Woodside, K. G. Taylor, and J . C. Deck, Carbohydr. Res., 27 (1973) 415-435. (128) M. Fialeyre, F. Lafuma, and C. Quivoron, J. Chim. Phys., 74 (1977) 701-706. (129) F. Lafuma and C. Quivoron, Can. J. Chem., 56 (1978) 2076-2085. (130) J. Umemura, G. I. Birnbaum, D. R. Bundle, W. F. Murphy, H. J. Bernstein, and H. H. Mantsch, Can. J. Chem., 57 (1979) 2640-2645. (131) H. Honig and H. Weidmann, Carbohydr. Res., 73 (1979) 260-266.

30

MOHAMED MATHLOUTHI A N D JACK L. KOENIG

in this molecular association. Moreover, intramolecular bonding contributes to the widening of the v ( 0 H ) band, so that the use of curve-fitting programs in such calculations becomes a necessity if elucidation of the contribution of each OH group to the broad u(OH) band is needed. g. Other Studies.-A tentative attempt to assemble and systematize the information acquired on the infrared spectroscopy of carbohydrates has been made.'32 The observed absorption bands of 38 phenoxyethyl and arylaminoethyl P-D-glucopyranosides were a ~ s e r n b l e d , 'but ~ ~ no assignments were proposed. It was that nonsulfated carbohydrates do not absorb appreciably in the region of vas(S=O). That is why investigations on the sulfate groups of heparin were localized in the 1400-950-cm-' range, which contains the strong absorption at 1230 cm-' associated with the antisymmetric stretching of S=O. Infrared spectroscopy was in the 1740-1640-cm-' range in order to differentiate between N-, 0-,and S-acetyl groups. It was found'33 that i.r. spectra are more indicative of the type and content of sulfate groups, and the Raman spectra more characteristic of the specific backbone structure of glycosaminoglycans. The 950-800cm-' range, where the vas C-0-S vibration is localized, is less easy to interpret, because of interference by the "fingerprint" vibrations of the saccharides and by that of the solvent. However, an i.r. in this region, of the sulfonic esters of some aldoses having the D-gluco, D-manno, D-galacto, and D-XY~O configurations showed that the observed variability of the absorption frequency for the sulfonic esters in the 900-800-cm-' region is to be assigned to factors other than configurational differences. Infrared and Raman spectroscopy are in current use fdr elucidating the molecular structures of nucleic acids. The application of infrared spectroscopy to studies of the structure of nucleic acids has been re~iewed,'~' as well as of Raman s p e c t r o s ~ o p y . 'It~ ~was noted that the assignments are generally based on isotopic substitution, or on comparison of the spectrum of simple molecules that are considered to form a part of the polynucleotide chain to that of the nucleic acid. The vibrational spectra are generally believed to be a good complementary technique in the study of chemical reactions, as in the of carbohydrate complexation with boric acid. In this study, the i.r. data demonstrated that only ribose forms a solid complex with undissociated H3B03,and that the complexes are polymeric. A Mesquida, Reu. Acud. Cienc. Exucrus, Fix-Quim. Nur. Zuragozu, 27 (1972) 121-127. F. Cabassi, B. Caw, and A. S. Perlin, Carbohydr. Rex, 63 (1978) 1-11. D. Horton and M. L. Wolfrom, J. Org. Chem., 27 (1962) 1794-1800. R. C. Chalk, M. E. Evans, F. W. Parrish, and J. A. Sousa, Curbohydr. Res., 61 (1978) 549-552. (135) M. Tsuboi, Appl. Spectrosc. Rev., 3 (1969) 45-90. (136) H. Fabian, A. Lau, S. Bohm, and R. Wetzel, Stud. Biophys., 80 (1980) 1-38.

(132) (133) (133a) (134)

VIBRATIONAL SPECTRA OF CARBOHYDRATES

31

The four 5,6-dideoxy-6-halo- 1,2-0-isopropylidene-3-0-methyl-a-D - x ~ ~ o hept-5-eno-l,4-furanurononitriles(bromo, chloro, fluoro, and iodo) were configurationally identifiedI3' from their i.r. spectra, among other spectral techniques. The binding of dextran B-1355 and of the monosaccharides methyl a-D-mannopyranoside and D-galactose to concanavalin A was i n v e ~ t i g a t e d 'by ~ ~ means of infrared, attenuated total-reflectance (a.t.r.) spectroscopy. The OH stretching mode of the polysaccharide was used as a measure of its binding. The i.r.-spectral data were shown to be sensitive to structure modification when the pH was varied from 6.1 to 9.0, or when urea or metal ions were added. Such chemical reactions as that of carbohydrate a-enones with iron carbonyls has been studied'39 by i.r. spectroscopy, and it was found from the i.r. spectra that each enone gives two diastereoisomers having the two possible orientations of complexation. Infrared spectroscopy has even been used in the of interstellar solid material; the bands observed in the 2-4-pm (5000-2500-cm-'), 8-13-pm (1250-770-cm-'), and 15-30-pm (667-333-cm-') ranges were compared to the known bands of cellulose. From this comparison, it appeared reasonable to infer the detection of polysaccharides in interstellar space. It was hypothesized140that such polymeric carbohydrates are formed by a biogenic processing of interstellar formaldehyde, and could be taken as an indicator of the evolution of prebiotic molecules. Thus, it is seen that noncomputer, spectral results have been used in numerous investigations on vibrational spectra-structure relationships. When such complex molecules as carbohydrates, which are sensitive to the environment and reveal configurational and conformational changes, as well as intra- and inter-molecular hydrogen-bonding, are dealt with, the noncomputer techniques, even though more qualitative and less rigorous than the calculation methods, remain quite useful in practice. 111. COMPUTATIONAL CALCULATION OF VIBRATIONAL

FREQUENCIES, A N D BANDASSIGNMENTS 1. Description of Methods

a. Calculation of Frequencies.-Calculation of the frequencies of vibration of carbohydrates constitutes a useful tool for the interpretation of their i.r. and Raman spectra. Although extensive material has been accumulated on the infrared spectra of mono-, oligo-, and poly-saccharides and their deriva(137) (138) (139) (140)

J. M. J. Tronchet and 0. R. Martin, Carbohydr. Res., 85 (1980) 187-200. M. Ockman, Biochim. Biophys. Acra, 643 (1981) 220-232. M . B. Yunker and B. Fraser-Reid, J. Org. Chem., 44 (1979) 2742-274s. F. Hoyle and N. V. Wickramazinghe, Nafure, 268 (1977) 610-612.

32

MOHAMED MATHLOUTHI AND JACK L. KOENIG

tives, and the laser-Raman results are becoming more and more available, comparatively few calculations of the vibrational spectra have been made, probably owing to their complexity. Their lack of symmetry elements, their great sensitivity to the environment (change in configuration and conformation), and the discrepancy between the potential energy of the groups of atoms in carbohydrates and in their closest models treated in the literature make it difficult to achieve good agreement between calculated and observed frequencies. Nevertheless, the improvements in the use of computers for solving the vibrational calculations are tending to lessen the gap. The data obtained from normal coordinate analysis of mono-, di-, and polysaccharides are of unquestionable interest in structure analysis of these products. The established method for calculating the vibrational frequencies of molecules is the Wilson G F method.27 In this method, the potential energy of a molecule is defined in terms of the force constants by a matrix F, and the kinetic energy, which depends on the geometry of the molecule, is defined by a matrix G. Using the methods of classical mechanics, the following equation may be derived. [GF- A E]L= 0,

(3)

where the eigenvalues A and the eigenvectors L are matrices of the vibrational frequencies and displacements, respectively, and E is the unit matrix. It is beyond the scope of this article to discuss the details of the solution ofthe secular equation (3); this may be found in a published text.23However, the steps of approach to the vibrational problem may be described, and some examples of simplification of the calculations given. The first step consists in deriving a set of internal coordinates ( r , 6 ) from the massadjusted, Cartesian coordinates, which are given by the crystallographic data. The advantages of the internal coordinates over the Cartesian coordinates were noted.23 They consist in a diminution of the size of the secular equation (3 N-6 coordinates instead of 3 N). The representation of the potential energy or force-constants matrix in terms of bond stiffness and resistance to bond-angle deformations makes these constants physically comprehensible. The transferability from one molecule to another of force constants associated with internal coordinates is made easier, but some difficulties arise in the expression of kinetic energy in internal coordinates, which are solved by the use of a computer program for transformation of the kinetic energy from Cartesian coordinates to internal coordinates. The second step consists in constructing the matrices G and F. Although G and F are symmetric, the G F product found in the secular equation is unsymmetric, requiring that G and F be separately diagonalized. A procedure that yields a symmetric, secular equation was proposed by Hannon

VIBRATIONAL SPECTRA OF CARBOHYDRATES

33

and coworker^.'^' This procedure consists in transforming the potentialenergy matrix, rather than the kinetic-energy matrix (as is usually done). The following relationship was utilized. r = Bx,

(4)

where B is the transformation matrix between internal coordinates and the mass-adjusted, Cartesian coordinates x. The potential energy in internal coordinates is 2 V = r‘Fr.

(5)

After transformation by use of Eq. 4, 2 V = x‘B’FBx,

(6)

and the inverse of the kinetic-energy matrix is unity, and so the secular determinant for mass-adjusted, Cartesian coordinates is [B’FB - A E] = 0.

(7)

In this form, the secular determinant is symmetric, making diagonalization easier, and saving considerable computer time; but, more importantly, it allows the solution of larger matrices on computers having limited memory storage. This simplification method was applied in the n.c.a. of cellulose29 and V-amyl~se.~’ In the case of these polymers, the symmetry coordinates are also expressed in Cartesian coordinates, and they are therefore called “external symmetry coordinates.” This is achieved owing to the transformation Xsym

=

ux,

(8)

which leads to the following, reduced secular equation: [UB‘FBU‘ - A El = 0.

(9)

It has been that, for long-chain polymers in an ordered conformation, the calculation of the normal modes is reduced by symmetry arguments to the determination of the vibrations of the repeat unit. The vibrations in a chemical unit are related to those in adjacent units by the secular equation through a phase angle 0, so that the form of the secular equation used in the previous calculations29~30 was [U( B)B’( B)F(B)B( B)U’(0 ) - A (B)E] = 0.

(1 0)

(141) M. J. Hannon, F. J . Boerio, and J. L. Koenig, J. Chem. Phys., 50 (1969) 2829-2836. (142) G. Zerbi, Appl. Specfrosc. Rev., 2 (1969) 193-261.

34

MOHAMED MATHLOUTHI AND JACK L. KOENIG

The eigenvalues, A( e), are related to the vibrational frequencies v( 0 ) by

A (e) = 4 . r r 2 ~q2, 2~(

(1 1 )

where c is the velocity of light. The most critical step in the normal-coordinate analysis is the transfer of force constants from simple molecules to the complex problem of carbohydrates. The use of data relative to such hydrogen-bonded molecules as carboxylic makes the calculations closer to approximating the stretching and bending of C-0-H in carbohydrates. It is often necessary to make some modifications of the force field, in order to take into account the interactions between different vibrations, or the influence of the trans or gauche forms.'43 The computation of frequencies, potential-energy distribution (p.e.d.), and the Cartesian displacement coordinates may be achieved by using a normal-coordinate analysis program, such as the one written by Boerio and Koenig.'@ The calculated results are generally compared to the observed frequencies, and assignments are proposed for the most prominent bands. This is not intuitively satisfying from the chemical viewpoint, but it allows easy description and visualization of a particular vibrational mode. In addition, the occurrence of group-frequency correlations suggests that force constants in internal coordinates may be transferable. The process of adjusting force constants to the observed frequencies is repeated several times, until only a few bands remain u n a ~ s i g n e d . However, '~~ a problem is posed in the case of carbohydrates by the fact that the number of internal coordinates exceeds the number of degrees of freedom. Indeed, there are 78 (24 stretching, 42 bending, and 12 torsion modes) vibrations of a-Dglucose'45 which is larger than the 66 (3 N -6) degrees of freedom. The excess coordinates are called redundant coordinates. This redundancy can lead to ambiguity in the calculation of the force field; only appropriate combinations can be ~ a l c u l a t e d .It~ ~was possible to take into account redundancies in a-and p - ~ - g l u c o s e ,and ' ~ ~to make assignments of frequencies that did not ignore the low frequencies where inter- and intra-molecular interactions take place. The number of force constants calculated in the general potential function is very large. For molecules having no symmetry, such as carbohydrates, this number is equal to 1 + 2 + 3 + . * . + ( 3 N - 7 ) + ( 3 N - 6 ) = (1/2)(3 N - 6 ) ( 3 N - 9 , so that it can be determined for small molecules only. For such large and complex molecules as sugars and their derivatives, additional information may be obtained from studies employing isotopes and model molecules. (143) J . J . Cael, J. L. Koenig, and J. Blackwell, Carbohydr. Rex, 32 (1974) 79-91. (144) F. J. Boerio and J. L. Koenig, J. Polym. Sci., Part A, 2 (1971) 1517-1523. (145) J. P. Huvenne, G . Vergoten, and G . Fleury, J. Mol. Strucf., 74 (1981) 169-180.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

35

b. Calculation of Intensities.-The experimental data show that tautomeric equilibria are associated with marked changes in intensity of the i.r. absorption bands or Raman scattered lines. Reliable results on the ratio of the a n ~ m e r s ' in ~ ~aqueous ~ ' ~ ~ solutions of D-glucose, or the relative amounts of furanoses and pyranoses in D-fructose solutions, have been based on the ratios of intensities of characteristic vibrations. It was noted'46 that some absorption bands change 10-20 times in i.r.-spectral intensity with transformation of tautomers. This is probably due to the fact that the change in geometry of the molecules yields very strong changes in the dipole moment of some characteristic groups of atoms. Consequently, analysis of the intensities of the vibrational spectra of the tautomers can be more effective than analysis of their f r e q ~ e n c i e s . ' ~However, ~ interpretation of the intensities is considerably more complex than that of the frequencies. Calculations of the intensities is more difficult, and leads to less accurate results, than the calculated frequencies, because of the relatively poor transferability of electro-optical parameters from one molecule to another, and the absence of a developed set of these parameters. It may be recalled is proportional to the square of change that the intensity of i.r. absorption Ik in the dipole moment. where p' = dp/dx, p is the dipole moment, x is the displacement coordinate, and C is a constant. For Raman scattering to occur, the electric field of the light must induce a dipole moment by a change in the polarizability of the molecule. The intensity of the scattered light is given by

where v is the frequency of the emitted radiation; P, the induced dipole moment; and c, the velocity of light. The selection rules only predict which modes are allowed in the i.r. or Raman spectra. The allowed modes can have extremely weak intensities, and not be observed, so that an additional difficulty arises in solving the intensity problem concerning the correlation between calculated and observed intensities. The discussion of intensities necessitates the quantum chemical description of infrared absorption and Raman ~ c a t t e r i n gSuch . ~ ~ a description helps in understanding the electromagnetic processes that occur in molecules, but (146) 0. B. Zubkova, L. A. Gribov, and A. N. Shabadash, Zh. Prikl. Spektrosk, 16 (1972) 306-312. (147) M. Mathlouthi and D. V. Luu, Carbohydr. Rex, 78 (1980) 225-233.

36

MOHAMED MATHLOUTHI AND JACK L. KOENIG

is not of much help in the practical calculation of intensities. These calculations may be based on electro-optical theory.I4' The calculation of the electro-optical parameters describing Raman intensities is not yet very advanced, because of the paucity of data. Nevertheless, some success was achieved in calculations of the intensity of infrared absorption. The results on trans and gauche bond-rotation in ethylene could be taken as a model for carbohydrates. Indeed, similar electro-optical parameters ( p C H , p O H , p C C , and p C 0 ) were calculated. This leads to the expectation that calculations of the intensity of the vibrational spectra of carbohydrates may be accomplished in the near future. In addition, the delicate problem of accounting for molecular interactions in calculating infrared intensities could be approached as it was for u(CCC) and v(C0) vibrations in a ~ e t 0 n e . This I ~ ~ will allow interpretation of weak, as well as strong, i.r. bands, in order to determine the structural properties of molecules. 2. Band Assignments

It is difficult to assign all of the observed i.r. and Raman vibrations of carbohydrates. The i.r. spectrum is particularly irregular, because it contains combination bands that may overlap with those due to fundamental modes, and interact with one another, leading to distortion of the shapes of the observed bands. Raman spectra show fewer irregularities, because combination bands in them are less important. However, even though the spectra of carbohydrates are complex, advantage can be taken of them by use of such techniques as isotopic substitution, or the model-compound approach. a. Isotopic Substitution.-When isotopic exchange is performed on a molecule, it might be assumed that the potential energy and the geometry of the molecule remain unchanged after substitution. However, the G matrix takes on different values as a result of the change in mass of various atoms. The isotopic substitution most frequently encountered in vibrational spectroscopy is hydrogen-deuterium exchange. The experimental techniques for exchange have been d e s ~ r i b e d .Hydrogen ~ ~ . ~ ~ atoms present in biological molecules may be classified as labile and nonlabile, depending on the ease with which they undergo exchange with aqueous solvents. Hydrogen atoms bound to oxygen, nitrogen, and sulfur are labile, and are exchanged much faster than nonlabile hydrogen atoms, those directly bound to carbon atoms. (148) L. A. Gribov, Intensity Theoryfor Infrared Spectra of Polyatomic Molecules, Consultants Bureau, New York, 1964. (149) S . Kh. Akopyan, M. A. Bionchik, V. B. Borisova, S. I. Luk'yanov, and L. A. Solov'eva, Zh. Fiz. Khim., 56 (1982) 1295-1297.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

37

fO-

H FIG. 2.-v(C-H)

X in a Hexose, Equivalent to v(X-H).

If a C-H vibration in a hexopyranose is considered, the system may approximate to an X-H vibration, where X represents the combination of all other atoms (see Fig. 2). The force constant between H and X is fX-H and the expression of the wavenumber in the Hookian approximation is

where p is the reduced mass

which gives

If deuterium exchange is made, the ratio of the wavenumber of the stretching vibration of the X-D group to that of X-H is given by

As may be seen from Eq. 17, the result of isotopic substitution is a shift of the X-H stretching vibration to lower wavenumber by a factor of l/h.In fact, the ratio of the observed frequencies v(X-D)/v(X-H) is often larger than the expected value of 0.707. The influence of anharmonic terms leads to small discrepancies, particularly in the case of vibrations involving hydrogen, where the amplitudes of vibrations are relatively large. Lowering of X-H bending vibration is not described by Eq. 17, as this is only applicable to stretching vibrations. Another rule that applies to isotopic substitution relates to the sum of the squares of the frequencies of isotopic molecules.23The basis of this rule

38

MOHAMED MATHLOUTHI AND JACK L. KOENIG

is that the sum of the squares of the frequencies is a linear function of the reciprocal masses of the atoms, so that, if several isotopic systems can be geometrically superimposed, with appropriate signs, in such a way that the atoms vanish at all positions, the corresponding, linear combination of the sum of the squares of the frequencies should also vanish. If a=Chi=4n2Xv:

(1 8)

i

then, for the water molecule, for example, we have U(

HOD) + U ( DOH) - U ( HOH) - U ( DOD) = 0.

(19)

When the isotopic molecules have different symmetries, the rule has to be applied independently to the frequencies of the subgroup common to all of the molecules. For the isotopes of water, the subgroup is C,, consisting in the identity and mirror-plane operations. The deuterium isotopes can also be used in calculating force constants for simple molecules. However, even for such simple molecules as HCN and DCN, the use of isotopes does not lead to a unique solution of the vibrational problem. It was emphasizedz3 that a certain chemical intuition and a “feel” for the relative magnitude of force constants is involved. Additional information could be taken from other isotopes (I3C, 15N,“O),and this helps in determination of a unique solution. However, such isotopes cause only small frequency shifts, so that frequency measurements must be extremely precise. It appears, then, that the use of isotopic substitution leads to some uncertainties in determination of force constants.

b. Models.-The approach to use in order to solve the vibrational problem of such large molecules as the carbohydrates is, first, to obtain data points (observed frequencies) for mono- and di-saccharides from simple molecules containing similar groups; and then, to take mono- and di-saccharides as models for polymeric carbohydrates. Moreover, simplification is needed for force-field calculations, because the force constants determined from the general force-field, even in the quadratic approximation, always exceed the observable vibrational frequencies, so that it is necessary to assume a model force-field by making certain approximations. This force field may be verified by its ability to reproduce independent experimental data. One of the simplified force-fields is the valence force-field, which is defined in terms of the forces resisting stretching, bending, or torsion of chemical bonds. Interaction force-constants or forces between nonbonded atoms are not considered in this approximation. It was found that the observed are more numerous than the frequencies calculated by assuming such a simple force-field. The difference between calculated and observed frequencies in

VIBRATIONAL SPECTRA OF CARBOHYDRATES

39

this approximation can be”’ of the order of f 10%. The valence force-field is, nevertheless, useful in assigning observed infrared bands and Raman lines to modes of vibration involving specific bond-stretching or anglebending coordinates. In addition, calculated force-constants have been found to be characteristic of the type of bond involved. For example, the C=C stretching force-constant is roughly the same in whatever molecule it is found. This observation is the basis for transferring force-constant values from one molecule to another of similar chemical structure. However, the vibrational spectrum of such molecules as carbohydrates, capable of intra- and intermolecular interactions, is sensitive to the local environment of the chemical bonds. Such interactions should be accounted for in the model force-field, so that the optimal model should tend to strike a balance between the simple model that neglects all interaction terms and the general, quadratic forcefield, which includes all interactions and is generally indeterminant. Improvement of simple models can be achieved by introducing those interaction force-constants that seem physically meaningful. However, the necessarily arbitrary nature of some assumptions makes a comparison of the published force-fields difficult. One of the most-used force-fields is the Urey-Bradley”’ force-field, which was developed and applied by Schimanouchi. It is generally known as the Urey-Bradley-Schimanouchi (UBS) force-field.”’ It consists in a mixed potential function, employing the principal bond-stretching and bond angle-bending, diagonal forceconstants of the simple valence force-field, with added central-force terms, namely, for repulsion between nonbonded atorn~.’~’ Another simplification of the vibrational problem consists in taking advantage of the local symmetry of a particular group (CH2,COH) in the molecule. For the methylene unit -CH2- (C2, local symmetry), the use of localsymmetry coordinates combines the valence-force constants to give methylene rocking, twisting, wagging, and bending coordinates. In the following Section, the application of the different modes of calculation to carbohydrates will be considered, and the contribution of isotopic substitution studies to the elucidation of the vibrational modes of these molecules will be shown. 3. Application to Carbohydrates

a. Calculation of Frequencies.-The carbohydrates most studied with n.c.a. were a- and P-D-glucose. The largest molecule to be treated for the first (150) T. Shimanouchi, in H. Eyring, D. Henderson, and W. Jost (Eds.), Physical Chemistry: An Advanced Treatise, Vol. 4, Academic h e s s , New York, 1971, p. 233. (151) H . C . Urey and C. A. Bradley, Phys. Rev., 38 (1931) 1969-1978.

40

MOHAMED MATHLOUTHI AND JACK L. KOENIG

time by calculation was" a-D-glucose, in 1972. Probably because of the economic importance of cellulose, its monomeric and dimeric models, P-D-glucose and cellobiose were actively investigated by different authors. 143.1 52-154 The calculations for a- and P-D-glucose were b a ~ e d ~ * * ' ~ ~ on the valence force-fields of isolated molecules, without consideration of the intermolecular interactions, except that the force constants for the stretching and deformations of the hydroxyl groups were taken from work involving hydrogen-bonded r n o l e ~ u l e sThe . ~ ~ conformation and vibrational spectrum of P-D-glucose were ~ a l c u l a t e dby ' ~ ~using an additive model of interatomic interaction (a.m.i.i.). In his normal-coordinate treatment, He transferHinenols4 used the Urey-Bradley-Schimanouchi f~rce-field.'~~ red the initial set of force constants from dimethyl ether, methyl alcohol, and cyclohexane. All of these author^'^^*^^^^^^^ took the atomic coordinates of P-D-glucose from the same X-ray diffraction work." The observed143 and calculated143~152*1s4 frequencies are listed in Table 111. As was emphasized,143at this stage of advance of the theoretical treatment, a rigorous, one-to-one correspondence between observed and calculated frequencies was not obtained. However, the agreement between the results was satisfactory. Discrepancies between the different calculated values are probably due to the differences between the model force-fields adopted and the initial force-constants. The latter set of data seems to be of major importance. Indeed, as stated by Andrianov and coworkers,1S2the calculations of Koenig and show slightly better agreement with experiment, due to the large number of parameters of the valence-force field compared with that used in Ref. 152. Moreover, some modifications to the transferred force fields42-46were made143in order to account for configurational and conformational peculiarities of P-D-glucose. For example, a force-constant value of 0.105 nN * nm was incorporated in order to describe the (bend-bend) interaction between the two HCC bends of the C-5-CH2-OH group, instead of the 0.012 nN * nm used previously. A value of -0.01 1 nN * nm was used to describe the bend-bend interaction between
VIBRATIONAL SPECTRA OF CARBOHYDRATES

41

and potential-energy distribution was possible, owing to the normal-coordinate analysis program written by Boerio and Koenig.la Calculated frequencies and potential-energy distributions are shown in Table IV for P-D-glucose. The discussed different vibrations of the ' ~ ~ an accurate i.r. spectrum obtained spectra below 1500 cm-'. H i n e n ~ ,using at the temperature of liquid helium, discussed the vibration and intermolecular interactions. Andrianov and coworkers'52 focused their attention on the low-frequency region (below 400 cm-I), when the twisting vibrations around C - 0 and C-C bonds are located. Cael and coworkers'43 interpreted the configuration- and conformation-sensitive modes between 1500 and 800 cm-'. H i n e n ~ noted ' ~ ~ that the observed spectra may be interpreted by a one-molecular model above 250 cm-', but, in the region below this value, a four-molecular model like the crystal unit-cell should be adopted if the effects of intermolecular interactions are to be accounted for. Such a model having 4 molecules results in 3 x 4 - 6 = 6 degrees of freedom, represented by A, + 2 BI, + B2, + 2 B3,. In fact, fourteen additional bands are which are caused by intermolecular vibrations inside and outside the unit cell. It was also remarked that an important coupling of frequencies occurs in this region. For example, 127 cm-' was assigned to C-C-0, C-C-C, and C-0-C bending modes coupled with C - 0 ring internal-motion modes. The twisting vibrations about the C-0 bonds have been found'52 to make a contribution of 90% to the potential energy of the vibration at 3 10 m-'; 40% correspond to the twisting vibration of the group 0-3-H-3 about the C-3-0-3 bond, and 25% to that of 0-2-H-2 around C-2-0-2; 99% of the calculated frequency at 307 cm-' corresponds to the twisting about C-0, so that one of the most important structural features was shown to be the twisting about the C-C and C-0 bonds of the heterocyclic ring (for numbering, see Fig. 3a).

(a)

Ib)

(C)

FIG. 3.-Numbering of Atoms (a), Atomic Displacements for the Calculated Frequencies of 901 cm-' (b) and 887 cm-' (c) for P-D-GlUCOSe.'43

MOHAMED MATHLOUTHI AND JACK L. KOENIG

42

TABLEI11

Observed and Calculated Frequencies for P-D-Glucose Observed f r e q ~ e n c i e s ' ~ ~ (cm-')" 1.r.

3350 m,br 3400 m,br 3350 s, br 3220 s, br 2978 w, sh 2950 w, sh 2934 m

1451 m

1425 1411 w

1372 s 1350 v, sh 1310 m 1275 m 1250 m,br 1225 m,br 1205 w, sh

1150s 1130 w, sh 1112m 1082 s 1050 w, sh 1035 s, br

Raman

2976 m 2945 m 2934 w, sh 2908 vs 2898 m 2880 m,sh 1479 vw 1448 m

1430 vw 1421 w, sh 1414 w, sh 1406 m 1383 w, sh 1370 s 1352 m 1329 w, sh 1306 rn 1277 w, sh 1263 m 1247 w, sh 1222 w 1204 w 1174 vw 1150m 1127 vs 1112vs 1110 w, sh 1078 w 1063 w 1050 vs

Calculated frequencies (cm-') (Ref. 143)

(Ref. 152)

(Ref. 154)

3399 3398 3398 3397 3397 2979 2945 2942 2937 2932 2930 2889 1492 1450 1444 1440 1429 1418 1410 1400 1381 1364 1338 1325 1307 1280 1254 1250 1228 1209 1176 1156 1150 1125 1106 1100 1083

3454 3454 3452 3452 3451 3000 2939 2934 2927 2920 2915 2882 1491 1465 1462 1450 1447 1438 1385 1357 1338 1332 1320 1314 1286 1267 1266 1258 1250 1170 1157 1148 1138 1132 1121 1087 1084 1066 1046 1019

3286 3260 3247 3228 3223 2983 2928 2919 2906 2901 2884 2855 1478 1474 1462 1451 1445 1444 1429 1423 1388 1374 1370 1352 1349 1345 1245 1230 1224 1221 1147 1121 1112 1104 1096 1085 1070 1065 1030 1014

1038

~~

(continued)

VIBRATIONAL SPECTRA OF CARBOHYDRATES

43

TABLE 111 (continued) Observed f r e q ~ e n c i e s ' ~ ~ (cm-')"

(Ref. 143)

(Ref. 152)

1006 vs 982 w, sh 941 vw 913 m 898 s

1017 989 973 90 1 887

714 w 628 w 589 w 575 m 521 vs 455 m 424 vs 402 s 346 w 329 vw 311 vw 297 w, sh 275 m

715 632 581 571 506

1000 936 874 653 600 556 538 493 427 383 373 342 312 310 307 302 294 292 278 269 248 242 141

1.r.

Raman

1015 s. br

915 m,sh 903 s 710 m 638 m 595 m 575 w, sh 523 w 460 m 422 w 403 w 355 w 323 m 300 m 292 w, sh 275 vw 262 vw

Calculated frequencies (cm-')

245 w 219 vw 212 vw 197 w, sh 182 w, sh 164 w, sh 123 w, sh

441

416 393 343 329 307 293 28 1 265 244 233 220 217 210 141 97 72 66

105

83 74

(Ref. 154)

987 945 907 703 662 63 1 610 594 576 514 559 537 489 419 384 368 326 313 283 273 248 241 143 96.1 72.9 71.8

Key: br, broad; m, medium; sh, shoulder; s, strong; vs, very strong; vw, very weak; w, weak.

From Table IV, it may be seen that two frequencies are calculated at 887 and 901 cm-' that could be attributed to an axial C-1-H vibration. In fact, many atoms were displaced for each vibration. The most prominent vibrations for these two calculated frequencies are shown in Fig. 3. It may be seen from Fig. 3b that the vibration at 901 cm-' contains coupling of CHI and C-1-H motions, whereas the calculated frequency of 887 cm-' exhibits, in Fig. 3c, coupling of CH2 and C-5-H groups, with no observed contributions from C-1-H or C-0-H bending. These observations, combined with

44

MOHAMED MATHLOUTHI AND JACK L. KOENIG TABLEIV Calculated Frequencies for P-D-Glucose, with Potential-Energy D i ~ t r i b u t i o n ' ~ ~ Calculated frequencies (cm-I)

Approximate potential-energy distributions'

Calculated frequencies (cm-I)

3399 3398 3398 3397 3397 2979 2945 2942 2937 2932 2930 2889 1492

OH( 100) OH( 100) OH(100) OH ( 100) OH( 100) CH(98) CH(98) CH(99) CH(99) CH(99) CH(100) CH(100) CCH(14), HCH(30) OCH(61) CCH(49), OCH(S5) CCH(57), OCH(29) CCH(SO), OCH(38) CCH(40). HCH(2O) OCH(35) CCH(61), OCH(34) CC H(22). OC H (45) CO H (36) CCH(46), OCH(63) CCH(48), OCH(38) COH(17) CCH(44), OCH(54) COH( 10) CCH(23), OCH(58) COH(24) CCH(26), OCH(58) COH( 14) CCH(48). OCH(17) COH(29) CCH(47), OCH(30) COH(26) CC(19), CCH(28) OCH(15). COH(37) CC(lI), CCH(30) OCH( 12), COH(42) CCH( 14), OCH(20)

1176

1450 1444 1440 1429 1418 1410 1400 1381 1364 1338 1325 1307 1280 1254 1250 1228

1156 1150

1125 1106 1100 1083 1038 1017 989 973 90 1 887 715 632 581 57 1

506 44 1

Approximate potential-energy distributions C0(47), CC(21) COH(20) C0(73), CC(I0) COH( 14) C0(55), CC(30) CCC(lI), CCO(l0) COH(14) CO(54). CC(53) CCO(lO), COH(14) CO(72). CC(17) COH(20) C0(37), CC(40) CCO(ll), COH(25) C0(47), CC(29) CCO(Il), COH(44) C0(45), CC(19) CC0(20), COH(14) CO(40). CC(47) CCO( 11) CO(42). CC(31) CCO(17) CO(42). CC(39) CC0(19), CCH(l0) CO(18). CC(20) CCO(lO), CCH(44) CO(44), CC(33) CCH( 19) CC0(48), CCH( 19) CC0(62), CCH(2I) CO(15), CCO(47) CCH(I5) CC( 1 l), CCC( 12) CCO(35). CCH( 19) OCO( 14) CO( 14), CC(22) CC0(39), CCH( 15) CC(12), CCC(23) CC0(30), CCH( 14)

(continued)

VIBRATIONAL SPECTRA OF CARBOHYDRATES TABLEIV Calculated frequencies (cm-')

Approximate potential-energy distributions"

1209

CO( 14), CC( 18) CCH(l2), OCH(16) COH(35) C0(11), CC(12) CCC(I7), COC(13) CC0(20), CCH( 10) CCC(19), CCO(44) CCH( 14) CC0(70), CCH( 11)

416

393 343 329 307 293 28 1

co(T ) b (11)

CC( 1 l), CCO(34) OC0(18), C 0 ( ~ ) ( 1 4 ) CC0(63), C 0 ( ~ ) ( 1 6 ) CC0(19), C 0 ( ~ ) ( 6 8 ) CC0(52), CCH(33) co( 12) CCC(19), COC(l0)

45

(continued)

Calculated frequencies (em-')

244 233 220 217 210 141 97 72 66

Approximate potential-energy distributions

CC0(25), Co(T)(21) CC0(34), CO(i-)(43) CCO(lO), C 0 ( ~ ) ( 7 7 ) CC0(15), C 0 ( ~ ) ( 7 4 ) CC0(13), C 0 ( ~ ) ( 7 6 ) CC0(17), C 0 ( ~ ) ( 7 8 ) CCC(I7), COC(21) CCH(I5), CCO(30) CCC(36), CCO(16) Cc(T)(40) CCC(26), CCO(30) cc(7)(32) CC(~l(86)

a The percentage may be greater or less than 100, because of contributions of off-diagonal terms in the force-constant matrix. * Torsional vibration.

deuteration studies, led the authors'43to assign the calculated vibration at 901 cm-I, rather than that at 887 cm-*, to C-1-H bending, observed at 898 cm-' in the Raman spectrum of P-D-glucose. Confirmation of the early results of the Birmingham ~ c h o o l , by ~.~ assigning the observed frequencies at 840 and 898 cm-' to C-1-H deformation in a- and P-D-glucose, respectively, was completed by revealing the important contribution, to these modes, of CH2 twisting and rocking motions. Change in the orientation of the C H 2 0 H group from gt to gg in a and P-D-glucose may occur, and produce frequency shifts. It was noted77that a correct analysis of the spectra of carbohydrates in the structurally sensitive region from 900 to 700 cm-' is possible only by consideration of the various dispositions of the CH,OH group. This group seems to be of particular importance in the discussion of polysaccharide spectra. Indeed, the region of frequencies from 1500 to 1200 cm-' contains highly coupled vibrations, including contributions of CHI, C-6-0-H, and C-5-C-6-H modes, and also of other C-0-H and C-C-H groups. However, the calculated frequencies at 1492 and 1429 cm-' for p - ~ - g l u c o s e show ' ~ ~ appreciable CHI bending contributions. The use of Cartesian displacement-coordinates, obtained from the normalized L matrix elements, permits graphical depiction of the components of atomic

MOHAMED MATHLOUTHI AND JACK L. KOENIG

46

(a)

lb)

(C)

FIG. 4.-Atomic Displacements for the Calculated Frequencies of (a) 1473 cm-' for Glucose, and (b) 1492 cm-' and (c) 1429 cm-' for P - D - G h ~ c o s e . ' ~ ~

(Y-D-

vibrations. It helps to see, as in Fig. 4, that, although the vibrations above 1430 cm-' are mainly due to 6(CH2), they are not pure CH, vibrations. Normal-coordinate analysis of a polymeric carbohydrate such as cellulose I is made easier owing to a favorable conjunction of circumstances. Indeed, the A and B (symmetric and antisymmetric to the screw axis) modes of cellulose I show negligible splitting, probably because of the fact that the repeating unit is so large that the inter-residue contributions are negligible with regard to the internal motions. The similarity of the A and B modes implies that, for most polysaccharide structures, calculations of the A representation alone will yield an adequate description of the individual vibrations. However, as seen in Table V for the modes below 1200 cm-', contributions from the glycosidic-bridge atoms result in differences between the p.e.d. of corresponding A and B species. In particular, the modes calculated at 1169 and 1170 cm-' show a high degree of mixing, and an important contribution of the inter-residue glycosidic oxygen atom (see Fig. 5 ) . This mode has been found to be common to most saccharides, and its assignment has been a matter of c o n t r o v e r ~ y It . ~has ~ been assigned'55 to a coupled vibration involving C - 0 stretching and C-0-H bending modes, and considered in another a r t i ~ l e "as ~ due to antisymmetric stretching in the C-0-C glycosidic linkage. These assignments are consistent with the p.e.d. results given in Table V. The calculations of Cael and coworkers29 were based on the isolated chain, without consideration of intermolecular effects, except that the force constants for the stretching and deformation (155) L. E. Segal, R. T. O'Connor, and F. V. Eggerton, 1.Am. Chem. SOC.,82 (1960) 2807-2812.

47

VIBRATIONAL SPECTRA OF CARBOHYDRATES

TABLEV CalculatedZ9Frequencies and Computed, Potential-Energy Distribution of Cellulose I Frequency (cm-')

Potential-energy distributions"

A modes

3398 3398 3398 296 1 2946 2941 2937 2933 2929 2868 1485 1434 1424 1372 1368 1355 1331 1327 1309 1299 1285 1282 1246 1239 1229 1206 1182 1169 1152 1141 1113 1098 1090 1055

1043 986 96 1 942 893 727 665 60 1

OH( 100) OH(100) OH(100) CH, asym. stretch (96) CH(98) CH(98) CH(99) CH(99) CH( 100) CH,, sym. stretch (100) CO(lO), HC0(57), HC,0,(41) HC-6-C(3 I ), HC-6-O(6 I ) HC-6-C(21), HCH(57) HCO( 19), HCC(67), COH( 11) HC0(21), HCC(71) HCO( lo), HCO(S5) CO(10), HC0(55), HCC(26), COH(16) HC0(20), HCC(70) HC0(66), HCC(34) HC0(67), HCC(67) CC(19), HCC(l5), COH(42) HC0(38), HCC(38), COH(12) CC(11), HCC( 13), COH(44), HC-6-O(20) CC( 12). HCC( lo), COH(25), HC-6-C( 14), HC-6-O(37) HC0(32), HCC(38), COH(30), 0-5-C-l-H(12) C0(13), HC0(16), HCC(32), COH(13), HC-6-O(14) CO(40). CC(23), CCC(lO), HCC(l4) C0(81), CC(10) C0(23), CC(61), CCO( 17), COH(14) C0(45), CC(53) C0(43), CC(30), CCO(18). HCO(10), COH(11) C0(49), CC(26), COH(13) C0(42), CC(44), COH(32) C0(60), C C ( l I ) , CCO( 19) CO(88), CC(10) C0(44), CC(20), CCO( 15), HC-6-C(11) CO(36). CC(41), CC0(21), HCC(I1) C0(61), CC(38), CCO( 10) CC(23), CCO( 14), HCC(lI), HC-6-C(47) CC0(31), COC(13), OCO(l5), HCC(I0) CC0(56), OCO(10), HCC(17) CO( 14), CC( 1 I ) , CCO(38) (continued)

MOHAMED MATHLOUTHI AND JACK L. KOENIG

48

TABLEV Frequency (cm-') 568 532 457 435 387 347 302 29 1 250 240 233 220 182 142 121 79 64 38 31

(Continued)

Potential-energy distributions" CCC(ll), CC0(36), COC(15). HCC(36) CC(lO), CC0(39), COC(16), HCC(22) CC(13), CCC(28), CC0(25), COC(14) CC( lo), CCC(l4), CC0(36), HCC(21) CC(ll), CC0(56), HCC(l0) CO(ll), CC(12), CCO(47) CC0(71), HCC(21) CC0(63), HCC(19) CCC(15). CCC(l5), C 0 ( ~ ) ~ ( 5 8 ) CO(7)(96) C0(7)(95) CCC(21), CC0(20), C 0 ( ~ ) ( 4 3 ) CC0(48), COC(ll), HCC(7)(10) CCO(41). OC0(23), cC(T)(lo) c c ( 1 1 ) , CCO(Sl), COC(14), CC(T)(lO) CcC(22), CCO(21), c C ( ~ ) ( 3 6 ) CC0(13), CC(7)(61) CC(7)(83) cc(7)(94)

B modes 3398 3398 3398 2961 2945 2942 2937 2932 2929 2868 1483 1434 1423 1372 1368 1355 1333 1326 1310 1297 1284 1282 1246 1242

OH( 100) OH( 100) OH( 100) CH2 asym. stretch (96) CH(98) CH(98) CH(98) CH(99) CH(100) CH,, sym.stretch (100) CO(lO), HC0(57), H-l-C-1-0-5(41) HC-6-C(3 1), HC-6-O(61) HCC(22), HCH(57) HC0(22), HCC(66), COH( 11) HC0(20), HCC(73) HCC(85), HCO(l0) CO(lO), HC0(50), HCC(38), COH(10) HC0(23), HCC(57), COH(16) HC0(72), HCC(34) HC0(58), HCC(40) CC(17). HCC(18), COH(37) HCO(37). HCC(33), COH(16) CC(1l), HCC(14), COH(43), HC-6-O(20) CC(12). HCC( 14), COH(21), HC-6-C(13), HC-6-O(31) (continued)

VIBRATIONAL SPECTRA OF CARBOHYDRATES

49

TABLEV (continued) Frequency (cm-') 1230 1205 1187 1170 1148 1137 1122 1097 1074 1060 1037 1008 964 956 897 669 621 572 546 526 448 427 386 362 356 299 288 242 240 237 231 192 159 92 94 59 14

Potential-energy distributions" HC0(34), HCC(36), COH(26), H-l-C-l-O-S(l2) HC0(17), HCC(34). COH(17), HC-6-O(15) C0(53), CC(21) C0(51), CC(37) CO(51), CC(31), CCC(16), CC0(16), COH(20) C0(53), CC(42), CCO(16) C0(40), CC(39), CC0(16), COH(l0) C0(57), CC(32), COH(29) C0(49), CC(33), CCO(13) C0(65), CC(30), COH(14) C0(39), C C ( l l ) , CC0(20), HC0(14), HCC(11) C0(46), CC(28), CCO(14) C0(48), CC(42), CCO(14) C0(58), CC(23), CCO(lS), COC(l0) CC(16), HCC(lS), HC-6-C(57) CC0(49), COC(17), HCC(21) CO(lO), CC0(38), OCO(lS), HCC(13) CCO(59) CC0(43), COC( 17), HCC(25) C0(17), CC(30), CCC(lO), CCO(21) CO(lO), CC(12), CCC(lS), COC(12), HCC(14) CC(lO), CCC(30), CC0(28), HCC(l0) CC0(45), HCC(25) CO(ll), CCC(14), CC0(43), HCC(16), CC(l0) CCC( 1I ) , CC0(49), COC( 14), HCC( 16) CCO(51) CCO(69) CCO(36). COC(19), HCC(lO), C 0 ( ~ ) ( 2 7 ) CO(~l(96) c0(4(91) CCO(11). C 0 ( ~ ) ( 7 7 ) CC(15), CCC(lS), CCO(40) CCC(16), CC0(46), HCC(10), C c ( ~ ) ( 1 3 ) CCC(19), CC0(37), COC(Il), CC(7)(18) c c c ( 2 7 ) , CC0(24), cc(T)(33) cc(T)(75) cc(T)(91)

The percentages may be greater or less than 100, due to contributions of off-diagonal terms in the force-constant matrix. T = torsional vibration.

of the C-0-H groups were taken from hydrogen-bonded molecules. Calculation and interpretation of the frequencies of the vibrational spectrum of cellobiose, the repeat unit of cellulose, was achieved by use of the additive model of interatomic interaction^."^ Frequencies below 700 cm-' were

50

MOHAMED MATHLOUTHI A N D JACK L. KOENIG

R

FIG. 5.-Atomic Displacements for the A and B Modes Calculated at (a) 1169 cm-' and (b) 1170 cm-', R e s p e c t i ~ e l y . ~ ~

attributed to dimeric-type modes for the 700-400-~m-~ range, dimeric lateral C-C-0 and torsional T ( C - 0 ) for both monomers and dimers in the 400250-cm-' region, and the region below 250 cm-' was assigned to torsional T(C-C) vibrations in the pyranose ring with contributions from C-5-C-6. The results obtained'53 by use of the a.m.i.i. were found to be in agreement with those given by the valence force-field model." The advantage of the interatomic-interactions model lies in the possibility of interpretation of the low-frequency range of the infrared spectrum. The 3500-2800-cm-' region has been generally less discussed than the vibrations below 1500 cm-'. It was noted'53 that the 3000-2800-cm-' range is characteristic of CH stretching. But, although the 3500-3000-cm-' region is unanimously assigned to the stretching vibrations of the hydroxyl groups, the agreement between calculated and observed frequencies in this range is somewhat less satisfactory than that obtained in the region below 1500 cm-'. This is probably due to the hydrogen bonding that cannot be explicitly incorporated into the potential f ~ r c e - f i e l d In . ~ ~fact, calculations cannot yield frequencies in good agreement with observed vibrations, as the initial set of force constants is not yet adjusted to this interaction problem. Additional work on alcohols, as a model system, may be beneficial, and may reveal the nature of these effects. This could be made easier by the availability of neutron-diff raction data, which give hydrogen-bond distances and angles with good accuracy.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

51

Another example where normal-coordinate analysis of the monomer was extended with a certain degree of success to the polymer is that of Va m y l ~ s e . ~Simplification ' of the calculations, and reducing of computer time, have been obtained by solving the secular determinant for only the A representation of the spectrum of the helical a-(1 + 4)-glucan. Conformation- and configuration-sensitive modes were disc~ssed.~' As for cellulose I (Ref. 29), the potential-energy distribution showed that most of the vibrational frequencies calculated at 1274 and 1266 cm-' are represented in Fig. 6. Both modes are characterized by OCH and CCH deformations of the ring-hydrogen atoms. In addition, the calculated frequency at 1274 cm-' contains large contributions from C H 2 0 Hdue to COH and OCH bending. The assignment of the frequencies is consistent with the proposed mechanism of V + B conversion of amylose, which involves helix extension and hydrogen-bonding at the CHzOH side-chain. The other COH-related modes have been d i s c~ s s e d , ~because ' of their importance in carbohydrate vibrational spectra. Frequencies with a significant contribution of C-0-H bending were calculated at 1388 and 1342 cm-' and in the regions 1280-1200 and 1120900 cm-'. The determination of C-0-H deformation modes is of particular interest in establishing the hydrogen-bonding network. However, the use of normal-coordinate analysis needs to be associated with such other techniques as deuteration and infrared dichroism studies, in order to resolve the problems of chain conformation and hydrogen bonding. Calculations of the conformations and frequencies of various monosaccharides (a and p anomers of o-glucose, o-allose, D-galactose, and D-

-

la1

FIG. 6.-Atomic 1266 cm-'.

(bl

Displacements for the Calculated Modes'" at (a) 1274cm-' and (b)

52

MOHAMED MATHLOUTHI A N D JACK L. KOENIG

gulose, and a-D-mannose and a-D-altrose) have been made156by use of the a.m.i.i. method. This model of calculation was found especially useful for the assignment of the observed vibrations in the low-frequency region, and for identifying the spatial structure of the sugars studied. The normalcoordinate analysis was also shown"' to be helpful in differentiating the polymorphic forms of cellulose I and 11. The model compound used in such an analysis may be a polyol, such as a 1,5-anhydropentit01.'~~ When the model is different from the molecule studied, as in this case, some discrepancies may appear between observed and calculated frequencies, but the classes of internal motions associated with the polymorphic change are readily distinguished. The suitability of the chosen model remains an important question to resolve. A progressive approach, such as that of Sivchik and Z h b a n k ~ v , "may ~ lead to a calculated result closer to the reality. These workers carried out calculations for 18-, 19-, and 20-atom molecular fragments characterized by stepwise introduction of C-1-0 and C-1-H bonds, in order to estimate the effect of the groups attached to C-1 on the spectral differences between a-and P-D-glucose. The difference in configuration between a and P anomers is associated with increasing the difference between vibrational frequencies in the 900-800-cm-' region when the number of atoms increases (see Table VI). Transferability of force constants, as well as adjusting of the model to the studied molecule, should be improved in order to reach the one-to-one correspondence between observed and calculated frequencies. Besides the calculation of frequencies by use of a simplified model of the secular equation, another kind of theoretical treatment was applied'60 to various carbohydrates, in order to determine the possibility of existence of rotational isomerism of the CH20H group. This treatment consisted of calculating the potential function of inner rotation of CH20H, which includes bond-orientation energy and the energy of steric interaction. The potential surfaces of the C H 2 0 H group in the rp, axis (rotation about C-6-0-6) and the rp2 axis (rotation about C - 5 4 - 6 ) were calculated for a-D-glucose and a-D-galactose. The values of 'pl and rp, at which the potential energy was the minimum were supposed to correspond to the stable conformations of the group ( ( p 2 values of -65 and 170" for a-D-galactose and (p2 = 80, 177, and 300" for a-D-glucose). The calculated orientations of the CH20H group of these two sugars were used in order (156) V. M. Andrianov, R. G . Zhbankov, and V. G . Dashevskii, Zh. Srrukr. Khim., 21 (1980) 42-47; Engl. Transl., 32-37. (157) R. H. Atalla, Appl. Polym. Symp., 28 (1976) 659-669. (158) L. J. Pitzner and R. H. Atalla, Spectrochim. Acta, Purr A , 31 (1975) 911-929. (159) V. V. Sivchik and R. G. Zhbankov, Zh. Prikl. Spekrrosk., 28 (1978) 1038-1045; Engl. Transl., 706-71 1. (160) R. G. Zhbankov, J. Polym. Sci., Part C, 16 (1969) 1629-1643.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

53

TABLEVI

F r e q u e n c i e ~ (in ' ~ ~cm-') of Molecular Fragments of in the 900-800-cm-' Range

(I-

and p-D-Giucose

Scheme

(-k0

(-)o-l

0 Fragment

Anomer

18-atom

19-atom

20-atom

C6H70S

C6H708

C6H808

837 875

837 887

24-atom 12 O6

c6

~~~~~~

a-D P-D

859 869

839 886

to interpret the discrepancies between their i.r. spectra in the 1500-1400-~m-~ region. An accurate determination of the rotational isomerism of the CHzOH group, combined with an interpretation of the i.r. intensities of the polysaccharides studied, led the author160to conclude that determination of the rotational isomer of the CH,OH group of the amorphous polymer helps in predicting its ability to crystallize. These results suggested that determination of the structure of carbohydrates may be clarified by comparison of such different results as conformation calculations and vibrational spectra as indicated in Scheme 1. b. Isotopic-substitution Studies.-Infrared spectra of deuterated carbohydrates were studied16' as early as 1958. It was possible to differentiate, from C-1-H and C-1-D stretching vibrations (which show a shift of -40 cm-I), the (Y and p anomers of D-glucose and of D-mannose. The deuteration technique was successfully applied16' to a series of free sugars, and their methylated and peracetylated derivatives, in order to characterize the anomers. The use of such techniques as normal-coordinate analysis and laser-Raman spectroscopy, associated with identification of characteristic vibrations by the deuterium-substitution method, enhanced the degree of (161) M. Stacey, R. H. Moore, S. A. Barker, E. J. Bourne, H. Weigel, and D. H. Whiffen, U.N. Int. Con$ Peaceful Uses At. Energy, Geneva, 2nd. 20 (1958) 251. (162) S. A. Barker, R. H. Moore, M. Stacey, and D. H. Whiffen, Nature, 186 (1960)307-308.

54

MOHAMED MATHLOUTHI AND JACK L. KOENIG

confidence in the proposed assignment^.^^ Classically, the interpretation of spectra of deuterium-labelled carbohydrates is based on the ratio of frequencies arising from the deuterated and nondeuterated groups, which should for deformation modes, as indicated be fifor stretching vibrations, and
(163) P. D. Vasko, J. Blackwell, and J. L. Koenig, Carbohydr. R e r , 19 (1971) 297-310.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

55

the vibrational spectra of carbohydrates by using deuterium substitution is particularly useful in interpreting the bands arising in the local-symmetry region (1500-1200 cm-I), because of the number of deformation modes located in this range of frequencies which contain a hydrogen atom (HCH, CCH, OCH, COH). The observed frequencies in this region are generally due to complicated, coupled modes of vibration that involve the C H 2 0 H group. The frequency variations observed'64 during the V + B conversion of amylose are also related to CH2, and COH modes from C H 2 0 H groups, as revealed by the Raman spectra of deuterated and nondeuterated Vhamylose. Deuterium substitution may contribute to elucidating the coupling of certain observed Raman lines; that is what was observed for the 1334-cm-' vibration in the Raman spectrum of V,-amylose, which gave rise, after deuteration, to a higher frequency (1414cm-') and a lower frequency (1290 cm-'). The lower frequency was presumed to come from the shifting of a COH mode resulting from the deuteration, and the higher frequency, from the decoupling of the CH2 and C-0-H motions. Study of the Raman spectrum of amylose in solution in Me,SO-d, enabled'64 elucidation of its conformation, which had been a matter of controversy. The lines observed at 1254 and 1334 cm-I, which are close to what was observed for the B-form, suggested that the chains of amylose are probably extended, with the C H 2 0 Hgroups hydrogen-bonded to solvent molecules, rather than involved in intramolecular linkages as in the V-form. These results were confirmed3' by use of computer calculations of atomic displacements and potential-energy distribution. Infrared spectra of glycosaminoglycans in D 2 0 and DCI were used'" for quantitative evaluation of the uronic acid and acetamido groups in this class of polysaccharide. The results showed that i.r. spectroscopy of compounds in D20 solution provides a simple, quantitative basis for examination of vas(C0;) and amide I bands in heparin and chondroitin 4-sulfate. In DCl, analysis was based on v(C02H)and amide I bands which were well resolved. The apparent acid-dissociation constants of the studied polysaccharides were estimated from the absorbance of v(C0;) or v(C02H) bands, or both, at different pH (pD) values. The major advantage of D 2 0 , which allowed such an i n ~ e s t i g a t i o n 'to ~~ be carried out, is its i.r. transparency in the carbonyl region (18001500 cm-I).

(164) J . J . Cael, J. L. Koenig, and J. Blackwell, Carbohydr. Res., 29 (1973) 123-134. (165) B. C a w , G. Scovenna, A. J . Cifonelli, and A. S. Perlin, Carbohydr. Res., 63 (1978) 13-27.

MOHAMED MATHLOUTHI AND JACK L. KOENIG

56

IV. FOURIER-TRANSFORM, INFRARED SPECTROSCOPY 1. Description of Method

Application of infrared spectroscopy to carbohydrates and their derivatives is well documented, as seen from the previous revie~s.’,’~,’’Our purpose is to mention innovations made in this field since the advent of computer-controlled, Fourier-transform infrared (F.t.4.r.) spectrometers. The most important element in an F.t.4.r. instrument is a Michelson interferometer (see Fig. 7). This interferometer consists of two mirrors and a beam splitter. When the light beam enters the interferometer, it reaches the beam splitter (BS) at a 45” angle. The beam splitter passes -50% of the radiation to a movable mirror (MM), and the other -50% is reflected to a fixed mirror (FM). The reflected beams from each mirror return to the beam splitter and to the detector. The two reunited beams will interfere constructively, or destructively, depending on the relationship between their path difference (x)and the wavelength of the light. When both mirrors (FM and MM) are positioned at the same distance from the beam splitter, x = O , and the light beams are identical. Under these conditions, all wavelengths of the radiation striking the beam splitter after reflection add coherently, to produce a maximum flux at the detector, and generate what is known as the “center” burst. As the movable mirror is displaced from this point, the path length in that arm of the interferometer is changed. This change in path length causes each wavelength of source radiation to interfere

1

FM

MM

r--’I I

1

I

1

I I

I I

;

I--,

I I

1 1

I

I

I

I

L,- J

-

1

\

\

Source or

emission

\\

-

\

\

Detector

FIG.7.-The Michelson Interferometer (BS= beam splitter, FM = fixed mirror, and MM = movable mirror).

VIBRATIONAL SPECTRA OF CARBOHYDRATES

51

lnterferogram

FIG. 8.-An

Interferogram.

destructively with itself at the beam splitter. The sum of the interfering fluxes yields, at the detector, an interferogram (see Fig. 8) which depends on the position of the movable mirror. The mirror displacement is accurately measured by a laser impinging upon the back of the mirror. The Fourier transform provides the relationship between the distance, x, and intensity I(x) measured in an interferogram and the desired spectral intensity I(v) at frequency v. The mathematical equation relating I ( v ) to I(x) is

j-, +m

I( v ) =

I(x) cos 27rxv dx.

The transform from the interferogram to the spectrum is carried out by the dedicated minicomputer on the instrument. The theory of Fouriertransform infrared spectroscopy has been treated, and is readily available in the literature.21’22”66Consequently, the advantages of F.t.4.r. dispersive spectroscopy will only be outlined in a qualitative sense: (i) The Fellgett or multiplex advantage arises from the fact that the F.t.4.r. spectrometer examines the entire spectrum in the same period of time as that required (166) R. J. Bell, Introducrory Fourier Transform Specrroscopy, Academic Press, New York,

1972.

58

MOHAMED MATHLOUTHI AND JACK L. KOENIG

for a dispersive instrument to examine a single spectral element. Theoretically, an F.t.4.r. spectrometer can acquire the spectrum from 0 to 4000 cm-I, with 1-cm-’ resolution, 4000 times as fast as a dispersive instrument having the same signal-to-noise ratio. This is quite an advantage as, for the same measurement time, an increase by a factor of -63 in signal-to-noise can be achieved with an F.t.4.r. instrument. (ii) The second advantage, named Jacquinot’s advantage, describes the larger throughput of the interferometer due to the ability to accept a large cross-section of radiation limited only by the size of the mirrors. This higher throughput is particularly important in the infrared region, where the signals are extremely weak as the infrared sources are weak. (iii) The third advantage, the frequency-accuracy advantage, comes from the fact that the frequencies on an F.t.4.r. instrument are internally calibrated by a laser, whereas conventional i.r. instruments exhibit drifts when changes in alignment occur. This is particularly important for coaddition of spectra to signal averages, as frequency accuracy is an absolute requirement in this case. Moreover, whereas it takes several minutes for a routine scan in a dispersive instrument, an interferometer can be scanned many times per minute. Application of F.t.4.r. spectroscopy to biological systems and carbohydrate mixtures or dilute solutions is of particular interest, because of the ease of analysis of data by use of such techniques as absorption subtraction or factor analysis. This is possible owing to the direct interfacing of the computer to the spectrometer, which allows arithmetic manipulation of the spectra in an “imaginative” way, as will be seen in the following Section. 2. Data-processing Techniques

a. Quantitative Analysis of Mixtures.-The digitized nature of the F.t.-i.r. spectrum, and the improvement of its signal-to-noise ratio, allow quantitative analysis of mixtures, and other treatments of data, by computerized methods. The logic pattern to be followed in developing spectral treatments is shown in Scheme 2. The classical approach to the analysis of mixtures by use of infrared spectroscopy consists in identifying specific, strong bands that belong to a suspected component, obtain a pure spectrum of the suspected component, and then remove those in the spectrum of the mixture that are due to the identified compound. The process is repeated for the remaining bands in the mixture spectra. Once the component spectra are known for a mixture, a series of calibration curves is produced. These curves relate concentration to absorbance, using Beer’s law. The concentration of the components of the mixture are then obtained by interpolation. The advantage of Fouriertransform, infrared spectroscopy is that components of a mixture may be

VIBRATIONAL SPECTRA OF CARBOHYDRATES

59

1 Unknown mixture

Computer

Absorbance spectra (Digital data)

Identification

Subtraction

Ratio method Recognition

Quantitative analysis Least-squares curve-fit Known mixture

I Report

SCHEME 2.-Computer-assisted

Treatment of Mixtures.

digitally subtracted for a two-component mixture, or the ratio method4’16’ is applied for more complex mixtures. This method consists in obtaining subtraction coefficients by obtaining the ratio for two spectra differing in relative concentration of each of the unknown components. The restriction for application of the method is that there should be regions of the mixture spectrum for which each component contributes, without interference by bands of the other components. However, for application of the ratio method, it is easier to assume that the mixtures consist of only two components. This method was successfully applied4 to mixtures of crystalline and amorphous phases, and to the characterization of trans and gauche orientational species. (167) T. B. Hirschfeld, Anal. Chem., 48 (1976) 721-728.

60

MOHAMED MATHLOUTHI A N D JACK L. KOENIG

Quantitative measurements are carried out by use of a single frequency in the conventional i.r. spectrum, but, with digitized spectra and a computer, the entire frequency spectrum of each component in a mixture can be fitted by curve-fitting techniques using such methods as least-squares refinements,’ which can yield an indication as to the precision of the fit. b. Factor Analysis.-The multivariate, statistical technique known as factor analysis has been applied6 to Fourier-transform infrared spectra in order to determine the number of components contributing to a spectral region. It was also used168for reduction of the amount of random noise present in a spectrum. Originally developed by sociologists, factor analysis of data is used where the number of independently contributing entities (factors) is required. The theory and feasibility of factor analysis applied to Fouriertransform infrared spectra have been examined.6 It was especially emphasized that the most beneficial effect of factor analysis for polymers is the detection of the number of rotamers contributing to the spectra. This effect was applied169 in order to measure the conformational composition in amorphous polymers, and to investigate the change in their structure under the effect of their thermal and mechanical history. c. Absorbance Subtraction.-The digital subtraction of F.t.4.r. spectra has been recognized as a useful tool in the determination of small spectroscopic differences in similar samples, and its utility for a large variety of spectroscopic problems was d e m ~ n s t r a t ed . ”~ In particular, the subtraction of the spectrum of water is required in order to examine the vibrational features of biological systems. Although subtraction is becoming a routine technique, its application needs a certain “skill” as many artifacts may be generated by inappropriate application. The difference spectrum is generally expanded, and this increase of sensitivity applies to the detection of sampling and photometric artifacts as well. The most common problem arises from attempts to effect digital absorbance-subtraction when the absorbances of the samples are too high. Ordinarily, the upper limit of absorbance for which subtraction is possible is 1.0. Subtraction of the spectrum of water may be accomplished by use of thin windows of calcium fluoride or barium fluoride having path-lengths of 2.5-1.5 pm, in order to keep the absorbance for the solution at 1.0 or less in the regions of interest. The same cell thickness must be used for the solvent. This yields a better result than the use of scaling factors of the computer to change a spectrum obtained at a different thickness. (168) P. C. Gillette and J. L. Koenig, Appl. Specrrosc., 36 (1982) 535-539. (169) J. L. Koenig and D. E. Kormos, Ann. N.Y. Acad. Sci., 371 (1981) 87-105. (170) J. L. Koenig, Appl. Spectrosc., 29 (1975) 293.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

61

Other processing techniques for analysis of F.t.4.r. data have been developed in order to obtain the maximum of information from the spectra. The advances made in time-resolved techniques, which sample only a portion of the interferogram, permit obtaining of spectra in the microsecond domain; this will lead to additional applications of F.t.4.r. spectroscopy such as the study of dynamic and kinetic processes. 3. Spectral Results

Spectra of various aqueous solutions, including 1.8 M D-glucose solution were measured’” with a Fourier-transform, infrared spectrometer. The data obtained for dilute solutions were found useful for the usual qualitative purposes, and also amenable to quantitative analysis, but the spectra of such concentrated solutions as 1.8 M D-glucose show distortions of the background at > 3000 cm-’, and negative, water deformation bands. These distortions were attributed to structural changes of water in the presence of the solutes. Polarized FA.-i.r. spectra of oriented, crystalline glycosaminoglycans have been r e ~ o r d e d , ”and ~ the dichroism data of the vibrational modes of the amide and carbonyl groups interpreted with respect to the particular molecular structures. The use of a Fourier-transform, infrared spectrophotometer having multiple-scanning (300) and signal-averaging capabilities permitted, by combination with a rotating-anode X-ray generator, relating the i.r. dichroism from thin samples of sodium hyaluronate, chondroitin 4-sulfate, and proteoglycan-hyaluronate complex to their chair conformation. Structural analysis of dextrans has been carried out by use of Fouriertransform, infrared-difference spe~trometry.‘’~ This technique is used when the spectral differences between the samples are small, which is the case for the i.r. spectra of polysaccharides. It consists in taking the spectrum of a simple polysaccharide as a “baseline” and expressing the spectral changes observed for the polysaccharides as deviations from the “standard” spectrum (in terms of a difference spectrum). This is possible only because of the spectral-subtractive capabilities of F.t.-i.r. spectroscopy. The data for different dextrans were adjusted (in absorbance) by reference to the linear dextrans. The spectral changes arise from discrepancies in their degree of branching. The determination of this degree of branching from the intensity of the difference-absorbance peak at 1090 cm-’ was achieved. As shown in (171) M . J. D. Low and R. T. Yang, Spectrochim. Acta, Part A, 29 (1973) 1761-1772. (172) J. J . Cael, D. H . Isaac, J. Blackwell, J. L. Koenig, E. D. T. Atkins, and J. K. Sheehan, Carbohydr. Rex, 50 (1976) 169-179. (173) F. R. Seymour and R. L. Julian, Carbohydr. Res., 74 (1979) 63-75.

62

MOHAMED MATHLOUTHI AND JACK L. KOENIG

0.1

0.2

0.3

0.4

0.5

0.6

0.7

lAn + 1)

-

FIG. 9.-F.t.-1.r. Difference, Absorbance Peak-height y (at 1080 cm-') in Absorbance Units vs. l / ( n + l ) , Where n is the Degree of Linearity in Terms of the Average Numbers of D-Glucopyranosyl Residues Between Branching Residues. (The circles correspond to different dextrans used.) (From Ref. 172.)

Fig. 9, there is a linear relationship between the diff erence-absorbance peak-heights and the number of linear units in the different dextrans. This technique of the diff erence-spectrum seems promising for the study of branching in microbial levans and dextrans. The amount of material needed'73 is small ( - 5 kg per sample) and the data-acquisition time is short (- 1 h). This is advantageous compared with 13C-n.m.r.spectroscopy, which yields the maximum information when 100-mg samples are available. When compared to g.1.c.-m.s. structural determination of permethylated derivatives, which is the only other method for the investigation of structure that employs small amounts of samples, F.t.4.r. spectroscopy was found"3 more profitable, as it requires much less time. The author^"^ concluded that F.t.4.r. absorbance-difference plots can be utilized in quantitative and structural analysis of various dextrans, such as those found in dental plaque or in industrial sucrose solutions. F.t.4.r. spectroscopy associated with deuteration and absorbance-subtraction techniques, as well as X-ray diffraction, have been applied'74 to investigation of the molecular structure and gelling mechanism of the bacterial polysaccharide curdlan. The F.t.-i.r. spectrum of the polysaccharide gel, after subtraction of water, was found to be similar to that of the dried-gel film after deuteration. These (174) W.S. Fulton and E. D. T. Atkins, Am. Chem. SOC.Symp. Ser., 141 (1980) 385-410.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

63

results that a high proportion of the hydroxyl groups and the interstitial water of crystallization are inaccessible to exchange with deuterium atoms. The hydrogen bonding between the water of crystallization and the triple helices form a micellar domain which dissolves partially to allow the gelation mechanism to occur. The bacterial polysaccharide xanthan was e~arnined'~'by F.t.4.r. techniques. From intensity measurements of bands at 1159, 1130-1 120, and 995 cm-', when the temperature was increased from 20 to 55", it was found that this polysaccharide shows a sigmoid transition at -40". This property was found comparable to the viscosity transition of xanthan observed when the temperature is raised. Very little change in the spectra of 1% xanthan solution in 1% KCl was observed in the same range of temperature. From these s t ~ d i e s , ' ~ ~it- 'is~ 'seen that Fourier-transform, infrared spectroscopy may be successfully applied to examining the structure, or to interpreting certain properties, of polysaccharides. This technique was applied176to analysis of the structure of chitins. Whereas conventional i.r. spectra do not permit much judgment as to the structure of chitin^,'^' because the positions of the bands are usually chosen arbitrarily, computeraided analyses provided precise data for amide I and amide I1 bands. It was that the amide-band frequencies, at 1661f 1 cm-I for amide I and 1626* 1.5 cm-' for amide 11, are characteristic of the lowest-energy conformation of a-chitin. This result was made possible by use of computer programs 178,179 to resolve the digitized infrared spectrum of the studied chitins into component bands in the 1700-1500-~m-~ range. Elucidation of the amorphous structure of freeze-dried sugars was achieved18' by using F.t.4.r. spectroscopy. Comparison of the spectra of quenched-melt, freeze-dried, and a saturated solution of sucrose (see Fig. 10) showed that the spectra of the solution and the lyophilized sample are comparable and that they differ from that of the quenched melt, especially in the 900-800- and 1200-1000-~m-~ regions. The better resolution of peaks in these regions is the sign of a higher degree of order. This result is in good agreement with previous conclusions181based on electron diffraction and d.t.a. techniques, which indicated that, in contrast to the quenched melt, which is completely orderless, the freeze-dried sample contains zones of crystallinity. This "native" order comes from the increase of organization (175) (176) (177) (178) (179) (180) (181)

J. Southwick, Ph.D. Thesis, Case Western Reserve University, Cleveland, 1981. A. Galat, J. Koput, and J. Popovicz, Acra Biochim. Pol., 26 (1979) 303-308. A. Galat and J. Popovicz, Bull. Acad. Pol. Sci. Ser. Sci. Biol., 26 (1978) 295-300. H. R. Zelsmann and Y. Marechal, Chem. Phys., 5 (1974) 367-381. J. Pitha and R. N. Jones, Can. J. Chem., 44 (1966) 3031-3050. M. Mathlouthi and J. L. Koenig, unpublished results. M. Mathlouthi, Ind. Aliment. Agric. (Paris),92 (1972) 1279-1285.

I

ss

D

FIG. 10.-F.t.-1.r. Spectra of Sucrose in Different Physical States. [Quenched melt (QM), freeze-dried (FD), and saturated aqueous solution (SS).]

VIBRATIONAL SPECTRA O F CARBOHYDRATES

65

Wavenurnber (cm-l ) 1400

1200

900

650

700

800

MA

I

a

1

6

7

8

I

9

10

11

12

13

I

1

14

15

Wavelength (prn)

FIG. ll.-Conventional, 1.r. Spectra of Freeze-dried (FD) and Quenched-melt (QM) Sucrose, and Amorphous Maltose (MA).

in concentrated solutions, as was demonstrated6' by use of X-ray diffraction. This kind of investigation was not possible with laser-Raman spectroscopy, because of fluorescence problems; conventional i.r. spectroscopy was insufficiently sensitive to reveal small differences of order between freezedried and molten sugars, as may be seen in Fig. 11. Conventional i.r. spectra were used" for estimating the purity of a sample of D-ribose. Comparison of the infrared spectra of pure and commercial D-ribose (see Fig. 12) to an F.t.4.r. spectrum 182-L82c(see Fig. 13) shows the high degree of precision of the Fourier-transform spectrum. In particular, the band at 1280cm-', utilized" for differentiation of pure and impure specimens, appears clearly in the F.t.-i.r. spectrum. The high quality of the F.t.4.r. spectrum (see Fig. 13) is evident in the region of CH and OH stretching (3600-2600 cm-'). It was possible, by comparison of this F.t.4.r. spectrum to the Raman spectra of solid and aqueous D-ribose, to assign (182) M. Mathlouthi, A.-M. Seuvre, and J. L. Koenig, Carbohydr. Res., 122 (1983) 31-47. (182a) Studies of metal complexes'82h*' of sugars and nucleotides by F.t.4.r. spectroscopy permitted identification of chelation sites and the hydrogen-bonding changes that occur upon metalation. (182b) H.A. Tajmir-Riahi, Carbohydr. Res., 122 (1983) 241-248. (182c) H.A. Tajmir-Riahi, Specrrochim. Acra. Pari A , 38 (1983) 1043-1046.

MOHAMED MATHLOUTHI A N D JACK L. KOENIG

66

Wavenumber (cm-1)

5000 100

2

2500

1800

1400 I . , . ,

3

4

5

6

7

_ .. 1200

8

900

1000 1

I

9

Wavelength

FIG. 12.-Conventional, 1.r. Spectra of Commercial

,

1

, .

1 0 1 1

800 1

.

750

1 2 1 3

700 1

1 4 1 5

(Vm) ( 9

. .) and Purified (-) P-~-Ribose.”

the observed bands with a certain accuracy, especially for the C-H vibrations.Ia2Classically, the region between 3600 and 2600 cm-’ is not discussed, because of the broadness of the CH and OH i.r.-absorption bands. Structural study of polysaccharides and other carbohydrates in solution or in the amorphous state has been significantly enhanced through the application of Fourier-transform, infrared spectroscopy. Among the advantages of this method may be mentioned the high quality of the spectra, and the “in-house” ability to interact with the computer, so that the digitized spectra may be stored and manipulated in such a way that additional information is obtained. The application of F.t.4.r. spectroscopy in the field of carbohydrate chemistry and biochemistry is still in its infancy,182abut

1600

1400

1200

FIG. 13.-F.t.-I.r.

800

1000 wavenumber

icmd 1

Spectrum of P-D-Ribose.

600

VIBRATIONAL SPECTRA OF CARBOHYDRATES

67

the future is promising. It is very probable that this technique, and the computerized treatment of data will become as popular as was conventional i.r. spectroscopy for the pioneering structural investigations of sugars and their derivatives. V. LASER-RAMANSPECTROSCOPY

Progress in the Raman spectroscopic study of carbohydrates became possible during the past few years owing to the introduction of laser sources. Before discussing the results of laser-Raman spectroscopy applied to carbohydrates, we shall give a brief recapitulation of the physical principles of the Raman effect. Experimental techniques of infrared spectroscopy have been described in previous re~iews,'*'~*'' but no such description has been given for the Raman method. That is why the Description Section, which follows, will include the physical fundamentals of the method, as well as the sampling techniques. 1. Description

a. Physical Principles of the Raman Effe~t.'*~-When an intense beam of monochromatic light strikes a sample, there is an interaction of the radiation with matter. Usually, two broad categories of interaction, scattering and resonance, may be distinguished. Scattering of most of the photons occurs elastically (Rayleigh scattering), but a few undergo inelastic scattering. The Raman effect arises from inelastic photon-scattering by a molecule. The inelastically scattered photons have different frequencies, and produce in the scattered radiation a spectrum of frequencies that constitutes the Raman spectrum of the molecule. When a photon interacts with a molecule in an excited energy-level, it may promote a transition to the ground state, or from the ground state to an excited, vibrational-energy level. In the first case, the photon gains energy, and it loses energy in the second. As the exchange of energy occurs between the vibrational-energy levels of the molecule, which are sensitive to the chemical nature and the structure of its constitutive groups of atoms, the Raman spectrum provides a probe for the identification and characterization of such complex molecules as carbohydrates. As may be seen in Fig. 14 when the incident radiation, of frequency v,, falls on the molecule, the molecule is raised to a virtual state. The only requirement of this virtual state is that it does not correspond to an electronicenergy level of the molecule. From this virtual state, the molecule can either (183) J. L. Koenig, Appl. Specrrosc. Rev., 4 (1971) 233-305.

MOHAMED MATHLOUTHI AND JACK L. KOENIG

68

1 -1r

Energy -.-

-

-

level. v

hW0

-V I

v =1

v

=o

Rayleigh

Rarnan Stokes

R arnan

An.t i- Stokes

FIG. 14.-VibrationaLenergy Levels; Rayleigh and Raman Scattering.

emit light, of energy hv,, and return to its ground state (E,), or it can emit light of energy h( v,- v), and return to its excited level (El). The emission with no shift of frequency (at v,) is the so-called Rayleigh line. It does not provide information of interest in this context. The other emission (at v, - v ) shifted by v the energy level of the molecule. If the molecule is initially in its excited state, it can emit the photon hv,, or return to the ground state with an emission of energy h( v,+ v). The lines occurring at ( v,+ v ) are called anti-Stokes lines, whereas those appearing at lower frequencies (v, v ) are called Stokes lines. Because, statistically, a larger number of molecules always exists in the ground state at normal temperatures, the Stokes lines are more intense than the anti-Stokes. The Stokes lines are usually the lines measured experimentally. In addition to the intensity and frequency of the Raman lines, the polarization character of the lines can be measured. In fact, what led Sir C. V. Raman to believe that he was observing a new phenomenon was the unique polarization properties of this “new radiation.” Usually, the observations are made perpendicular to the incident beam, which is planepolarized, as at (a) in Fig. 15. The “depolarization ratio” p is defined as the intensity ratio of the two polarized components of the scattered light that are respectively parallel and perpendicular to the direction of the (polarized) incident beam when the polarization of the incident beam is perpendicular to the plane of propagation and observation ( p = Ill/Il-).

VIBRATIONAL SPECTRA OF CARBOHYDRATES

69

FIG. 15.-Polarization Directions of Beams in Raman Spectroscopy.

Theoretically, 0 s p s 314, depending on the nature and symmetry of the vibration. Nonsymmetric vibrations give depolarizations of 3/4. Symmetric vibrations give p ranging from 0 to 314. Accurate values of p are important for determining the assignment of a Raman line to a symmetric or an asymmetric vibration. The information given by Raman spectroscopy is complementary to that obtained from infrared s p e ~ t r a . ' ~In ~ .general, '~~ the more symmetrical the molecule, the greater the differences between the infrared and Raman spectra. Raman scattering arises easily from nonpolar groups in the molecule, because they are easily polarized, whereas polar groups that contain dipole moments absorb infrared light strongly. The complete picture of the vibrational pattern of a molecule can only be obtained by using both techniques. However, an additional interest in Raman spectroscopy arises (184) J. L. Koenig, J. Polym. Sci., Purr 0, 6 (1972) 59-177.

70

MOHAMED MATHLOUTHI A N D JACK L. KOENIG

from the weak Raman scattering of the most important and most common solvent for carbohydrates, namely, water. Only limited regions of the vibrational spectra are available in the infrared, due to the intense infrared absorption of water. Among the advantages of Raman spectroscopy when applied to the chemistry and biochemistry of carbohydrates and their derivatives may be noted the accessibility of the low-frequency modes, which are sensitive to conformational changes and the sensitivity to such homonuclear bonds as C-C, C=C, and N-N. In general, the signal-to-noise ratio for Raman spectra decreases as the molecular weight increases. This is due to the sensitivity of the Raman effect to the density of the sample. In addition to this effect, it has been found that, for polysaccharides, many vibrational modes cannot be separated; the maxima observed can involve the merging of several neighboring maxima. Other experimental advantages, and some difficulties, will be listed after description of the sampling techniques. b. Instrumentation, and Sampling Techniques.-The experimental arrangement used to excite and detect Raman spectra is relatively simple. It is presented in block form in Fig. 16. It includes a light source (currently, a laser). The laser beam passes through a narrow-band pass filter, and is Mo noc hroma to r

Photomultip liar

Sample chamber

FIG. 16.-Block

Diagram Indicating the Components of a Raman Spectrometer.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

Back transmission

Front reflection

Clear pellet transmission

Drilled pellet transmission

71

F ront-surface reflection of powder

Solution transmission in a capillary or vertical tube

FIG. 17.-Sampling Techniques for Solids and Solutions.

focused onto the sample, which is located in a sample chamber where it is correctly disposed to allow transmission or reflection (see Fig. 17) of light. The light scattered by the sample is collected by a lens and focused onto the entrance slit of a “high-performance’’ light-dispersion system which is a double or triple monochromator. The output of the monochromator is detected by a “high-performance’’ photomultiplier tube. The photoelectron pulses from the photomultiplier tube are counted, passed through a discriminator barrier, and recorded on a strip-chart recorder as a function of wavelength or frequency. The properties required of a laser as a light source are directionality, coherency, intensity, monochromaticity, and polarization. The beam can be focused to small spots, allowing the study of minute samples and surfaces. Large values of output power allow examination of dilute solutions and

72

MOHAMED MATHLOUTHI A N D JACK L. KOENIG

weakly scattering solids. Because laser beams have small half-widths and are highly polarized, the resolution of nearly overlapping bands and the accurate measurement of depolarization ratios are enhanced. The most common lasers are continuously operating, gas (Arf, Krf, or He-Ne) lasers. Typical characteristics of the lasers commonly used are summarized in Table VII. The illuminating chamber is built to facilitate the focusing of the beam on the sample. The sampling techniques used are shown in Fig. 17, the method adopted depending on the transparency of the sample. For clear, solid samples, right-angle scattering is used. With translucent specimens, a hole drilled into the pellet is often helpful. With opaque samples, frontsurface reflection is required. Powdered samples can be examined by frontsurface reflection from a sample holder consisting of a hole in the surface of a metal block inclined at 60" to the beam. For liquid samples, the laser beam may be focused into a capillary or other tube containing the sample. Capillaries are also used for powder samples. Difficulties arise when the sample absorbs the laser beam, or when fluorescence occurs (which sometimes completely obscures the Raman spectrum). One of the easiest and most successful methods for decaying any fluorescence is to expose the sample to the laser beam for a moment before recording the Raman spectrum. It is recommended that the sample be spun, in order to prevent the heat effect caused by the laser beam. Development of laser sources was followed by the use of special monochromators that can resolve the more-intense, elastically scattered light (Rayleigh line) from the weak, inelastically scattered, Raman signal. The requirement of frequency matching in the double or triple monochromators presents a challenging, coupling problem for frequency-scanning systems. For detection of the small number of scattered photons, modern photomultiplier tubes having low internal noise and high gain are used. The amplification method employed is generally direct-current amplification.

TABLEVII Characteristics of Lasers Used in Laser-Raman Spectroscopy

Laser He-Ne Kr+ Ar+

Wavelength (om)

Typical output power (mW)

632.8 647.1 488.0 514.5

65 200 500 500

VIBRATIONAL SPECTRA OF CARBOHYDRATES

73

The photoelectron pulses from the phototube are detected and processed individually through an adjustable gating system, and subsequently integrated over a predetermined period of time. The output is recorded, or treated by a computer. Among the experimental advantages of laser-Raman spectroscopy may be mentioned the ease of sampling, the small amounts (down to 1 mg) of sample required, and the use of aqueous solutions and glass holders without any disadvantage to the Raman spectrum. As the Raman-scattering intensity is a linear function of the concentration of the scattering species, low concentrations of impurities do not generally interfere with the spectrum of the specimen studied. Finally, in Raman spectroscopy, the complete frequency range (4000-10cm-') can be obtained in a single run on one instrument, which is not the case for infrared spectra. Some of the experimental difficulties of Raman spectroscopy are associated with fluorescence, the heat generated by the intense laser beam, and, sometimes, the alignment of the scattered radiation. 2. Aqueous Solutions

Raman spectroscopy has played a role in the understanding of the structure of sugars in aqueous solutions. The ease with which the spectra are obtained allowed the early investigation^'^ to be carried out for aqueous solutions. The primary advantage of Raman spectroscopy over i.r. spectroscopy is the low interference of liquid water; the 2000-200-cm-' region of the vibrational spectrum is completely accessible with either D 2 0 or H20. Thus, aqueous solutions of carbohydrates can be studied for isomeric equilibria, H-bonding, and conformational changes. The effect of such physical factors as concentration, temperature, and pH may be investigated. However, the OH-stretching region, from 3800 to 2800 cm-', gives rise to a broad band, from the water molecule, which completely obscures the carbohydrate OH bands, and renders the Raman spectra of aqueous solutions of sugars difficult to interpret. Differences between aqueous D-fructose on the one hand, and aqueous D-glucose and sucrose on the other, have been in the 1700-100cm-' region. As may be seen in Fig. 18, the spectrum background of D-fructose solutions exhibits only a maximum at 1640 cm-I, corresponding to the bending of H 2 0 , whereas the spectra of D-glucose and sucrose solutions show, besides the bending band of HzO, a staggered band between 1000 and 400 cm-I, probably due to the librations2 movement of HzO. The differences are attributed to the molecular interactions between the aqueous solvent and the sugars. These interactions are probably H-bonds of various natures (water-sugar, sugar-sugar, or water-water) and strength.

74

MOHAMED MATHLOUTHI AND JACK L. KOENIG

S

1

.

1

1

1

1

1

1

1

,

1

1

1

1

1

Fru

1700

1500

1300

1100

900

700

500

300

W a v o n u m b o r (crn-l)

FIG. 1 8 . 4 e n e r a l Aspect of Laser-Raman Spectra of D-Fructose (Fru), D-Glucose (Glc), and Sucrose (S) in Aqueous Solution. (The curve - . - indicates the H,O libration background.)

VIBRATIONAL SPECTRA OF CARBOHYDRATES

75

Comparison of aqueous solutions and solid samples of 1-thio-P-Dhexopyranosides was made."' It was observed that the carbohydrate bands were broadened in aqueous solutions, due to interaction of the hydroxyl groups of the carbohydrate with water. Moreover, bands at 1659 cm-' from the in-plane vibration of an N-acetyl group, or at 1650 cm-' from a carboxyl group, are completely masked by the water band at 1648 cm-'. This led the authors185to suggest that analysis of N-acetyl and carboxyl groups in a carbohydrate must be conducted with a solid sample, as the conclusions obtained from aqueous solutions are less reliable. However, it may be observed that increase of concentration of the aqueous solution, or curvefitting treatment of the overlapped band at 1648 cm-', could be helpful in solving this problem. Similarities were observed1n4between the spectra of carbohydrates in aqueous solution and in the solid state. In both cases, the spectra of D-glucose, maltose, cellobiose, and dextran were found similar in the region of 1500-700 cm-', but below 700 cm-', each carbohydrate examined has distinct features in its Raman spectrum. The similarity in the spectra in the 1500-700-~m-~ range is quite important, as it indicates that, in this region, assignments for these carbohydrates will be relevant in studies of such polymers of D-glucose as cellulose and amylose. The region of the Raman spectrum below 700 cm-' has potential as an identification for the molecules studied. This is particularly valuable in view of the difficulties encountered in this region with the infrared spectra of aqueous solutions. The assignment of frequencies in aqueous solutions is possible by use of deuteration methods that allow differentiation of COH, CH2, and ring modes, especially in the crowded region of 1500-1200cm-'. Most of the Raman results on carbohydrates were obtained with aqueous solutions. 3. Results

Compared to the extensive Raman investigations of nucleic acid and protein structures, relatively few studies of carbohydrates have been made by laser-Raman spectroscopy. Laser-Raman spectra of some mono- and di-saccharides28.~43."47.~82.'85 have been obtained. Generally, the Raman study of monomers and dimers of sugars, having known structures and geometries, has had as its objective the elucidation of the conformation of the corresponding polysaccharides. One of the earliest application^^^'" of laser-Raman spectroscppy in the carbohydrate field was the detection of the C=N vibration in such spectra of oximes, at 1665-1650cm-'; in the (185) A. T. Tu, J. Lee, and Y. C. Lee, Carbohydr. Rex, 67 (1978) 295-304. (185a) D. Horton, E. K. Just, and B. Gross, Carbohydr. Res., 16 (1971) 239-242.

76

MOHAMED MATHLOUTHI AND JACK L. KOENIG

i.r. spectra, these compounds show weak or negligible absorptions. LaserRaman spectra have also been used for identifying, and investigating, the different forms of the same sugar. 147*182*'86-188 An analysis of the results of laser-Raman spectroscopy, and a summation of the assignments proposed, will be made for the mono- and di-saccharides on the one hand, and for the oligo- and poly-saccharides on the other. a. Mono- and Di-saccharides-Laser-Raman spectra of ~ - f r u c t o s e , ' ~ ~ D-glucose, and sucrose187at different concentrations in water were recorded, and assignments of the main frequencies observed were proposed. These assignments were based on earlier work on the vibrational spectra of sugars, the physical properties of the aqueous solutions, and determination by other techniques of the composition of D-fructose and D-glucose solutions as regards different isomers. The point of view adopted in this was that of the biologist, for whom it is less important to know the contribution, to the Raman line, of each of the modes of vibration than the assignment of the most probable vibration to that line. The assignments proposed for the bands observed in the laser-Raman spectrum of a 20% aqueous solution of D-fructose are shown in Table VIII. As the Raman intensity is proportional to the mass of the scattering molecules, it was possible to calculate the proportions of the fructofuranoses and fructopyranoses from the ratio of intensities of characteristic modes of vibrations. The C-C vibration was found to be one of the most characteristic. The furanoid ring, which is the more compact, was presumed to have a higher internal energy, and higher frequencies, for the same modes of vibrations, than the pyranoid. Assignment of v(C-C) at 874cm-I for the furanoid ring, and at 826 cm-' for D-fructopyranose, was proposed. The ratio 1(874)/1(826) = 0.69 : 1 gave a proportion of 59% of pyranoses and 41% of furanoses, which is good agreement with the results obtained by other techniques. The 3700-2700-cm-' region is less easy to interpret than the region below 1700 cm-'. Nevertheless, comparative study of intensities of the C-H vibrations permitted differentiation of D-fructose from D-glucose and sucrose. It was found'87 that the asymmetrical vibration vas(C-H) for CH2 in Dfructose is stronger than the symmetrical vibration; the opposite was observed for D-glucose, and the spectrum of sucrose exhibits almost the same intensity for the two vibrations. This result (see Fig. 19), which could

(186) H. Susi and J. S. Ard, Cnrbohydr. Res., 37 (1974) 351-354. (187) M. Mathlouthi and D. V. Luu, Cnrbohydr. Res., 81 (1980) 203-212. (188) A. T. Tu, W. K. Liddle, Y. C. Lee, and R. W. Myers, Carbohydr. Res., 117 (1983) 291-297.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

77

TABLEVIII Bands Observed" in the Laser-Raman Spectrum of D-Fructose in Aqueous Solution cm-'

I

P

1640 1460 1376 1266 1186 1150 1086 1068 986 922 874 826 712 636 530 460 428 344

17 65 29 59 19 27 76 84 24 16 54 78 33

0.52 0.69 0.35 0.67 0.67 0.66 0.47 0.36 0.57 0.64 0.10 0.12 0.12 0.20 0.46 0.42 0.43

lOOb

41 23 41 12

0.25

3449

187

0.26

3267

185

0.12

2988 2946 2908 2800

54.5 lOOb

55.5 10

0.31 0.19 0.27 0

Assignments

u,(OH) in (H,O) u,(OH) in (H,O) u,(OH) in (CH,OH) u,(OH) in (CH,OH) 4CH) u,(CH) in (CH,) u,(CH) in (CH,) combination of S(CH2)+ 4 C H A

Key: I = relative intensity; p = depolarization ratio; us= symmetrical stretching-mode; uas= asymmetrical stretching-mode; S = bending mode; and 7 =twisting mode. Taken as reference.

be important from an analytical point of view, was a t t r i b ~ t e d "to ~ different local orientations of the CHzOH groups in the three sugars. Assignments of the frequencies observed1" in the spectra of D-glucose in aqueous solutions permitted determination, from the ratio of intensities of characteristic modes of vibrations, of the proportions of a and p anomers at equilibrium, which were 32 and 68%, respectively. The particular behavior (189) M. Mathlouthi, Dr. Sciences Thesis, University of Dijon (1980).

S

F ru

I

. . . . . , . , 3700

3500

3300

3100

I

2900

l

l

2700

Wavenumber (crn.1 ) FIG. 19.-Laser-Raman Spectra of D-Fructose (Fru), D-Glucose (Glc), and Sucrose (S) in the 3700-2700-cm-' Region. (Note the relative intensities of the C-H stretching-vibrations.)

VIBRATIONAL SPECTRA OF CARBOHYDRATES

79

of each of the a- and P-D-glucoses in the aqueous environment (short and long-range hydration) was discussed. This behavior particularly affected perturbation in its variation the depolarization ratio, p, which with concentration for the modes assigned to the P anomer. The results of a study of the laser-Raman spectra of sucrose are summarized in Table IX. The most important features observed'87 when the concentration was varied consisted in shifts of the frequencies of the CHIOH group, which are sensitive to intra- and inter-molecular hydrogen-bonding. In addition, the association of D-glucose with D-fructose that leads to the TABLEIX Bands Observed" in the Laser-Raman Spectrum of Sucrose in Aqueous Solution cm-'

I

P

1628 1456 1366 1340 1266 1130 1110 1064 920 836 746 640 600 548 528 470 456 416 374

164 33.6 45.7 37.8 25 68 60 89 25

0.56 0.93 0.67 0.72 0.71 0.53 0.20 0.3 1 0.34 0.10 0.38 0.24 0.45 0.25 0.17 0.10 0.23 0.84 0.25

S(H0H) S(CH2) w(CH2) r(CH2) dCH2) S(C0H) u( C - 0 ) endo u(C-0)exo S(C-H) l4C-C) S(CC0)endo Fru S(CC0)exo Fru S(OC0,) S(CC0)endo Glc S(CC0)exo Glc S(CCC) FN S(CCC) Glc S(0-H-0) S(C0C)

0.27 0.23 0.18 0.12 0.24

v,(C-H) in CH2 v,(C-H) in CH, u(C-H) Fermi resonance of ( H 2 0 ) u(OH) sucrose ua(OH)H2O

2912 2944 2982 3272 3324 345 1

lOOh

21 35.7 31.4 60.7 69 38 36.4 17.8 40 98.9 lOOb

66.3 168.4 shoulder 186.7

Assignments

Key: I = relative intensity; p = depolarization ratio; us= symmetrical stretching mode; u,, = asymmetrical stretching-mode; 7 =twisting mode; S = bending mode; r = rocking mode; w = wagging mode; endo = endocyclic; ex0 = exocyclic; Fru = D-fructosyl group; Glc = D-glucosyl group. Taken as reference.

80

MOHAMED MATHLOUTHI A N D JACK L. KOENIG

formation of the sucrose molecule was found to have a most important effect on the structure of the D-glucosyl part. The vibrations from the D-glucosyl ring exhibit shifts of -12 cm-’, whereas no important modification of the D-fructosyl modes was observed. The major differences between the spectra of sucrose and the monosaccharides that form it are found below 1600 cm-I, in the region of the glycosidic-ring vibrations. The frequencies of the vibrations S(C-C-C) and S(C-0-C) were found to be higher for sucrose than the same modes in the monosaccharides. This was interpreted as due to an increase of the energy of the vibrations after the “dimerization,” which acts more by virtue of the higher cohesion in the disaccharide than by the mass effect (which normally leads to lower frequencies). Comparison o f the Raman spectra of crystalline a-D-glucose and its aqueous solution revealedZSthat the differences in frequency (between the solid and the solution) are less than the experimental error (*4 cm-I). This suggested that there is little or no intermolecular coupling between the vibrational modes of the four molecules in the unit cell of the crystal. The in the 3000-2800-cm-’ and Raman spectrum of P-D-glucose was 1500-100-cm-’ regions. The Raman lines observed were sharp and well resolved, and they permitted verification of most of the calculated frequenof the spectra of D-glucose, cellobiose, and maltose was cies. A made in order to elucidate the structure of polysaccharides of the D-glucan type. Identification of the bands due to C-0-H and C-H deformations was obtained by use of deuteration. Thus, the Raman lines at approximately 1349, 1071, 1021, and 913 cm-’ were assigned to modes related to C-0-H groups, and those at 1404, 1360, 1250, 1076, 1047, 911, and 836 cm-’, to C-H-related modes. The Raman spectra of a-lactose monohydrate, P-lactose in the crystalline state, a-lactose-&lactose mixture, and equilibrated lactose in aqueous solution have been investigated.’s6 It was found that the spectra are very sensitive to small structural changes, and this suggested that Raman spectroscopy should be used as a method for identification of closely related isomers. Model molecules for hyaluronic acid, an important biological polymer found in synovial fluid, skin, umbilical cord, and connective tissue, were studied’” by use of laser-Raman spectroscopy. The spectra of a-D-glucose, D-glucuronic acid, 2-amino-2-deoxy-~-glucoseHCI, and 2-acetamido-2deoxy-a-D-glucose were compared, and the vibrations due to C02H, -CHz, NH:, and =NH groups identified. Assignments of the observed frequencies were based on the deuterium-exchange technique. The same method was

-

(190) C. Y. She, N. D. Dinh, and A. T. Tu,Biochim. Biophys. Acla, 372 (1974) 345-357.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

81

for the study of methyl a- and P-D-glucoside and hyaluronic acid. The methylated monomers were used in order to investigate the glycosidic linkages present in hyaluronic acid. An extensive discussion of the differences observed in the spectra, especially the 842- and 890-cm-' lines, respectively characteristic of a and P anomers, was given. The heteropolymer hyaluronic acid was found'" to contain only P-glycosidic linkages, as the 840-cm-' line was absent from the spectrum, and the 896-cm-' line was very distinct. Considerable differences in the v ( 0 H ) region were interpreted as due to the different types of hydrogen bonding in the anomers studied. Laser-Raman spectroscopy was also applied'85 to the study of 1-thio-@-Dhexopyranosides. These sugar derivatives may be used as models for celladhesion studies, for induction of glycosidases, or for affinity chromatography. The glycosidic linkage in these components involves a sulfur atom instead of the glycosidic oxygen-atom, and this is of interest in the study of the characteristic, anomeric lines. It was found that all 1-thio-P-glycosides investigated exhibit a distinct band at 891 f 7 cm-', which is characteristic of an axial C-H deformation and is independent of whether an oxygen or a sulfur atom is attached to C-1. Raman spectroscopy has several inherent advantages over i.r. spectroscopy, among which is the fact that some modes, such as C-N, C=S, and S-H vibrations, yield strong Raman lines, whereas they give rise to very weak i.r. absorption. This property was utilized'88 in order to examine the C=N stretching vibrations of per- O-acetylated aldohexopyranosyl cyanides having 1,2-truns and 1,2-cis configurations. It was possible to correlate the value of v(C=N) with the stereochemistry of the anomeric cyano group. A was published that dealt with the Raman spectra of seven monosaccharides, two disaccharides, one trisaccharide, and three polysaccharides in phosphate-buffered solutions whose pH values were varied from 6.0 to 8.5, at a constant ionic strength of 0.1, and in various HCl solutions of pH 0.8 to 5.0. Of the thirteen sugars studied, only fructose 1,6-bisphosphate (FruP,) displayed changes in band height with change in pH. The ratio of the l-phosphate to the 6-phosphate group was derived from the ratio of the 982- to 1080-cm-' bands. It was found'92 that the phosphate groups of FruP2 are hydrolyzed at pH 0.8, and that the other sugars examined do not exhibit degradation in the whole range of pH, except at pH 8.0. b. Oligo- and Poly-saccharides.-Vibrational modes of carbohydrate polymers are complex. That is why most of the spectroscopic work on polysaccharides has been based on examination of the vibrational modes of simpler carbohydrates. In this context may be mentioned a laser-Raman spectro(191) A. T. Tu, N. D. Dinh, C. Y. She, and J. Maxwell, Stud. Biophys., 63 (1977) 115-131. (192) T. W. Barrett, Specrrochim. Acra, Part A, 37 (1981) 233-239.

MOHAMED MATHLOUTHI A N D JACK L. KOENIG

82

scopic studyIg3of cyclomaltohexaose and related compounds. In this work, careful examination of the spectra of maltose, maltotriose, and cyclomaltohexaose permitted the suggestion that the trisaccharide could have a fixed conformation similar to half of the conformation of the hexaose. An investigation of the polymorphic forms of a m y l ~ s e ,and ' ~ ~a structural study of dextran [( 1 + 6)-a-~-glucan],used Raman spectroscopy, and the Raman spectra of cyclomalto-hexa- and -heptaose were also r e ~ 0 r d e d . I ~ ~ Most of the vibrational modes observed in the amylose spectrum were assigned to vibrations occurring in the individual residues, with only a few additional lines arising from coupling of modes between residues. Some of the additional lines, such as that at 940 cm-', are coupled modes involving cooperative vibrations of the glycosidic oxygen-atom and the ring atoms. The change in frequency of the line at 940 cm-' was correlated with extension of the helix. The frequency observed for the skeletal mode was 949, 946, and 936 cm-' for cyclomaltoheptaose, and V- and B-amylose, respectively. The shifts in frequency are consistent with the conversion of V- into Bamylose by extension of the 61 helix. The experimental data obtained by using the Raman effect are consistent with the normal-coordinate analysis of V-amyl~se.~'Raman spectra of amylose have also been studied for in Me,SO-d,. It was observed that the V form is absent from the solution; this is interesting, as the V structure is formed when films are cast from this solvent. Characteristic lines of B-amylose in the solution were observed at 1254 and 1334 cm-', probably because of the similarity between the hydrogen bonding of the CHzOH group in the solvated, random amylose and in B-helices. Progress in the i.r. and Raman spectroscopy of polysaccharides was reviewed by B l a ~ k w e l l . 'In ~ ~this review, work on polymorphic forms of amylose, on oriented films of glycosaminoglycans, and on bacterial polysaccharides was summarized. The usefulness of the vibrational spectra in determining the conformations of polysaccharides was shown. Changes in the conformation of the different forms of cellulose have been investigated 157.1 58,195,196 by use of Raman spectroscopy. Celluloses I and I1 were found"' to have different, and distinct, molecular-chain conformations. No assignments of the frequencies were proposed, but the correlation between the spectra and the structure of celluloses was discussed. The major differences in the Raman spectra were observed below 800cm-', in the (193) (194) (195) (196)

A. T. Tu, J. Lee, and F. P. Milanovich, Carbohydr. Res., 76 (1979) 239-244. J. Blackwell, Am. Chem. Soc Symp. Ser., 45 (1977) 103-113. R. H. Atalla and B. E. Dirnick, Carbohydr. Res., 39 (1975) 61-63. R. H. Atalla, R. E. Whitmore, and C. J. Heimbach, Macromolecules, 13 (1980) 17171719.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

83

skeletal and ring-vibrations region. The features occurring between 1500 and 800 cm-', which are due to -CH2, -CH, and -COH deformations differ primarily in relative intensity. The changes in the spectra upon conversion from cellulose I into I1 have been used'95 as the basis for developing an index of the degree of conversion in partial-mercerization experiments. Orientation in native cellulose fibers was derived'96 from the modification of the Raman intensities in the spectra of single fibers, recorded with changes in the polarization of the incident, exciting radiation. The native-cellulose chains were found to have a coherent arrangement wherein the molecules possess a lamellar organization, rather than the fibrillar aggregation generally accepted. The Raman spectra of 2-acetamido-2-deoxy-~-glucopyranose (GlcpNAc), its oligomers, and its p -(1 +4 ) polymer, chitin, were re~ 0 r d e d . Assignments I~~ of the observed frequencies were proposed. Interpretation of the Raman and infrared'76 spectra, in the amide-vibrations region, from 1700 to 1600cm-', led to the conclusion that chitins from Antarctic krill and crayfish have the so-called a structure, and that oligomers of GlcpNAc form crystals similar to those of a-chitins. Raman scattering was also used in a study of branched poly~accharides.'~~ Comparison of the i.r. and Raman spectra of starch and glycogen was made for the whole range of frequencies (4000-50 cm-'). Hydrogen bonding and hydration of these polysaccharides were discussed. It was suggested that, in starch, all of the hydroxyl groups are, essentially, randomly solvated, and exhibit wide, solvated, broad i.r. bands, whereas, in glycogen, the anisotropically solvated OH groups give rise to distinct i.r. bands in the 1050-970-cm-' region. It was proposed that this region could serve as an approximate criterion of polysaccharide h eter ~ g e n e ity .'~ ~ c. Other Results.-A Raman-spectral approach to investigation of the complex-formation of cations of Group 11, and of borate anions, with saccharides was adopted by Williams and Atalla.'99 These authors took, as models of nonionic polysaccharides, such different polyols as ethylene glycol, cyclohexanediols, 1,5-anhydro-~-ribitol,and a number of inositols. The model systems in aqueous solution were examined by Raman spectroscopy in the presence and absence of the ions. Changes occurred in the spectra, especially in the 1300-800-~m-~ region, that were interpreted as due to chelation of the polyols. Different levels of interaction were observed. Low-energy interactions occurred with a favorable configuration of hydroxyl

(197) A. Galat and J. Popowicz, Bull. Acad. Pol. Sci., Ser. Sci. B i d , 26 (1978) 519-524. (198) A. Galat, Acra Biochim. POL, 27 (1980) 135-142. (199) R. M. Williams and R. H. Atalla, Am. Chem. SOC.Symp. Ser., 150 (1981) 317-330.

84

MOHAMED MATHLOUTHI AND JACK L. KOENIG

groups, and resulted in small changes of the spectra, suggesting there were only minor modifications of the polarizabilities of the adjacent OH groups. High-energy interactions took place with borate ions, and provoked changes in the relative intensities of the bands. It was suggested’99that new molecular species were formed, giving highly coupled, Raman vibrations. Calcium complexes of sucrose were studied”’ by Raman spectroscopy. Comparison of the spectra permitted differentiation of two types of complexation according to the method of preparation, which consisted either in adding CaO to a sucrose solution, or in making neutral, with NaOH, a solution of sucrose plus CaC12. Raman-spectral studies of cerebrosides in the solid and gel phases have been reported.201 Assignments of frequencies, and comparison of peak heights for characteristic vibrations, allowed elucidation of the conformation of both the chain and head-group portions of these molecules. Interpretation of the spectral data was found in agreement with calorimetric and X-ray structural data. The resonance Raman effect, which is characterized by an enhancement of the normal Raman intensity when the exciting radiatidn approaches an electric absorption band of the scatterer, was applied”’ to the investigation of the iodine complexes of amylose and agarose, and to other iodinecontaining complexes. The iodine chain-length in the amylose complex was calculated from the spectral results to be -28 atoms. The inclusion matrix of the iodine chain was found not to have an observable effect on the Raman spectra of the complexing polymers. The iodide-starch complexes were also studied by Handa and coworkers,203using resonance Raman spectroscopy (r.R.s.). Study of the vibrations in the low-frequency region that are associated with the D-ribosyl ring in nucleosides was achievedzo4 with F.t.4.r. and Raman spectroscopy. Numerous other investigations of nucleosides, nucleotides, and nucleic acids have been conducted by use of laser-Raman spectroscopy; this work has been reviewed by different authors. 136*205-207 The spectral (200) C. Francotte, J. Vandegans, D. Jacqmain, and G. Michel, Sucr. Belge, 98 (1979) 137-144. (201) M. R. Bunow and I. W. Levin, Biophys. J., 32 (1980) 1007-1022. (202) M. E. Heyde, L. Rimai, R. G . Kilponen, and D. Gill, J. Am. Chem. SOC.,94 (1972) 5222-5227. (203) T. Handa, H. Yajima, and T. Kajiura, Biopolymers, 19 (1980) 1723. (204) C. P. Beetz, Jr., and G. Ascarelli, Spectrochim. Acta, Part A, 36 (1980) 525-534. (205) W. L. Peticolas, Ado. Raman Specrrosc., 1 (1972) 285-295. (206) K. A. Hartmann, R. C. Lord, and G. J. Thomas, Jr., in J. Duschesne (Ed.), Physical and Chemical Properties of Nucleic Acids, Vol. 2, Academic Press, London, 1973, pp. 1-89. (207) P. R. Carey and V. R. Salares, Adu. Infrared Roman Spectrosc., 7 (1980) 1-58.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

85

information found in these reviews on the sugars of nucleic acids was discussed in a studyIE2of D-ribose and 2-deoxy-~-erythro-pentose;it proved possible to identify, from the spectral results, analogs of the sugars studied, and to point out the influence of the aqueous environment on their structures. It may be concluded, from the analysis of the Raman results, that the information provided by Raman spectroscopy is, in essence, similar to that of infrared spectroscopy. The exploitation of the data, namely, the frequencies and intensities due to the molecular vibrations, is of a certain benefit in giving some insight as to the conformations of carbohydrates, and their interactions with the environment. As laser-Raman spectroscopy is applicable to solids, as well as to aqueous solutions, the linear relationship between Raman intensities and mass concentrations, and the specificity and high quality of the spectra experimentally obtained, make this technique particularly promising in investigations of the chemistry and biochemistry of carbohydrates.

VI. CURRENT PROBLEMS One of the most important problems that has been actively studied during the past few years is the hydration of biological molecules, especially carbohydrates, and the effect of hydration on the conformation of the solute molecule, as well as the effect of the latter on the “water structure.” Different theoretical and experimental methods have been utilized, and the discrepancies between the results, expressed as numbers of hydration, are considerable. In addition, the water molecule is a reactant in a number of biochemical reactions. The kinetics of these reactions is influenced both by the conformation of the carbohydrate and the structure of the water. These questions will be discussed, with particular reference to the contribution of the vibrational, spectroscopic information to an understanding of such complex mechanisms. 1. Water and Aqueous Solutions

Vibrational, spectroscopic studies of water and carbohydrate solutions have been performed, in order to provide information on the nature and variety of hydrogen bonding between molecules (see Sections II,3 and V,2). It is generally accepted2087209 that i.r.- and Raman-spectral results concerning (208) N. J. Hornung, G . R. Choppin, and G . Renovitch, Appl. Spectrosc. Rev., 8 (1974) 149- 18 1. (209) G . E. Walrafen, in W. A. P. Luck (Ed.), Structure of Water and Aqueous Solutions, Verlag Chemie, Marburg, 1973, pp. 300-321.

MOHAMED MATHLOUTHI AND JACK L. KOENIG

86

water structure are controversial. Indeed, vibrational spectroscopists seem to be divided in advocacy of mixture2” or continuum’” models of water structure. The effect of sugars on water structure has often been designated by the term “structure maker.” This concept was found’” misleading when applied to infrared-spectral studies of aqueous solutions, and it was suggested that it should be discontinued. However, in the solvation of D-glucose and sucrose, studied’I3 by infrared spectroscopy, an enhancement of the water structure by these solutes was shown. The ratio of the integrated Raman intensities of the 175-152-cm-’ ~’~ of bands for water and -2 M sucrose solution ~ e r m i t t e d demonstration the “structure making” effect of sucrose on water. The investigation of solute-solvent interactions in aqueous solutions of D-fructose, D-glucose, and sucrose by comparison of characteristic Raman frequencies of water and the sugar components was achieved!’ It was shown that variation of the ratio of the intensities of these characteristic bands as a function of the concentration cannot be interpreted in a simple way. The hydrogen bonds that may occur have various natures and strengths. 1.r. absorption intensity and the frequency of v ( 0 - H ) stretching vibrations give some insight as to the hydrogen bonding in water and aqueous solutions. Thus, when OH stretching bands undergo a diminution of intensity and an upward shift of peak frequency, this indicates a weakening of hydrogen bonding.’” A shift of 40 cm-’ of the OH stretching-vibration was observed’16 in the spectrum of a solution of dextran in D 2 0 containing 3.5 M KCI, revealing a decrease in the energy of the hydrogen bonds and a conformational change of the polysaccharide. Consequently, vibrational spectra, and, particularly, Raman data on the shape, the intensity, and the frequency of OH bands, should be helpful in the elucidation of weak-energy hydrogen-bonds in liquids.*”

2. Molecular Structure The vibrational spectra of carbohydrates were summarized in the preceding Sections with reference to molecular structure. It was especially shown G. E. Walrafen, J. Chem. Phys., 48 (1968) 244-251. T. T. Wall and D. F. Hornig, J. Chem. Phys., 43 (1965) 2079-2087. S. E. Jackson and M. C. R. Symons, Chem. Phys. Letf., 37 (1976) 551-552. 0. D. Bonner and G. B. Woolsey, J. Phys. Chem., 72 (1968) 899-905. G. E. Walrafen, J. Chem. Phys., 44 (1966) 3726-3727. M. Falk and H. R. Wyss, 1. Chem. Phys., 51 (1969) 5727-5728. V. P. Panov, A. M. Ovsepbjan, V. V. Kobyakov, and R. G. Zhbankov, Zh. Prikl. Specfrosk, 29 (1978) 62-68, Engl. Transl. (1979) 803-808. (217) J. P. Perchard, C. Perchard, A. Burneau, and J. Limouzi, 1. Mol. Sfnrcf., 47 (1978) (210) (211) (212) (213) (214) (215) (216)

285-290.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

87

that i.r. and Raman results may be interpreted in terms of handedness, orientation, and degree of crystallinity of saccharides. The geometry of hydrogen bonding in carbohydrates is still difficult to determine precisely. However, the use of X-ray diffraction data, together with infrared results, permitted Kanters and coworkers218to elucidate the geometry of the hydrogen bonding in p-D-fructopyranose. Raman and infrared spectra in the 0 - H stretching region permitted33identification of four bands corresponding to four 0 . 0 distances obtained from X-ray analysis of a 3,6-dideoxyp- o-hexopyranoside monohydrate. In order to improve the information provided by the i.r. or Raman bands, or both, in the region of OH stretching, these broad bands must be fitted with their Gaussian or Gaussian-Lorentzian components. This operation necessitates use of high-quality spectra, and computer treatment of the data. The techniques of Fourier-transform, infrared and laser-Raman spectroscopy described herein are readily computerizable, and could provide precise insight into the molecular structure of carbohydrates. Different models of curve fitting were u ~ e d ” ~ *in* treat’~ ments of carbohydrate spectra. One of the most promising fields of application of computer-aided, spectroscopic techniques is study of the hydration of saccharides.

- -

3. Molecular Interactions The stability of a conformation and the interactions of carbohydrates are largely influenced by water. The term “interactions” is generally utilized to designate the inter- and intra-molecular hydrogen-bonding that occur between water and the sugars. One of the most studied manifestations of these molecular interactions is hydration. It has been shown,220by techniques other than i.r. and Raman spectroscopy, that sugars are capable of short- and long-range hydration. The short-range order of water molecules in the immediate vicinity of the carbohydrate molecule is called the “hydration shell.” The number of water molecules in the hydration shell is an intrinsic property of the sugar. A number of hydration of 21 for sucrose, and of 10 for D-glucose, were found2” from the i.r. spectra of these sugars at 25”, compared to the i.r. spectrum of water at higher temperatures. The same method of determination of hydration numbers was applied221to eight different sugars at 25”. The influence of temperature and concentration on the hydration number (218) J. A. Kanters, G. Roelofsen, B. P. Alblas, and 1. Meinders, Acra Crysfallogr., Seer. B, 33 (1977) 665-672. (219) D. K. Buslov and L. J. Brazhnik, Zh. Prikl. Spekrrosk., 36 (1982) 157-159. (220) M. A. Ryazanov, Zh. Fiz. Khim., 52 (1978) 1313-1315. (221) J. L. Hollenberg and D. 0. Hall, J. Phys. Chem., 87 (1983) 695-696.

88

MOHAMED MATHLOUTHI AND JACK L. KOENIG

was studied. However, this procedure for obtaining the hydration numbers has been criticized,222because it involves contradictory assumptions as regards the species of water of hydration. Carbohydrates form relatively few stable hydrates in the solid state, despite their high solubility.223 The long-range order of water in the presence of saccharides plays an important role in the understanding of their properties. The effect of sugars on the water structure seems to be specific for each sugar. Indeed, a laserRaman, spectroscopic study of the effect of traces to lo-*, w/w) of D-fructose, D-glucose, and sucrose on the structure of water allowed224the conclusion that D-fructose shows a behavior different from that of the other sugars. Whereas D-glucose and sucrose enhance the water-water association, D-fructose was found to have a structure-breaker effect. 4. Structure-Properties Relationships

In order to understand the properties of carbohydrate molecules, not only in terms of chemical reactivity, but particularly their physiological aspects (for example, elucidation of the sweet taste of some members) and physical properties, it is necessary to take into account their configuration and structure. Vibrational spectroscopic information is particularly useand ful in this field. The results on the c o n f ~ r m a t i o n ~of ~ . amylose ’~~ amylopectin, for example, could be utilized to explain their industrial properties, such as thickening, or water retention in certain food-processes. The laser-Raman r e s ~ l t s on ~ ~D-fructose, .~~~ D-glucose, and sucrose in aqueous solutions were usedz2’ as a basis for a molecular interpretation of their relative sweetness. The role of water in the intensity and duration of the sweet-taste sensation was derived from the water structure induced by the sugars, and determined by the intensity and depolarization ratio of the Raman OH bands. The hydrolysis of sucrose catalyzed by invertase was found226sensitive to the “folding” of the molecule when the concentration was increased. The intramolecular hydrogen-bonds of sucrose, and the state of association of water, that helped in understanding the mechanism of reaction were given by X-ray6’ and I a ~ e r - R a m a ninvestigations ~~ of sucrose solutions at different concentrations. The “structure-breaker” effect of D-fructose revealed by the Raman spectra of its dilute solutions could explain the fact (222) J. Jayne, J. Phys. Chem., 87 (1983) 527-528. (223) G . A. Jeffrey, Acc. Chem. Res., 2 (1969) 344-352. (224) M. Mathlouthi and D. V. Luu, Absrr. Int. Symp. Carbohydr. Chem., XIrh, Vancouver, B.C., Canada, 1982, 11. 31. (225) M. Mathlouthi, Food Chem., 13 (1984) 1-16. (226) D. Combes, P. Monsan, and M. Mathlouthi, Carbohydr. Res., 93 (1981) 312-316.

VIBRATIONAL SPECTRA OF CARBOHYDRATES

89

that this sugar is not a cryoprotector, whereas D-glucose and sucrose have a cryoprotective effect on frozen, living cells. Many other examples demonstrating clearly that the elucidation of the structure helps in understanding the properties of carbohydrates could be given. However, the intra- and inter-molecular interactions in carbohydrates are complex. For their determination, they need application of different techniques, among which, the computerized spectroscopic techniques seem to be particularly interesting. From the point of view of efficacy, it is desirable that an effort be made to achieve cooperation between structuralists working on theoretical calculations and improvement of spectroscopic techniques, and phenomenologists describing the chemical and biochemical behavior of carbohydrates, and their structures. This wish could be realized were an increasing number of investigations on the biological and technological properties of carbohydrates supported by vibrational-spectroscopic and normal-coordinate results.

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ADVANCES IN CARBOHYDRATE CHEMISTRY A N D BIOCHEMISTRY, VOL. 44

MONOSACCHARIDE ISOTHIOCYANATES AND THIOCYANATES: SYNTHESIS, CHEMISTRY, AND PREPARATIVE APPLICATIONS

BY ZBIGNIEW J. WITCZAK Department of Biochemistry, Purdue University, West Lafayetre, Indiana 47907

I. INTRODUCTION Considerable attention has been directed toward the synthesis of various types of heterocyclic derivatives of sugars, among them, nucleoside analogs'** having potential, antibacterial and antitumor proper tie^.^ Although several synthetic approaches have been developed, only a few appear to have sufficient versatility for the construction of a variety of heterocyclic systems. Among these are approaches that employ heterocyclic elaboration upon sugar isothiocyanates: Thiocyanates' and isothiocyanates6 are important reagents in heterocyclic chemistry, and undergo many reactions, such as nucleophilic additions and cycloadditions. The reactions of isothiocyanates with various nucleophiles indicate the strong electrophilic character of the -NCS group. The electron-withdrawing strength of the carbon atom in the -NCS group is most important for these reactions. Nucleophiles attached to a labile hydrogen atom that is able to protonate

a nitrogen atom can react with isothiocyanates, whereas the electronegative residue bonds to the carbon atom of the -NCS group. (1) S. Hanessian and A. G. Pernet, Adv. Carbohydr. Chem. Biochem., 33 (1976) 111-188; G. D. Daves and C. C. Cheng, Prog. Med. Chem., 13 (1976) 303-349. (2) S. R. James, J. Carbohydr. Nucleos. Nucleot., 6 (1979) 417-465. (3) For surveys, see (a) R. J. Suhadolnik, Nucleoside Antibiotics, Wiley-Interscience, New York, 1970; (b) Nucleosides as Biological Probes, Wiley-Interscience, New York, 1979. (4) H. Ogura, H. Takahashi, K. Takeda, M. Sakaguchi, N. Nimura, and M. Sakai, Heterocycles, 3 (1975) 1129.

(5) R. G. Guy, The Chemistry of Cyanates and Their Thio Derivatives, Wiley-Interscience, New York, 1977, pp. 819-886; for a brief review on monosaccharide thiocyanates, see Z . J. Witczak, Heterocycles, 20 (1983) 1435-1448. (6) L. Drobnica, P. Kristian, and J. Augustin, in Ref. 5, pp. 1003-1221.

91

Copyright @ 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.

ZBIGNIEW J. WITCZAK

92

R-N=C=$+HX

-

R-NH-C-X

II

:S:

In contrast, the mechanism of cycloaddition of isothiocyanates is quite different. The -NCS group reacts with appropriate reagents to form 1,2-, 1,3-, and 1,Ccycloadducts. It may be assumed that one of the polar resonance-structures of the NCS group contributes predominantly to the resonance hybrid, and the compounds react through either the C=S or the C=N bond. R-N-&S

c ,

R-N=C=S

+

c ,

R-N=C-S

-

The ability of the isothiocyanate group to participate in both of the aforementioned reactions provides an attractive approach to nucleoside analogs and other nitrogen and sulfur heterocycles. Sugar thiocyanates are generally good precursors for the synthesis of thio and deoxy sugars; they are readily synthesized by a variety of methods from the corresponding halides.'.' These methods often parallel those used for preparing the corresponding halides, reflecting the pseudohalide character of the thiocyanate group? This character of the anion differs, however, from that of halide anions, in that thiocyanate is an ambident" nucleophile. -S-CEN

-

S=C=N.:

The resonance hybrid has the charge distribution shown.'' Consequently, kinetically controlled reactions of the thiocyanate anion with organic compounds (among them halides) may lead to the thiocyanates by nucleophilic -0.7108

-0.1934

................................

-0.4826

N

attack of the sulfur atom, to the isothiocyanates by nucleophilic attack of the nitrogen atom, or to a mixture of the two. The thermodynamically more-stable isothiocyanate may also be formed by a secondary, isomerization reaction. In common with other ambident species,'* the relative KJK, of the sulfur and nitrogen atoms of the thiocyanate nu~leophilicity'~ anion may depend on the interplay of different factors, including the solvent, the catalyst, counter-ions, the temperature, the nature of the leaving group, (7) E. Fischer, Ber., 47 (1914) 1377-1393. (8) A. Miiller and A. Wilhelms, Ber., 74 (1941) 698-707. (9) P. Walden and L. F. Audrieth, Chem. Reu., 5 (1928) 339-359. (10) N. Kornblum, R. A. Smiley, R. K. Blackwood, and D. C. Iffland, 1. Am. Chem. SOC., 77 (1955) 6269-6280. ( 11) E. L. Wagner, J. Chem. Phys., 43 (1965) 2728-2735. (12) W. J. Le Noble, Synthesis, (1970) 1-6. (13) A. Fava, A. Iliceto, and S. Bresadola, J. Am. Chem. SOC.,87 (1965) 4791-4794.

MONOSACCHARIDE ISOTHIOCYANATES A N D THIOCYANATES

93

the concentration, and the structure of the organic compound (particularly the geometry of the molecule). Such physicochemical methods as i.r.14 and ‘H-n.m.r.” spectroscopy permit rapid detection of isothiocyanate coproducts; these may be readily removed by chemical or chromatographic methods.I6 Knowledge of sugar isothiocyanates and thiocyanates is growing steadily, and now constitutes an expanded class of derivatives that had hitherto seldom been described as key intermediates to the various classes of heterocyclic derivatives of sugars. This article collates information on the reactivity of sugar isothiocyanates and isomeric thiocyanates, and illustrates some of the chemical properties that have contributed to the synthesis of nucleoside analog^,'^-'^ and thio and deoxy sugars. 11. MONOSACCHARIDE ISOTHIOCYANATES

1. Method of Synthesis of Sugar Isothiocyanates

The classical, Fischer7 synthesis of sugar isothiocyanates involves treatment of an acylated glycosyl halide with an inorganic thiocyanate in a polar solvent. Depending on the reactivity of the halide and the reaction conditions¶ either a thiocyanate or an isothiocyanate is formed directly. R-SCN R--X+SCN-<

1 R-NCS

Sugar thiocyanates rearrange more or less readily into the corresponding i s o t h i o ~ y a n a t e s . ~ According * ~ ~ - * ~ to the first comprehensive article24concerned with the isomerization of organic thiocyanates into isothiocyanates, ~ The SN1 mechanhalides may react either by an SN1 or an S N mechanism. ism gives rise to the thiocyanate, which may isomerize to an isothiocyanate. (14) G. L. Caldow and H. W. Thompson, Spectrochim. Acta, 13 (1958) 212-215; E. Lieber, D. N. R. Rao, and J. Ramachandran, ibid., 296-299. (15) A. Mathias, Tetrahedron, 21 (1965) 1073-1075. (16) L. A. Spurlock, R. K. Porter, and W. G. Cox, J. Org. Chem., 37 (1972) 1162-1168. (17) H. Ogura, H. Takahashi, K. Takeda, and N. Nimura, Nucleic Acids Rer, S2 (1976) 7-10. (IS) H. Ogura and H. Takahashi, Heterocycles, 6 (1977) 1633-1638. (19) H. Ogura and H. Takahashi, Heterocycles, 8 (1977) 125-146. (20) R. J. Ferrier and N. Vethaviyasar, Chem. Commun., (1970) 1385-1387. (21) R. J. Ferrier and N. Vethaviyasar, J. Chem. Soc., C, (1971) 1907-1913. (22) R. D. Guthrie and G . J. Williams, Chem. Commun., (1971) 923-924. (23) R. D. Guthrie and G. J. Williams, J. Chem. SOC.,Perkin Trans. 1, (1972) 2619-2623. (24) M. Renson, Bull. Soc. R. Sci. Likge, (1960) 78-93; Chem. Absrr., 55 (1961) 9253J:

ZBIGNIEW J. WITCZAK

94

Isomerization of organic thiocyanates to isothiocyanates is also discussed in other article^.*^-^' The first sugar isothiocyanate reported, namely, 2,3,4,6tetra-0-acetyl-P-D-glucopyranosyl isothiocyanate (2) was synthesized by Emil Fischer' in 1914 by treatment of 2,3,4,6-tetra-O-acetyI-a-~glucopyranosyl bromide (1) with silver thiocyanate in anhydrous xylene. It was also prepared by Miiller and Wilhelms' by thermal isomerization of the corresponding thiocyanate 3, obtained by treatment of 1 with potassium CH20Ac

tr"

AcO CH20Ac

AgsY 2

OAc

1'31

AcOQBr

CH20Ac OAc 1

OAc 3

thiocyanate in acetone. Fischer and coworkers3' similarly prepared 2,3,4-tri0-acetyl-6-bromo-6-deoxy-a-~-glucopyranosyl isothiocyanate. Interestingly, 1,3,4,6-tetra-0-acetyl-2-am~no-2-deoxy-a-~-glucopyranose hydrobromide (4) reacts with silver thiocyanate3' to give 2-acetamido-3,4,6tri-0-acetyl-2-deoxy-~-~-glucopyranosyl isothiocyanate (5), in a reaction which must involve an O + N acetyl migration. CHzOAc

CH20Ac

=

AcOQOAC

AcoQcl

NHIBr 4 (25) (26) (27) (28) (29) (30) (31)

CH~OAC

NHAc 5

NHAc 6

0. Billeter, Helv. Chim. Acta, 8 (1925) 337-338. 0. Mumm and M. Richter, Ber., 73 (1940) 843-860. A. Fava, A. Iliceto, A. Ceccon, and P. Koch, J. Am. Chem. Soc., 87 (1965) 1045-1049. U. Tonellato, 0. Rosseto, and A. Fava, J. Org. Chem., 34 (1969) 4032-4034. F. Micheel and W. Lengsfeld, Chem. Ber., 89 (1956) 1246-1253. E. Fischer, B. Helferich, and P. Ostmann, Ber., 53 (1920) 873-886. F. P. van de Kamp and F. Micheel, Chem. Ber., 89 (1956) 133-140.

MONOSACCHARIDE ISOTHIOCYANATES A N D THIOCYANATES

95

The same isothiocyanate 5 was obtained when 6 was used as the starting material.32For this 0 + N acyl migration, a mechanism has been proposed that involves the oxazoline intermediate 8, which results after dehydration of the initially formed 1,2-(orthoacety1)amide (7). The oxazoline derivative subsequently reacts with the thiocyanate ion at C-1, with Walden inversion.

&71= CH,OAc

CH20Ac

CH~OAC

-

AcoQ$.cs

AcO

H 2 N-F-OH H,N-$-oH

cs

AcoQ

N=C-Me

I

Me

NHAc 5

8

7

The Fischer method' for preparation of sugar isothiocyanates is general; however, many modifications have been introduced, using, for example, nonpolar solvents and silver t h i ~ c y a n a t e , ~ ' -lead ~ ~ t h i ~ c y a n a t e ,or ~~ ammonium t h i ~ c y a n a t eand , ~ ~ potassium thiocyanate in acetonitrile in the presence of tetrabutylammonium hydrogen~ulfate.~~" Another route to sugar isothiocyanates involves thermal isomerization of the corresponding thiocyanates. Ferrier and Vethaviyasar20'2' and, later, Guthrie and W i l l i a r n ~reported ~ ~ . ~ ~ the allylic rearrangement of unsaturated thiocyanates to isothiocyanates; see formulas 9-14. CH2R'

CH2R'

R'

R' = H, R2 = SCN, R3 = O M S R' = SCN, R2 = H, R3 = OMS 11 R ' = H , R 2 = S C N , R 3 = N 3 9

10

12 R' = H, R2 = NCS, R3 = OMS 13 R' = NCS, R2 = H, R3 = O M S 14

R' = H, R2 = NCS, R' = N3

Guthrie and Irvine3' also developed an original synthesis of unsaturated isothiocyanates, starting from a mixture of the unsaturated azido sugars 15 (32) F. Micheel, H. Peterson, and H. Kochling, Chem. Ber., 93 (1960) 1-3. (33) H. Ogura and H. Takahashi, Heterocycles, 17 (1982) 87-90. (34) V. A. Kulshin, R. G. Macharadze, S. E. Zurabyan. M. L. Shulman, and A. Ya. Khorlin, USSR Pat. 666,182 (1979); Chem. Abstr., 91 (1979) 141,177d. (34a) M. J. Camarasa, P. Fernandez-Resa, M. T. Garcia-Lopez, F. G. d e las Heras, P. P. Mendez-Castrillon, and A. San Felix, Synthesis, (1984) 509-510. (35) R. D. Guthrie and R. W. Irvine, Carbohydr. Rex, 82 (1980) 207-224.

ZBIGNIEW J. WITCZAK

96

and 16. Treatment of these azides with triphenylphosphine in carbon disulfide afforded the isothiocyanate 17, which was also prepared in 32% yield by an independent route by the boron trifluoride-catalyzed reaction of 18 with potassium thiocyanate in acetonitrile. The reaction probably CH~OAC

N3

/L

15

0. CH~OAC

AcO

CH~OAC

KSCN,MeCN BF, Et,O

CH~OAC I

NCS

0

AcO

17

18

16

proceeds by way of an oxocarbonium ion of type 19, by attack of a nucleophile, in the presence of acids, at C-3. Similarly, the formation of 17 CH~OAC

19

has been observed in the reaction of 20 with potassium thiocyanate in acetonitrile in the presence of boron trifluoride etherate.36 Previously, the reaction of 3,4,6-tri-O-acetyl-l,5-anhydro-2-deoxy-~arabino-hex-1-enitol (D-glucal triacetate) (20) with lead thiocyanate in a mixture of acetic acid, acetic anhydride, and carbon tetrachloride had been reported by Igarashi and H ~ n m a . ~ They ’ observed formation of isothiocyanate 21 as the main product, accompanied by a mixture of four isomeric thiocyanates.

6) CH~OAC

PbtSCN),, AcOH

AcO

Ac,O. BrICCI,

CH~OAC

.

A c O Q N c s SCN

20

21

(36) K. Heyns and R. Hohlweg, Chem. Ber., 111 (1978) 1632-1645. (37) K. Igarashi and T. Honma,J. Org. Chem., 32 (1967) 2521-2530.

MONOSACCHARIDE ISOTHIOCYANATES AND THIOCYANATES

97

Ramjeesingh and Kahlenberg" reported the synthesis of 6-deoxy-6isothiocyanato-D-glucopyranose(24). They employed the common method of synthesis of aryl isothiocyanates by treatment of amine 22 with carbon disulfide in the presence of dicyclohexylcarbodiimide (DCC). CHZNCS

22

CHZNCS

24

23

Another synthetic sequence, leading to 1,3,4,6-tetra-O-acety1-2-deoxy2-isothiocyanato-ff-~-glucopyranose (26), proceeds by treatment of the corresponding 1,3,4,6-tetra-0-acetyl-2-am~no-2-deoxy-~-~-glucopyranose hydrochloride (25) with t h i ~ p h o s g e n e A . ~similar ~ approach was employed CH~OAC L O

CHzOAc L O

O WoAc W CSCI,, CaCO,,

AcO

CH$&,H,O

Ac

AcO

I

I

NCS

NHz.HCI

25

26

for the preparation of a cellulose isothiocyanate. This sequence proceeded by treatment of cellulose with 2,4-diisocyanatotoluene,followed by hydrolysis, and reaction of the resulting amine with thiophosgene?' 2. Sugar Isothiocyanates as Intermediates in the Synthesis of Nucleoside Analogs

a. Reaction with Ammonia and Amines.-The chemistry of sugar isothiocyanates dates back to the classic paper by Emil Fischer in 1914 in which he described the conversion of isothiocyanates into thioureido deriva.~ then, these thioureido derivatives tives by the action of a m m ~ n i a Since have become important intermediates in synthetic approaches to nucleoside Naito and analogs, and their chemistry was discussed by I. Goodman:' Sano" first described the synthesis of l-(tetra-O-acetyl-~-~-glucosyl)-2(38) (39) (40) (41) (42)

M. Ramjeesingh and A. Kahlenberg, Can. J. Chem., 55 (1977) 3717-3720. J. C. Jochims and A. Seeliger, Terrahedron, 21 (1965) 2611-2616. P. Gemeiner, J. Augustin, and L. Drobnica, Carbohydr. Rer, 53 (1977) 217-222. I. Goodman, Ada Carbohydr. Chem, 13 (1958) 215-236. T. Naito and M. Sano, Chem. Pharm. Bull., 9 (1961) 703-714.

ZBIGNIEW J. WlTCZAK

98

thiothymine (29), using as the starting material 1-(tetra-0-acetyl-P-Dglucosyl)-2-thiourea (27) which, in a reaction with 3-methoxy-2-methylacryloyl chloride, gave the intermediate 28. Cyclization of 28 occurred spontaneously in the presence of dilute ammonia, with the formation of 29.

CH~OAC S

I

AcOCH2 CI-C-C=CH

OAc

28

27

OAc

NH~OHI

I N K

2

OAc 29

l-P-~-Ribofuranosyl-2-thiothymine was synthesized similarly43 from the corresponding 2,3,5-trLO-benzoyl-~-ribofuranosyl isothiocyanate. Ukita and coworkers43 improved this method in the preparation of ~-P-Dribofuranosylthymine. 0

NC/ I

BzOCwCS NCS

AcO AcO

HCOAc

BzO

30

NCS

OBz 31

I I HCOAc I HCOAc I

AcOCH

CH,OAc 32

(43) T. Ukita, A. Hamada, and M. Yoshida, Chem. Phorm. Bull., 12 (1964) 454-459.

MONOSACCHARIDE ISOTHIOCYANATES AND THIOCYANATES

99

Ogura and T a k a h a ~ h i ' ~detailed ,'~ the reaction of the sugar isothiocyanates 2, 30, 31, and 32 with ammonia and amines, as well as with various classes of hydrazines. The reaction of sugar isothiocyanates with various classes of a r n i n e ~ , ~ hydra~ines,~~-'* ~-~' and hydrazides has been reported by (44)T. Osawa, J. Org. Chem., 31 (1966) 3839-3840. (45) R. Bognlr and L. Somogyi, Chem. Ber., 99 (1966) 1032-1039. (46) R. BognPr, L. Somogyi, L. Szilagyi, and Z . Gyorgydeak, Carbohydr. Res., 5 (1967) 320-328. (47) G. L'abbt, S. Toppet, A. Willcox, and G. Mathys, J. Heterocycl. Chem., 14 (1977) 1417-1418. (48) J. Szczerek and T. Urbanski, Carbohydr. Res., 7 (1968) 357-360. (49) K. K. De, G. T. Shiau and R. E. Harmon, J. Curbohydr. Nucleos., Nucleot., 2 (1975) 171-176. ( 5 0 ) R. E. Harmon and G. L. Heise, Abstr. Pap. Am. Chem. Soc. Meet., 186 (1983) 22. (51) L. A. Ignatova, Yu. E. Kazantsev, and B. V. Unkovskii, Zh. Org. Khim., 5 (1969) 1792-1794; Chem. Abstr., 72 (1970) 21,268~. (52) A. D. Shutalev, L. A. Ignatova, and B. V. Unkovskii, Khim. Geterofsikl.Soedin., (1982) 269; Chem. Abstr., 96 (1983) 218,169r. (53) A. D. Shutalev, L. A. Ignatova, and B. V. Unkovskii, Khim. Geterotsikl. Soedin., (1982) 825-829; Chem. Absrr., 97 (1982) 163,356n. (54) H. Takahashi, N. Nimura, and H. Ogura, Chem. Pharm. Bull., 27 (1979) 1130-1136. ( 5 5 ) H. Takahashi, N. Nimura, and H. Ogura, Chem. Pharm. Bull., 27 (1979) 1147-1152. (56) H. Dorn, Angew. Chem, Int. Ed. Engl., 3 (1964) 308. (57) H. Dorn and M. Schutt, Chem. Ber., 97 (1964) 3246-3255. (58) H. Dorn and M. Schutt, Chem. Ber., 97 (1964) 3256-3261. (59) H. Dorn and H. Welfle, Pharmozie, 22 (1967) 558-561. (60) H. Ogura, I. Furuhata, K. Iwaki, and H. Takahashi, Nucleic Acids Res., s10 (1981) 23-26. (61) H. Ogura, H. Takahashi, and 0. Sato, Nucleic Acids Res., s 8 (1980) 1-4. (62) H. Ogura, H. Takahashi, and 0. Sato, J. Carbohydr. Nucleos. Nucleot., 8 (1981) 437-443; D. Cech, J. Konig, and B. Meinelt, Z. Chem., 22 (1982) 58-59; J. Fuentes Mota, C. Ortiz Mellet, F. Segura Ramos, A. Pradera Adrian, and A. Cert Ventula, An. Quim., 79C (1983) 221-224; J. Fuentes Mota, C. Ortiz Mellet, and A. Pradera Adrian, ibid., 8OC (1984) 48-53. (63) C. Gmernicka-Haftek and W. Wieniawski, Acta Pol. Pharm., 24 (1967) 253-259; Chem. Abstr., 68 (1968) 69,232~. (64) W. Wieniawski, G. Gmernicka-Haftek, J. Dzierzydska, and D. Jastalska, Pol. Pat. 63,828 (1971); Chem. Abstr., 76 (1972) 154,102r. (65) A. A. Tashpulatov, V. A. Afanasev, M. Lidaks, N. M. Sukhova, J. Popelis, and J. Rakhrnatullaev, Khim. Geterofsikl. Soedin., (1983) 170-174; Chem. Abstr., 99 (1983) 22,81814. (66) W. Wieniawski and C. Gmernicka-Haftek, Diss. Phann. Pharmacol., 20 (1968) 41 1-417; Chem. Abstr., 780 (1969) 58,174~. (67) W. Wieniawski, C. Gmernicka-Haftek, M. Korbecki, and E. Walczak, Acta Pol. Pharm., 30 (1973) 255-260; Chem. Abstr., 80 (1974) 83,464e. (68) W. Wieniawski and M. Wojtowicz, Acta Pol. Pharm., 31 (1974) 287-291; Chem. Abstr., 82 (1975) 171,353g. (69) W. Wieniawski, C. Gmernicka-Haftek, and M. Korbecki, Pol. Pat. 77,769 (1975); Chem. Abstr., 84 (1976) 106,008h.

100

ZBIGNIEW J. WITCZAK

many ~orkers.'~-'~Condensation of isothiocyanate 2 with 1,3,4,6-tetra-Oacety~-2-amino-2-deoxy-~-~-glucopyranose afforded the thioureido derivative 33 in 58% yield.44Similarly, condensation of 2 with a protected sugar having a free primary hydroxyl group has been reported.44 CHZOAC

OAc 33

Analogously, Bogn5r and coworker^^^*^^ prepared the same thiourea derivative 33, and synthesized some other N-glycosyl heterocyclic derivatives, such as thiazole 36, 1,3,4-thiadiazole 38, and 1,2,3,4-thiatriazole 39, using as the starting material isothiocyanate 2, or its ureido (34)or semicarbazide (37) analogs. It is noteworthy that treatment of semicarbazide 37 with a nitroso acid at 0" afforded the 1,2,3,4-thiatriazole derivative 39 by addition and cyclization. The same 1,2,3,4-thiatriazole derivative 39 was obtained by treatment of isothiocyanate 2 with hydrazoic acid. W. Wieniawski, M.Korbecki, and M.Wojtowicz, Pol. Pat. 85,567 (1976); Chem. Abstr., 87 (1977) 184,897~. C. Gmernicka-Haftek and W. Wieniawski, Acta Pol. Pharm., 34 (1977) 23-27; Chem. Abstr., 88 (1978) 51,1241. E. t a d a and W. Wieniawski, Acta Pol. Pharm., 34 (1977) 29-32; Chem. Abstr., 88 (1978) 5 1,111 m. M.Wojtowicz and W. Wieniawski, Acta Pol. Pharm., 34 (1977) 149-155; Chem. Abstr., 88 (1978) 62,575s. Z. Nowakowska and W. Wieniawski, Acra Pol. Pharm., 35 (1978) 189-193; Chem. Abstr., 90 (1979) 157,110s. M.Wojtowicz and W. Wieniawski, Acta Pol. Pharm., 34 (1977) 575-580; Chem. Abstr., 89 (1978) 110,253~ M.Wojtowicz and W. Wieniawski, Acta Pol. Pharm., 35 (1978) 37-40; Chem Abstr., 90 (1979) 152,534~. C. Gmernicka-Haftek, Z. Nowakowska, M.Wojtowicz, J. Bogacka, and B. Borkowski, Acta Pol. Pharm., 36 (1979) 139-145; Chem. Abstr., 93 (1980) 47,0950. B. Borkowski, M. Wojtowicz, C. Gmernicka-Haftek, Z. Nowakowska, and J. Bogacka, Acra Pol. Pharm., 39 (1982) 55-60; Chem. Abstr., 99 (1983) 5938r. D. Gumieti and C. Gmernicka-Haftek, Acta Pol. Pharm., 38 (1981) 667-672; Chem. Abstr., 99 (1983) 5939s.

MONOSACCHARIDE ISOTHIOCYANATES A N D THIOCYANATES

OAc

101

OAc 35a R = M e 35b R = P h

34

36a 36b

R=Me R=Ph

CH~OAC

31

2

NI

CH

OAc

OAc

I

OAc

7

,0:7 '

39

AcO/JH OAc 38

r a b b i and coworkers4' employed the aforementioned approach for the preparation of some model 1,2,3,4-thiatriazoles for examination of their 13 C-n.m.r. spectra. They also resolved the confusing problem of prototropic tautomerism of 1,2,3,4-thiatriazole 39. Methylation of 39 with diazomethane N=N

'

i

N=N

d

/

\

NYS- HNKS H

AR

N\

R

39

R = 2,3,4,6-tetra-0-acetyl-cu-~-g~ucopyranosy~

afforded, in the ratio of 7 :3, a mixture of two products that were identified as 39a and 39b on the basis of their I3C-n.m.r. spectra; they respectively

ZBIGNIEW J. WITCZAK

102

/N=N

N Me’

N=N

/

\

\

MeNKS N\ R

\R 39s

39b

showed a C-5 resonance at 6 181.2 and 156.4 in comparison with a characteristic signal of model compounds ( R = Ph, Ts).~’On the other hand, treatment of isothiocyanate 31 with an excess of hydrazoic acid in ether for one week at room temperature afforded 5-(2,3,5-tri-o-benzoyl-~-~ribofuranosylamino)-4-thia-l,2,3-triazole (3942) in 78% yield. A characteristic signal for C-5 observed at S 177.4 in the I3C-n.m.r. spectrum confirmed

HN

I

R R = 2,3,5-tri-O-benzoyl-P-~-ribofuranosyl 39c

the amino form of 39c. Szczerek and U r b a n ~ k reported i~~ the synthesis of the N-D-glucopyranosylthiocarbamoylderivatives 43-45 by treatment of 40-42. isothiocyanate 2 with the 5-alkyltetrahydro-5-nitro-1,3-oxazines

At

OAc

OAc

2

R1=Me 41 R’=Et 42 R’=Pr 40

43

44 45

Harmon and coworkers49synthesized a number of N-deacetyl- N- (per-0acetyI-D-glucopyranosylthiocarbamoyl)-(methylthio)colchicines (48-50), as potentially effective antileukemic agents, by condensation of N deacetyl(methy1thio)colchicine 46 with the corresponding isothiocyanates 2, 30, and 47 in benzene. Harmon and Heiseso also reported the novel synthesis of pyridine nucleoside analogs, using, as the starting material, peracetylated isothiocyanates 2 and 30. Condensation of the isothiocyanates with the sodium enolate of an aldehyde in N,N-dimethylformamide proceeds regiospecifically, with the formation of l-glycosyl-3-formyl-2-( 1H )pyridinethiones 51 and 52. Synthesis of the N-glycosyl compounds 55-58 was reported by Russian author^.^^-^^ The synthetic approach employing

MONOSACCHARIDE ISOTHIOCYANATES A N D THIOCYANATES

Me0

S=C=N-R Me0 1

-

Me0

0 SMe

SMe

46 2

48 49

R = 2,3,4,6-tetra-O-acetyl-P-D-gh1copyranosy~

30 R = 2,3,4-tri-0-acetyl-aY-~-arabinopyranosyl 47 R = 2,3,4-tri-O-acetyl-ar-~-arabinopyranosyl

50

CHZOAC

OAc

54

2 R'=H, R ~ = O A ~ 53 R I = OAC, R~= H

OAc

OAc

55 56

57 58

103

ZBIGNIEW J. WITCZAK

104

the previous methodology for synthesis of the D-galacto derivative34 56 starts from the corresponding isothiocyanates 2 and 53. The existence of a tautomeric equilibrium involving prototropic, ringchain tautomerism was established on the basis of the u.v., i.r., and 'H-n.m.r. spectra of the synthetic compounds 55 and 56.

I

-NH

OAc 62

Me

I

NMe

OAc NH

63

OAc

HN-N

59

CH~OAC

OAc

OAc 65

66

0

MONOSACCHARIDE ISOTHIOCYANATES A N D THIOCYANATES

105

The synthesis of a number of substituted thioureides by treatment of isothiocyanates 2 and 30-32 with aniline, 2-aminopyridine, l-adamantylamine, benzylamine, 2-aminobenzimidazole, 2-aminobenzothiazole, obromoaniline, 2-aminopyridine, 5-amino-1-phenylpyrazole-4-carboxylic acid, and various classes of sulfamines, in benzene or xylene solution, has been reported.54 The reaction of isothiocyanate 2 and 30 with 2-amino-4-picoline has also been reported.55 Dorn and c o ~ o r k e r sreported ~ ~ - ~ ~the synthesis of a number of substituted thioureido derivatives by treatment of isothiocyanates 2 and 5 with 2-phenylethylamine, bis(2-~hloroethyl)amine, and 2-(4ethoxypheny1)- 1-phenylethylamine. Other interesting examples for application of similar ideas have appeared in the literature.60-62For example, hepta-0-acetyl-P-lactosyl isothiocyanate33*60(59)has been reported to react with 1,3,4,6-tetra- O-acetyl-2-amino-2deoxy-P-D-glucopyranose (60)or 6-amino-1,3-dimethyluracil(61) to yield the 1,3-diglycosylthiourea (62) or N-glycosylaminoisothiazolo[3,4dlpyrimidine (63). Reaction of isothiocyanate 59 with the acylhydrazine 64 afforded intermediate hydrazinothiocarboxylamide 65 which, on treatment with acetic anhydride-phosphoric acid, yielded the hepta-0-acetyl-Plactosyl-1,2,4-triazole (66). A similar approach has been employed for the 69-74, using as starting synthesis of the glycosylimidazoline-2-thiones61~62 materials isothiocyanates 2, 30, and 31. Reaction of 2, 30, and 31 with the chloroalkylamine hydrochlorides 67 and 68 in the presence of dry pyridine, triethylamine, or anhydrous sodium acetate in acetonitrile respectively aff orded6'@ the imidazolidine-2-thiones 69-74. The reaction most probably proceeds by way of the intermediate thioureide, which, under the stated

2 R = 2.3,4,6-tetra-O-acetyl-/3-D-glucopyranosyl 30 R = 2,3,4-tri-O-acetyI-cr-~-arabinopyranosyl 31 R = 2,3,5-tri-O-benzoyl-P-D-ribofuranosyl

L

L

J

I R 69 7 j n=l 71

n=3

ZBIGNIEW J. WITCZAK

106

reaction-conditions, cyclizes, with the formation of imidazoline-2-thiones 69-74. Similarly, treatment of 2, 30, and 31 with 2-aminoethanol afforded thioureides 75-77, which readily cyclized6' to the iminothiazolidines 78-80 on treatment with thionyl chloride at 0-5". R-N=C=S

H,N(CH,),OH b

CbHb

R-NH-C-NH(CH,),OH II

s

2

30

75 16

31

11

I

C HZ-C H 2 0 H

n H N y S N

1

R

SOCI,

I

HN\ t

C-SH

II I

N

R

18 79 80

In a series of publications and patents,63,64p66-79 Wieniawski and coworkers reported the results of investigations of the reaction of sugar isothiocyanates with amines and various substituted hydrazides of heterocyclic acids. The reaction of isothiocyanate 2 with 2,6-diaminopyridine 81 was described,63 and the formation of dithioureide 82 as the sole product was observed. The

synthesis of a number of Schiff bases (83a-c) derived from D-glyCOSyl thio~emicarbazide~~'~~ and L-arabinosyl thioserni~arbazide~~ has also been reported.

0

MONOSACCHARIDE ISOTHIOCYANATES AND THIOCYANATES

107

CH~OAC

Hj-NH-N=CHR

AcO OAc 83a

R = C,H,OMe-o

83b R = Q 83c

R = f i NO,

0

Treatment of isothiocyanate 2 with nicotinoyl hydrazide in boiling 1,4dioxane afforded 84 in 85% yield; this cyclized on treatment with yellow mercury(I1) oxide in to oxadiazole 85. The mass spectrum of, and 'H-n.m.r. data for, 85 were also reported?' Interestingly, in this case, the cyclization of 84 occurred preferentially, with the formation of the 1,3,4-oxadiazole derivative 85, in contrast to literature" detailing similar

HsO

EtOH

OAc 85

cyclization of the hydrazinothiocarboxamide 65. Treatment of isothiocyanate 53 with nicotinic acid hydrazide in anhydrous benzene7' afforded thiosemicarbazide 86. Deacetylation of 86, followed by cyclization-desulfurization with mercury( 11) oxide, afforded the oxadiazole derivative 87.

OAc 53

OAc 86

ZBIGNIEW J. WITCZAK

108

NH CH2OH

OH 87

Analogously, thiosemicarbazides 91-96, derived from D- and L-arabinose, have been synthesized.'* Treatment of isonicotinic acid hydrazide (97) with 0 I1

R=2-pyridyl 89 R = 3-pyridyl 90 R = 4-pyridyl 88

R4

R6

HiNHNH:-RII

RQ R2

1\4

R6

91,92 R=2-pyridyl 93,94 R = 3-pyridyl 95,% R = 4-pyridyl

isothiocyanate 2 afforded thiosemicarbazide 99 which, on treatment with mercury(11) oxide afforded o ~ a d i a z o l e ~ '103. - ~ ~Thin-layer chromatography CH2OAC N=C=S

RiQ

-!DN

NHNH

97

Q!-

R2

OAc

+ N H N H2

NI*R'

98

2 R ' = H , RZ=OAc

NYo

53 R' = OAc, R2 = H

CH~OAC J ( ,R

HjNHNHi-R'

R2 OAc 99

ioo

R~ = OAC} R3 = 4-pyridyl R' = OAC.R~= H R I = H,

101 R' = H, R2= OAC] R3 = 2-pyridyl 102 R' = OAc, R2 = H

OAc 103 104 105 106

MONOSACCHARIDE ISOTHIOCYANATES A N D THIOCYANATES

-

+H2NNHC-R3

II

53

0

109

CH~OAC R

G

?

H CjNH N HR'

R2

107 R 3 = 0 m

OAc

111

R'=OAc, R 2 = H , R 3 =

Q

of thiosemicarbazides 99-102 has been reported.74 The galactopyranoside analog 104 of 103 has been prepared.73376 Analogously, the oxadiazoles 105 and 106 have been ~ y n t h e s i z e d . ~ ~ A similar approach has been applied in the synthesis of 2-fury1 and 2-quinolyl derivatives of the ~-glucopyranosylthiosemicarbazides~~ 109112, which have been used as the starting materials in the synthesis of the corresponding 1,3,4-0xadiazole deri~ative,'~by a method previously 0

0

N BS

I Me

I

Me 114 115 116

117 118 119

A1

HpNHN

I

Me

1I3

I

PhMe

R-N=C=S 2 R = 2,3,4,6-tetra-0-acety1-/?-~-glucopyranosyl 30 R = 2,3,4-tri-O-acetyl-a-~-arabinopyranosyl 31 R = 2,3,5-tri-O-benzoyl-/?-~-ribofuranosyl

Me 120 121 122

I

H

ZBIGNIEW J. WITCZAK

110

employed in the synthesis of similar compound^.'^ 2-Hydrazinopyridine reacted with isothiocyanates 2, 30, and 31, respectively, to give analogous thiocarboxamides.80'81 Similarly treatment of isothiocyanates 2, 30, and 31 with 6-hydrazino1,3-dimethyIuracil (113) in acetonitrile solution yielded N-glycosyl-2( l,3-dimethyl-2,4-dioxopyrimidin-6-ylhydrazino)thiocarboxamides 114116, respectively.80'8' Ring closure of 114-116 with N-bromosuccinimide (NBS) in methanol proceeded readily, to yield 2-(glycosylamino)-6,8dioxopyrimido[4,5-e]1,3,4-thiodiazines117-119 which, under reflux in toluene, underwent ring contraction to the pyrazole derivatives 120-122, Reaction of isothiocyanates 2, 30, and 31 with the acyl- or aroyl- and alkyl-hydrazine derivatives8' 123a, 123b, 124a and 124b has also been R-N=C=S 2 R = 2,3,4,6-tetra-O-acety~-~-o-giucopyranosyl 30 R = 2,3,4-tri-O-acetyl-a-~-arabinopyranosyl 31 R = 2,3,5-tn-O-benzoyl-~-~-ribofuranosyl RI-NHNH,

I

123a R ' = M e 123b R ' = P h R-NHC-NHNHR'

II

124a R ' = M e 124b R'=Ph R-NHC-NHNHC-R'

II S

S

125a or b 126a or b 127a o r b I . Ac,O 2. H,PO,

128a o r b 129a or b 130a or b

II

0

131a or b 132a or b 133a o r b I . MeONa 2. AcZO

134a o r b 135a o r b 136a or b

(80) M. Ogura, H.Takahashi, and M. Sakagushi, Nucleic Acids Res., s5 (1978)251-254. (81) H.Ogura, H.Takahashi, and E. Kudo, 1.Curbohydr. Nucleos. Nuclear., 5 (1978)329-341.

MONOSACCHARIDE ISOTHIOCYANATES A N D THIOCYANATES

111

reported to occur, with formation of the corresponding thiosemicarbazides l25a or b, 127a or b, and 131a or b. Intermediates 125a or b to 127a or b were treated with acetic anhydride-phosphoric acid, to yield the l-oglucosyl-l,2,4-triazole derivatives 128a or b to 130a or b, respectively. Treatment of 131a or b to 133a or b with methanolic sodium methoxide or acetic anhydride afforded the glycosylamino- 1,3,5thiadiazoles 134a or b to 136a or b. By a similar approach, amidines 137a-e react with isothiocyanates 2,30, and 31 in the presence of triethylamine,s2~83 to give the corresponding R-N=C=S R = 2,3,4,6-tetra-O-aCetyl-P-D-glUCOpyranOSyl 30 R = 2,3,4-tri-O-acetyl-cr-~-arabinopyranosyl 31 R = 2,3,S-tri-0-benzoyl-P-~-ribofuranosyl 2

EtlNl

NH,.HX

HN=l.-Rl

R'=SMe R'=H R'=Me R'=OMe e R'=NH,

137a b c d

/

R-NHC-N=C,

R~

(

E

'

3

y

N A N

R'

II S

NH2

138a-e 139a-e 140a-e

Y N-R1

AN>

SA N / N H

S

I

I

R

R

142a-c, e 143a-e 144s-e

141a-q e

R-NHC-N=C,

SII 138b, c 139c 140b,c

/

R1

NHz (R' = H, Me) (R'=Me) (R' = H, Me)

SOCl

A

N'

P' ANH I

CHCI,, 0"

R 145

(82) H. Ogura, H.Takahashi, and 0. Sato, Nucleic Acids Rex, s6 (1979)13-16. (83) H. Ogura, M. Takahashi, and 0. Sato, Chem. Pharrn. Bull., 29 (1981)1838-1842.

ZBIGNIEW J. WITCZAK

112

glycosylisothiobiurets 138a-e to 140a-e. On treatment with N-bromosuccinimide (NBS), the latter compounds cyclize to the corresponding glycosyl1,2,4-triazole-3-thiones142a-c and e, 143a-e, and 144a-e. On the other hand, treatment of 138a-e and 140a-e with ethyl orthoformate afforded the corresponding glycosyl-s-triazines 141a-141d and glycosyl-5-azothiocytosine 141e in good yield. It is noteworthy that treatment of 138b or c and 140b or c with thionyl chloride in chloroform solution under cooling afforded the corresponding glycosyl-1,2,4,6-thiatriazineS-oxide derivative 145 in moderate yield.s4 Valentiny and coworkerss5reported the synthesis of nucleoside analogs by an approach similar to that previously employed by Wieniawski and C O W O ~ ~ ~ ~asS well , ~ ~ as’ ~ Ogura ~ * ~ and ~ - ~ ~ coworkers. 54,55,60-62,80-84This sequence starts from 2, which, on treatment with hydrazides 146a-e in 1,4-dioxane, affords the corresponding thiosemicarbazides 147a-e. Thermal cyclization of 147a-e under basic conditions (sodium methoxide) yielded 3-alkyl-5-thioxo-l,2,4-triazoline derivatives 148b-e, respectively. In the case of 147a (R= H), the formation of 149 was observed. CH~OAC

OAc 147a-e

146a R = H b R=Me c R=Et d R=i-Pr e R=OEt

MeONa

OH

OH

148b-t

KovSE

and

coworker^^^

149

also

prepared

N-(tetra-0-acetyl-P-D-

glucopyranosyl)-2-thioxo-1,3-thiazolidin-4-one(150) by treatment of (84) H. Ogura, H. Takahashi, and 0. Sato, Chem H a m . Bull., 29 (1981) 1843-1847. (85) M. Valentiny, A. Martvoil, and P. KovPE, Collect Czech. Chem. Commun., 46 (1981) 2197-2202.

$

MONOSACCHARIDE ISOTHIOCYANATES AND THIOCYANATES

0 II

2

+ HSCHzC-OH

113

AcO OAc 150

isothiocyanate 2 with thioglycolic acid in boiling xylene. This reaction is useful for characterizing isothiocyanates, and is well known for distinguishing them from thiocyanates, which, under similar conditions, give N-acyldithiocarbamate~.~’~~~ b. Reaction with Amino Acids and Carboxylic Acids.-Reactions of sugar isothiocyanates with amino acids have been reported by a number of a ~ t h o r s .The ~ ~ earliest - ~ ~ work, that of Haring and Johnson8’ on the synthesis of D-glucosylhydantoin 151 and its thio congener 152, involved the condensation of starting isothiocyanate 2 with glycine ethyl ester in pyridine

CHZOAC

OAc 151 X = O 152 X = S

(86) (87) (88) (89) (90)

H. L. Wheeler and H. F. Merriam, J. Am. Chem. Soc., 24 (1902) 439-448. K. M. Haring and T. B. Johnson, 1. Am. Chem. Soc., 35 (1933) 395-402. F. Micheel and W. Brunkhorst, Chem. Ber., 88 (1955) 481-486. A. Klemer and F. Micheel, Chem. Ber., 89 (1956) 1242-1246. H. Takahashi, K. Takeda, N. Nimura, and H. Ogura, Chem. Phurm. Bull., 27 (1979)

1137-1142. (91) H. Takahashi, N. Nimura, and H. Ogura, Chem. Phurm. Bull., 27 (1979) 1143-1146. (92) S. E. Zurabyan, R. G. Macharadze, and A. Ya. Khorlin, All Union ConJ Chem. Biochem. Carbohydr., 6th. Moscow, 1977, pp. 51-52. (93) S . E. Zurabyan, R. G. Macharadze, and A. Ya. Khorlin, Izu. Akud. Nauk SSSR, Ser. Khim., (1979) 877-880; Chem. Abstr., 91 (1979) 91,887n. (94) S. E. Zurabyan, R. G. Macharadze, and A. Ya. Khorlin, Bioorg. Khim., 4 (1978) 1135-1136; Chem. Abstr., 89 (1979) 180,263g.

ZBIGNIEW J. WITCZAK

114

solution. Subsequent deacetylation and hydrolysis of the ester, followed by cyclization by treatment with hydrochloric acid, afforded 151 in moderate yield. S ~ ~ - the ~ ~ In a series of articles, Micheel and C O W O ~ ~ ~ ~ reported reaction of isothiocyanate 2 with DL-alanine methyl ester, which afforded the corresponding substituted thioureides 153a-c in, however, very poor yield. Twenty years later, Ogura and coworkersg0improved this reaction by using as the solvent benzene containing a catalytic amount of pyridine, and obtained the same thioureides 153a-c in high yields. CH~OAC

C02Et

OAc 153s R ’ = M e b R’ = CH,CHMe,

c

R’=CH2Ph

In contrast, isothiocyanate 32 reacts with glycine, as well as with palanine, in tetrahydrofuran (THF) solution with the formation of the corresponding N-(carboxyalky1)-D-gluconamides 154a-d by nucleophilic attack9’ of the amino group on the “hard” site, with simultaneous elimination of a molecule of thiocyanogen. Hoiever, the reaction of 4aminobutanoic acid with isothiocyanate 32 occurred at the “soft” site, with 0

ovN=C=S\/ L

I I AcOCH I HCOAc I HCOAc I

HCOAc

CH,OAc

32

+ H,N(CH,),CO,R’

I I AcOCH I

TH F

NH(CH2),,CO2R’

HCOAc HCOAc

I I

HCOAc CH,OAc 154s R ’ = H , n = I b R’=Et, n = 4

c ~ ‘ = ~ , n = 2 d R’=H,n=5

the formation of thioureides of the type 153a-c. Anthranilic acid also reacts with isothiocyanates 2, 30, and 31 in benzene solutiong’ under reflux with the simultaneous formation of thioureides 155- 157 and the glycosyl-2thioquinazolin-4-one derivatives 158-160, in the ratio of 1 :7. Interestingly, in the presence of zinc chloride, the ratio was changed to 3 : 8. Thioureides 155a or b to 157a or b are also very readily cyclized to the glycosyl-2-

* ~ ~ - ~

MONOSACCHARIDE ISOTHIOCYANATES A N D THIOCYANATES

115

155a o r b 156s o r b 157a o r b

1

ZnCI,

R’= H or Et

H 158 159 160

thioquinazolin-Cone derivatives 158- 160 under the influence of zinc chloride in nonpolar solvents. Analogously, the reaction of isothiocyanates 2,30,and 31 with the 3-aminopyrazole-4-carboxylic acid 161 in the presence of zinc chloride afforded the corresponding glycosylpyrazole[4,3-c]-4thiopyrimidin-6-ones” 162-164. Similarly, treatment of isothiocyanate 2

+

R-N=C=S 2 30

ZnCl

H2NQco2H

H

1

R 162

161

31

163 164

with 2-amino-3-(ethoxycarbonyl)pyridine(165) gave only 1-[3-(ethoxycarbonyl)pyridin-2-yl]-3-(2,3,4,6-tetra0-acetyl-P- D-glucopyranosyl)thiourea (166) in good yield.

2 + EtOzcQ

HzN 165

-

0 CH20Ac

E t o * c ~

OAc

HC 5 II N H

AcO OAc 166

ZBIGNIEW J. WITCZAK

116

On the other hand, the condensation of isothiocyanates 2, 30, and 31 with 6-aminopenicillamic acid (167), as well as ampicillin (168), in tetrahydrofuran or N,N-dimethylformamide solution in the presence of triethylaminey led to formation of only the corresponding thioureides 169-174. H ' H R-NHg-NFSy

+

R-N=C=S

' DMF

"C 0 R 167 R ' = H 168 R' = CH2COPh

2

30

S

0 170 R ' = H 171 1691

31

Me --Me

:I

'\C02R'

173 R'=CH,COPh

Khorlin and c o ~ o r k e r s reported ~ ~ - ~ ~ that the reaction of isothiocyanates 2 and 53 with acetic acid, as well as benzoic acid, in the presence of triethylamine, leads to the formation of N-glycosylacetamides 175 or 176 in 35% yield, and, as coproducts, 1,3-bis(glycosyl)ureas (177 and 179) and 1,3bis(glycosy1)thioureas (178 and 180). This method has been employed in CH~OAC 2 53

CH20Ac R

3 Et,N

1

O H I C = X

R2 OAc

I OAc

1,

175 R' = H, R2 = OAc 176 R' = OAc, RZ= H

the synthesis of substituted amides 184-188 by condensation of isothiocyanates 2,5,53, 181, and 182 with benzyl N- (benzyloxycarbonyl)-~-aspartate~~ (183). CH~OAC

2 5 53 181 182

R'=H,R2=R3=OAc Z = PhCH20C0 R' = H, R2 = OAc, R3 = NHAc R'=R'=OAc,R'=H R' = R3 = OAc, R2 = P-D-Galp(OAc),-( 1 + O ) R' = H, R3 = NHAc, R2 = p-~-Glcp(OAc),-(1 +0)

CH20Ac

184 185 186 187 188

(95) A. Ya. Khorlin, S. E. Zurabyan, and R. G. Macharadze, Curbohydr. Res., 85 (1980) 201-208.

MONOSACCHARIDE ISOTHIOCYANATES A N D THIOCYANATES

117

Novel methods for reversed-phase, pressurized liquid-chromatographic resolution of nonesterified amino acid enantiomers by the formation of diastereoisomers using two chiral reagents, namely, 2,3,4,6-tetra- 0-acetyl-PD-glucopyranosyl isothiocyanate (2) and 2,3,4-tri-O-acetyI-a-~arabinopyranosyl isothiocyanate (30),have been reported?6397

c. Reaction with Enamines.-The reaction of monosaccharide isothiocyantes 2,30,and 31 with ethyl 3-aminocrotonate (189) lead^'^^^^ to the formation of 3-amino-2-glycosyl(thiocarbamoyl)crotonates190-192,together with 4(ethoxycarbonyl)-5-(glycosylamino)-3-methylisothiazoles (193-195)in the H,

,,C02Et C

R-N=C=S 2

30 31

II

/c\ H2N 189

Me

-

i C,C02Et

RHNC,

II

/c\

H2N

190 191 192

M e S k H R +

N-S

Me 193 194 195

ratio of 2: 1. Products were separated by column chromatography, and distinguished by ‘H-n.m.r. spectroscopy, mainly on the basis of the -NH and -NH2 group signals, which, for ring-opened intermediates, showed a characteristic doublet at S 9.92-11.88 due to -NH, and a broad singlet at 6 8.85-10.80 due to -NH2. Moreover, according to a previous observation,” the thioureides 190-192 are readily cyclized to 193-195 by heating in acetonitrile. Interestingly, isothiocyanate 32, under the same conditions as before, reacts with enamine 189 to form 5-(carbethoxy)-6-methyl-2( 1,2,3,4,5-penta-O-acetyl-~-g~uco-pentitol-l -yl)pyrimidine-4-thione (196), whereas, with 6-amino-1,3-dimethyluracil(61),it affords 4,5,6,7-tetrahydro4,6-dimethyl-5,7-dioxo-2-( 1,2,3,4,5-penta-O-acetyl-~-gluco-pentitol-l -yl)pyrirnid0[4,5-d]pyrimidine-8-thione”~~* 197. The course of this reaction could be explained by nucleophilic attack of the isothiocyanato group on C-5 (hard site) of enamine 61,with the formation of 199 by way of intermediate 198. However, this occurs only in N,N-dimethylformamide or acetonitrile solution. The formation of the additional products 2-glycosyliminothiazolo[4,5-d]pyrimidine-4,6-dione (201)by way of intermediate 200, by attack of the isothiocyanato group on the 6-amino group (soft site), was observed when the reaction was performed in oxolane solution. On the other hand, treatment of isothiocyanates 2,30, (96) N. Nimura, H . Ogura, and T. Kinoshita, J. Chromatogr., 202 (1980) 375-379. (97) T. Kinoshita, Y. Kasahara, and N. Nimura, J. Chrornatogr., 210 (1981) 77-81. (98) H. Ogura, H. Takahashi, and K. Takeda, Chem. Pharm. Bull., 29 (1981) 1832-1837.

ZBIGNIEW J. WITCZAK

118

H

1-

CO,EL \ /

HCOAC

AcOCH I 1 HCOAc

H,N

I

I

189

HCOAc

I

CH~OAC

0

11

A

R-C-N=C=S 32

H ~ O A ~

I I

AcOCH HCOAc

I I

HCOAc CH20Ac 196 1

MeN

6,

N q H HCOAc

Me

I

Me

61

I HCOAc I

HCOAc I CHzOAc

AcOCH I

1

HCOAC

I I

HCOAc CHzOAc 197

198

199

2

I

Me 61 R = 2,3,4.6-tetra-O-acetyl-PD-glucopyranosyl-

S II

M e N 5

N'O

I Me

NHCNHR

-+

M0

I

b

I

Me 200

201

N

1

R

MONOSACCHARIDE ISOTHIOCYANATES A N D THIOCYANATES

119

and 31 with enamine 61 in N,N-dimethylformamide solution for 4 h at 70-80" gave 3-(glycosylamino)-5,7-dimethylisothiazolo[3,4-~]pyrimidine4,6-diones 199,204, and 205 in good yields.55The mechanism of this reaction that was proposed by Ogura and was additionally supported by the results of the reaction of the appropriately amino-protected derivatives (206) of 1,3-dimethyluracil with isothiocyanates 2, 30, and 31, which yielded the same kind of derivatives (after deprotection by hydrogenolysis with hydrogen in the presence of Pd-C in methanol solution).

Me 198 202 203

R-N=C=S 2

\

RHN

I

Me

Me

206

P h H2CH N A N

I

A.

Me 207 208 209

d. Reaction With Diamines and Diazomethane.-Such diamines as ophenylenediamine (210), 2,3-diaminopyridine (211), and 5,6-diamino-1,3dimethyluracil (212) react readily with the isothiocyanates 2,30, and 31, to form the corresponding thioureas;' 213-215,219-221, and 223-225. Cyclodesulfurization of these thioureides by using methyl iodide in oxolane (THF) solution9*affords (g1ycosylamino)benzimidazoles216-218 and N-glycosyl3-deazapurine (222), as well as (g1ycosylamino)theophyllines 226-228. Cyclodesulfurization of thioureide 213 with lead acetate or yellow mercuric oxide in methanol, followed by acetylation with acetic anhydride, affords two products, acetylaminobenzimidazole and (glycosy1amino)benzimidazole 216; however, the latter was formed in very poor yield.99 The (99) H. Takahashi, N. Nimura, N. Obata, H. Sakai, and H. Ogura, Chem. Pharm. Bull., 27 (1979) 1153-1158.

ZBIGNIEW J. WITCZAK

120

S

I1

-RTNHR ONHCNHR NH2 213 214 215

210

219 220,221

I Me

212

216 217 2 18

222

R = 2,3,4,6-tetra-O-acetyl-PD-glucopyranosy l

4

I Me 223,224,225

I

Me 226,221,228

possible mechanism of the cyclodesulfurization proceeds by way of Salkylation of the intermediate thioureide with methyl iodide, leading exclusively to the S-methylthiopseudourea. Subsequent, intramolecular, nucleophilic elimination of methanethiol affords the fused glycosylimida~ole.~' It is noteworthy that N-bromosuccinimide (NBS) oxidations1 of thioureides 223-225 in methanol as the solvent affords the respective glycosyl-5,7-dioxopyrimido[5,4-e]~s-triazine-3-thiones~~~'~' (229-231). Similarly, thioureides 233 and 234 (derived from lactosyl isothiocyanate 59), as well as maltosyl isothiocyanate (232),afford the 2-glycosyltriazine-3thione derivatives 235 and 236, respectively.'" Another interesting reaction of diamines with isothiocyanates is the formation of 1,3,5-triazepine-2-thionederivatives by the action of various classes of diamines on 2,3,4,5,6-penta-O-acetyl-~-gluconyl isothiocyanate (32).The reaction has been performed in acetonitrile or N,N-dimethylformamide solution with o-phenylenediamine, diaminomalononitrile, 5,6-diamino-l,3-dimethyluracil, 4,5-diamino-2,6-dithiopyrimidine, 4,5diamino-2-thiopyrimidine, and 4,5-diaminopyrimidine, and afforded (100) H. Ogura, H. Takahashi, and K. Ohokubo, Nucleos. Nucleor., 1 (1982) 147-154.

MONOSACCHARIDE ISOTHIOCYANATES A N D THIOCYANATES S

O

MeN%NHc-NHRII

oAN

I

MeOH NBS

-

NH2

H

M e N y A y s oAN

N’

I

R = 2,3,4,6-tetra-O-acetyl-~-D-glucopyranosy~-

LR

I

Me

Me 223 224 225 233

121

H

229 230 231 235

R = 2,3,4-tri-O-acetyl-cr-~-arabinopyranosylR = 2,3,5-tri-O-benzoyl-P-o-ribofuranosyl R = 2,3,4,6-tetra-O-acetyl-P-~-galactopyranosyl-( 1 + 4)2,3,6-tri-O-acetyl-P-~-g~ucopyranosyl 1 + 4)234 R = 2,3,4,6-tetra-0-acetyl-~-D-glucopyranosy~-( 2,3,4-tri-0-acetyl-P-D-glucopyranosyl

236

1,2,3,4,5-penta-0-acetyl-D-gluco-pentitol- l-yl)-1,3,5-triazepine-2-thiones (239a-c) by way of the intermediate thioureides 238a-c. 0, ,N=C=S \C

S

II

I

NHC-NH

HCOAc

I

I

4

AcOCH I

NH2

HCOAC

I

HCOAc I

CH~OAC

237a R 1 = R 2 = H

R1

I I

HCOAc

AcOCH

b R1=SH,R2=H c R~=R~=SH

HC~OA~

I I

HCOAc 32

CH~OAC 238a-c

H

ACOCH

I I HCOAc I HCOAc

CH~OAC

239a4

Reaction of isothiocyanates 2, 30, and 31 with diazomethane, as well as with ethyl diazoa~etate,6’*~~ in 1,Cdioxane affords the corresponding glycosylamino-1,2,3-thiadiazoles(240 and 241) in moderate yields. However,

ZBIGNIEW J. WITCZAK

122

0

+

R-N=C=S

R'-CH-I~N

2

R' = H,C02Et

30

-

Z I

R' RHN

240 241

31

the isomeric glycosylamino-1,3,4-thiadiazole (38) had been synthesized by Bognlr and coworkers4 by the action of nitrous acid on the corresponding semicarbazide (see Section 142,a). Under the same conditions as for isothiocyanates 2, 30, and 31, the isothiocyanate 32 reacted with diazomethane with the formation of 2-(penta-O-acetyl-~-gluco-pentitol-lyl)-4-0xathiazolone~~ (242) in 92% yield.

c-

+

CH2-NEN

HCOAC

I

HCOAC -

AcOCH I HCOAC

I I

HCOAc CH20Ac 32

+

,CH,-N=N

I

AcOCH I HCOAC

I I

HCOAc CH~OAC 242

e. Miscellaneous Reactions.-It has been found that aryl and alkyl isothiocyanates are able to react with such reducing agents as triethyl phosphite,"' triethylphosphine,"* or triphenyltin hydride,lo3 to form the corresponding isocyanides. However, when treated with tributyltin hydride in benzene solution, in the presence of the radical initiator azobis(isobutanonitri1e) (AIBN), monosaccharide isothiocyanates 2 and 5 yielded a mixture of the corresponding isocyanides 243a and 243b, and 1,5-anhydro-~-glucitolderi~atives''~244a and 244b in the ratio of 1 :3. (101) T. Mukaiyama, H. Nambu, and M. Okamoto, 1. Org. Chem., 27 (1962) 3651-3654. (102) A. W. Hofmann, Ber., 3 (1870) 761-772. (103) D.H. Lorenz and E. J. Becker, J. Org. Chem., 28 (1963) 1707-1708. (104) Z. J. Witnak, Tetrahedron Lett. (1986) 155-158.

o='=' 0

MONOSACCHARIDE ISOTHIOCYANATES A N D THIOCYANATES

C,HBu,SnH ,, AIBN

AcO

c'

123

+

AcO

2 R~=OAC 5 R'=NHAc

2438 R'=OAc b R'=NHAc

2448

b

These examples of radical-induced reduction of the isothiocyanato group indicate that the intermediate isocyanides 243a and 243b also undergo reduction, to 1,5-anhydro-~-glucitolderivatives 244a and 24413. Previously, the reduction of monosaccharide isocyanides by tributyltin hydride had been r e p ~ r t e d , ' ~ ~and - ' ~ 'their chemistry has been discussed."' 111. MONOSACCHARIDE THIOCYANATES 1. Method of Synthesis of Sugar Thiocyanates

a. S NDisplacement ~ Reactions of Sulfonyloxy Groups by Thiocyanate Ion in Hexopyranoses.-The first sugar thiocyanates were synthesized by Fischer and by treatment of 2,3,4,6-tetra-0-acety~-a-~-glucopyranosy~ bromide (1) with potassium thiocyanate in anhydrous acetone, whereas the isomeric isothiocyanate 3 had been synthesized earlier by treatment of 1 with silver thiocyanate in anhydrous xylene. Muller and Wilhelms' applied the Fischer method" to the preparation of 6-deoxy-6-thiocyanato-a-~glucopyranosyl bromide, and examined the problem of the isomerization of the thiocyanate to the corresponding isothiocyanate. The synthesis of both the methyl a- and /3-glycoside of 6-deoxy-6-(thiocyanato)-~-glucose~ has been reported. The authors* also first applied nucleophilic displacement of the p-tolylsulfonyloxy group in 245 by the thiocyanate ion in anhydrous acetone during 10 h in a sealed tube at 130" for the preparation of 1,2,3,4tetra-O-acetyl-6-deoxy-6-(thiocyanato)-~-~-glucose (246), an intermediate for disulfide 247,formed by treatment of 246 with sodium methoxide.

(105) D. H. R. Barton, G. Bringmann, G. Lamotte, R. S. H. Motherwell, and W. B. Motherwell, Tetrahedron Lett, (1979) 2291-2294. (106) D. H. R. Barton, G. Bringmann, G. Lamotte, W. B. Motherwell, R. S. H. Motherwell, and A. E. A. Porter, J. Chem. Soc.. Perkin Trans. 1, (1980) 2657-2664. (107) D. H. R. Barton, G. Bringmann, and W. B. Motherwell, J. Chem SOC.,Perkin Trans. 1. (1980) 2665-2669. (108) Z . J. Witczak, J. Carbohydr. Chem., 3 (1984) 359-380.

ZBIGNIEW J. WITCZAK

124

CH20Ts

CHz-S

CHZSCN

-AcoQAc-

AcOQAc OAc

OAc

H o Q j

-

OH

2

The foregoing method of preparation of sugar t h i o ~ y a n a t e s ' ~by ~ ~S" ~N ~ displacement of a sulfonyloxy group has been reported by many workers. 111-143 Displacement of sugar sulfonate groups by various nucleo(109) (110) (111) (1 12) (113) (114) (115) (116) (117) (118) (119) (120)

(121) (122) (123) (124) (125) (126) (127) (128) (129) (130) (131) (132) (133) (134) (135) (136) (137) (138)

J. Stanek and L. Tajmr, Chem. Listy, 52 (1958) 551-553; Chem. Abstr., 53 (1959) 4146. J. Stansk and L. Tajmr, Collect Czech. Chem. Cornrnun., 24 (1958) 1013-1016. A. C. Richardson, Carbohydr. Res., 10 (1969) 395-402. D. H. Ball and F. W. Parrish, Adu. Carbohydr. Chem., 23 (1968) 233-280; Adu. Carbohydr. Chem. Biochern., 24 (1969) 139-197. J. Hill, L. Hough, and A. C. Richardson, ploc. Chem. Soc., (1963) 346-347. J. Hill and L. Hough, Carbohydr. Res., 8 (1968) 398-404. A. F. Cook and W. G. Overend, J. Chem. Soc., C, (1966) 1549-1556. L. N. Owen and P. L. Ragg, J. Chem. Soc., C, (1966) 1291-1296. S . D. Gero, Tetrahedron Lett., (1966) 3193-3198. S. D. Gero and R. D. Guthrie, J. Chem. SOC.,C, (1967) 1761-1762. N. K. Kochetkov, E. J. Budovskii, V. N. Shibaev, and Yu. Yu. Kusov, Izu. Akad. Nauk SSSR, Ser. Khim., (1970) 404-411; Chem. Abstr., 73 (1970) 25,804k. N. K. Kochetkov, V. N. Shibaev, Yu. Yu. Kusov, and M. F. Troitskiy, Izu. Akad. Nauk SSSR, Ser. Khim., (1973) 425-430; Chem. Abstr., 79 (1973) 18,964n. L. Vegh and E. Hardegger, Helu. Chim. Acra, 56 (1973) 1792-1799. J. Hill, L. Hough, and A. C. Richardson, Carbohydr. Rex, 8 (1968) 19-28. B. Castro, Y. Chapleur, and B. Gross, Bull. Soc. Chim. Fr., (1973) 3034-3039. B. Castro, Y. Chapleur, and B. Gross, Carbohydr. Res., 36 (1974) 412-419. B. Gross and F. X. Oriez, Carbohydr. Res., 36 (1974) 385-391. R. A. Boigegrain and B. Gross, Carbohydr. Res., 41 (1975) 135-142. W. E. Dennis, Ph.D. Thesis, Wayne State University, 1966; Dissertation Absrr., American Doctoral Diss. 1966-1969, p. 50. J. P. Dickerson, Ph.D. Thesis, Wayne State University, 1966; Dissertation Abstr., American Doctoral Diss. 1966-1969, p. 50. J. E. Christensen and L. Goodman, J. Am. Chem. Soc., 82 (1960) 4738-4739. J. E. Christensen and L. Goodman, J. Am. Chem. Soc., 83 (1961) 3827-3834; L. Goodman, ibid., 86 (1964) 4167-4171; Chem. Cornrnun., (1968) 219-220. R. D. Guthrie, Chem. Ind. (London), (1962) 212. R. D. Guthrie and D. Murphy, J. Chem. Soc., (1965) 6666-6668. C. I. Gibbs and L. Hough, Carbohydr. Rer, 18 (1971) 363-371. A. Klemer and G. Mersmann, Carbohydr. Res., 12 (1970) 219-224. L. A. Reed, 111 and L. Goodman, Carbohydr. Res., 94 (1981) 91-99. J. C. P. Schwarz and K. C. Yule, Proc. Chem. Soc., (1961) 417. D. M. G. Hull, P. I. Orchard, and L. N. Owen, J. Chem. Soc., Perkin Trans. 1, (1977) 1234-1239. K. Tokuyama, Bull. Chem. SOC.Jpn., 37 (1964) 1133-1137.

MONOSACCHARIDE ISOTHIOCYANATES AND THIOCYANATES

125

philes was discussed by Richardson,"' as well as by Ball and Parrish,"' taking into consideration all of the factors influencing reactivity in reactions, particularly the geometry of the molecule and the nucleophilicity of the attacking group. StanCk and Tajmrlog*l'oapplied this method for preparation of the quinovose derivatives 250 and 251. CHZSCN

CH20Ts

CH3

C,H,,O. 2 h. 130"

R3 245 248

R3

R'= R3=OAc; R2 = H R' = H, R2 = OAc, R3 = OTs

246 249

R3

R' = R3 = OAc, R2 = H R' = H, R2 = OAc, R3 = OTs

250 R' = R3.= OAc, R2 = H 251 R' = H, R2 = OAC, R3 = OTs

Hough and coworker^"^ prepared methyl 4,6-dideoxy-4,6-di(thiocyanato)-a-D-galactopyranoside(253a) by the S N displacement ~ of the methanesulfonyloxy group in 252a with thiocyanate ion in N, N-dirnethylformamide. Intermediates 253a and 254a could, clearly, also be used for preparing the deoxy sugars 255 and 256 by desulfurization with Raney nickel. Similarly, methyl 2-acetamido-3-O-acetyl-2-deoxy4,6-di-O-(methylsulfonyl)-a-~-glucopyranoside~~~ (252b) undergoes S N ~

0 CH20Ms

MsO

R~=OH b R2=NHAc

254a

OMe "-?.QOMe

R2

252s R' = R2 = OBz b R' = OAc, R2 = NHAc

R' 253a b

\t-? Raney Ni

OMe

R2

255 R ' = R ~ = O H 256 R' = R2 = OBz

(139) (140) (141) (142) (143)

K. Tokuyama, M. Kiyokawa, and M. Katsuhara, J. Org. Chem., 30 (1965) 4057-4060. C. H. Bolton, L. Hough, and R. Khan, Carbohydr. Res., 21 (1972) 133-143. J. Defaye and J. Hildesheim, Carbohydr. Res., 4 (1970) 145-156. L. Goodman, Adu. Carbohydr. Chem., 22 (1967) 109-175; see p. 158. P. A. Risbod, T. S. Philips, and L. Goodman, Carbohydr. Res., 94 (1981) 101-107.

ZBIGNIEW J. WITCZAK

126

nucleophilic displacement with thiocyanate anion, to give the corresponding 4,6-dideoxy-4,6-di(thiocyanato)-ru-~-galactopyranosidederivative 253b, together with the disulfide 254b, derived from the dithiocyanate 253b by hydrolysis, followed by oxidative ring-closure. Cook and Overend"' repor~ of a sulfonyloxy group on C-4 in 257 with ted the S N displacement thiocyanate ion, to yield the 4-substituted galacto derivative 259, which was an intermediate to the deoxy sugar 260, obtained by desulfurization with CHZOH

CHzOH

OBz

CHZOH

OBz

257 R'=OTs 258 R' = O,SC,H,Br-p

OH

259

260

Raney nickel. Displacement of the p-bromophenylsulfonyloxy group in 258 gave 259 in improved yield (55%) after heating for only 2.5 h. Also, Owen and RagglI6 attempted, without success, the same reaction at C-4 of the corresponding P-D-galactoside and other 4-0-p-tolylsulfonylP-D-galactosides. However, Gero"' and Gero and Guthrie"* prepared (262)in 56% yield methyl 4-deoxy-4-(thiocyanato)-a-~-glucopyranoside by S N displacement ~ of the methylsulfonyloxy group by thiocyanate ion in N,N-dimethylformamide during 46 h at 140". They confirmed by 'H-n.m.r. spectroscopy the gluco configuration in the 4C1( D) conformation. Kochetkov and c ~ w o r k e r s , ~ ~as' ~well ~ ~ ' as Vegh and Hardegger,I2' also prepared the 4-C-thiocyanate (262)as a starting material for the preparation"' of uridine 5'-(4-deoxy-~-xylo-hexosyldiphosphate), as well as of 4 - t h i o - a - ~ glucopyranosyl phosphate"' and the corresponding deoxy sugarlo6 260. CH20Bz

MsoQ

&POMe CH~OBZ

OMe

-

NCS

OBz

OBz

26 1

262

Under conditions similar to those described in the l i t e r a t ~ r e , "and ~ in contrast to previous report^,"^ the 2,3-diacetate and 2,3-dibenzoate of 252a and 263, methyl 4,6-di-O-(methylsulfonyl)-a-~-glucopyranoside, afforded the corresponding 4,6-dideoxy-4,6-di(thiocyanato)-cu-~-galactopyranosides121s122 (253aand 264),together with a small proportion of the

MONOSACCHARIDE ISOTHIOCYANATES AND THIOCYANATES

127

thiocyanates (265 and 266), as well as d i ~ u l f id e "~ 254, which is probably formed by hydrolysis of dithiocyanate 264. CHzSCN

NcsQoMe I

R=

ooMe +' 253a R1= R2 = OBz 264 R ~ = R ~ = O A C

CHzSCN

N~SQOM~ O A c

MsO

+254

RZ

266

252a R1= R2 = OBz 263 R' = R2 = OAc

265

Treatment of the disulfonate 267 under the same conditions affords a mixture of the mono- and di-thiocyanates (268 and 266) in the ratio of 1 : 1, but in only 50% yield (because of excessive decomposition of the starting material). Ferrier and Vethaviyasar2032'reported the thermal rearrangement CHZSCN

Tsog)Me 4 NCSQAC

CH~OTS

&)Me

OAc 266

DMF.3h. 160"

Tsoa CHZSCN

OAc 267

OAc

268

of the 2,3-unsaturated thiocyanates 9 and 10 to 3,4-unsaturated isothiocyanates; see Section I I , l . The starting thiocyanates 9 and 10 were prepared by

ZBIGNIEW J. WITCZAK

128

S N displacement ~ of a methanesufonyloxy group on C-4 by thiocyanate ion under unusual conditions (in N,N-dimethylformamide, for 72 h at room temperature) for 9, and in N,N-dimethylformamide for 16 h at 45" for 10. It is noteworthy that the S N displacement ~ in these reactions favored the CHZOMs

MsOO

O

CHZOMs

E

t

NcsGoE

DMF. KSCN 72 h. 20"

269

9

CHZOMS

CHZOMs

M s o a o E t

DMF, KSCN 16h.45'

.

NCS&OE

270

t 10

secondary, allylic position, in contrast to previous literature reports'13 detailing similar nucleophilic displacements of the 4,6-di-0- (methylsulfonyl) described the rearrangement of derivative 252. Guthrie and Williams22923 allylic thiocyanates, using, as the starting compound, ethyl 6-azido-2,3,6t~deoxy-4-O-(methylsulfonyl)-a-~-threo-hex-2-enopyranoside (271). Isothiocyanate 14 was an intermediate in the preparation of a derivative (272), of the antibiotic sugar purpurosamine C, namely, 2,6-diamino-2,3,4,6tetradeoxy-D-threo-hexose, by reduction and acetylation. CHzNa

I

CHIN,

I _

-r\

DMF.96 h, r.t.

MsOW

O

E

9

b

t

U

O

E

t

271

CHZOMs 1. H,, RIC. EtOH

2. Ac,O.MeOH

272

14

Interestingly, all attempts to introduce an azido group at C-6 in 13 by use of sodium azide in N,N-dimethylformamide failed. Instead of the azido derivative, the crystalline derivative 273, containing no thiocyano, isothiocyano, methylsulfonyl, or azido group (as indicated by i.r. and 'H-n.m.r. spectra), was ~btained.'~

MONOSACCHARIDE ISOTHIOCYANATES AND THIOCYANATES

129

CH~OMS 41

10 +

sc N 13

6-2

N

\ N //" 273

An approach to 2-deoxy-2-(thiocyanat0)-D-glucopyranosederivatives has been reported by Igarashi and H ~ n m a . ~This ' reaction proceeds in the presence of acetic acid and acetic anhydride by addition of thiocyanogen to 3,4,6-tri-O-acetyl-~-glucal(20), with the formation of a mixture of isomeric thiocyanates (274 and 275) and isothiocyanate 21 (3% yield), as (276). well as 3,4,6-tri-0-acetyl-2-(thiocyanato)-~-glucal CH~OAC

H~COAC 274

HSCN, CCI,

21 t

AcOH, Ac,O

AcO

20

H~COAC

OAc

AcO CH~OAC

SCN 276

A AcO c o

S SCN O A

c

275

It is noteworthy that all attempts to convert 275 into the corresponding D-glucosyl bromide with hydrogen bromide in acetic acid were unsuccessful, because the reagent was also attached to the 2-(thiocyanato) group. Treatment of 275 with titanium tetrachloride in chloroform afforded crystalline 3,4,6-tr~-O-acetyl-2-deoxy-2-(thiocyanato)-a-~-glucosyl chloride (277)

ZBIGNIEW J. WITCZAK

130

g), CH~OAC

275

AcO

TiCI,

+276

SCN 277

together with 3,4,6-tri-O-acetyl-2-(thiocyanato)-~-glucal (276). However, treatment of 277 with diethylamine in anhydrous benzene at room temperature afforded 276 in 76% yield.37 On the other hand, methanolysis of 277 in the presence of silver carbonate and silver perchlorate gave the corresponding glycoside 278 in 83% yield, as well as the 2-thiocyanato-~-glucal triacetate 276 in 11YO yield. Desulfurization of 278 with Raney nickel afforded 3,4,6-tri-O-acetyl-2-deoxy-@-~urubino-hexopyranoside (279) in 56% yield.37

0 CH~OAC

277

Ag,CO,, MeOH AgCIO,

*

M e +216

AcO

k N 278

YNi CH~OAC

gyMe

AcO

279

In contrast to the behavior of 275, treatment of 274 with titanium tetrachloride gave only 3,4,6-tri-O-acetyl-2-deoxy-2-(thiocyanato)-cu-~mannosyl chloride (280). Methanolysis of 280 gave a mixture of anomeric CH~OAC

OAc S

274

CH~OAC

CHZOAC

CHCI,

280

2810, b /key

CH2OH

HO&Me 2820. b

Ni

MONOSACCHARIDE ISOTHIOCYANATES AND THIOCYANATES

131

glycosides 281a and 281b, which was separated by preparative, thin-layer chromatography. Desulfurization of 281a and 281b with Raney nickel afforded the corresponding deoxy derivatives, 282a and 28213,r e ~ p e c t i v e l y . ~ ~ On the other hand, reduction of the thiocyanato group in 278 with lithium aluminum hydride, followed by acetylation, afforded a mixture of anomers 283a and 283b in the ratio of 1 :9 (a:p ) , and the coproduct 284 in 5% yield. CHI

I I

CH~OAC U

LIAIH,

278

Ac20,C,H,N

*

O

M

HCSAc e + AcOCH I HCOAc

AcO

I I

HCOAc

SAC 283a, b

CH~OAC 284

Treatment of 278 with mercaptoacetic acid in anhydrous benzene yielded methyl 3,4,6-tri- 0-acetyl-2-S- ( N-acetylthiocarbamoyl)-2-thio-~-~-glucopyranoside (285) and methyl 3,4,6-tri-0-acetyl-2-thio-2-S-(thiocarbamoyl)p-D-glucopyranoside (286)in 75 and 14% yield, re~pectively.~~ Castro and

oMe OMeOM' CH~OAC

CH~OAC

CH~OAC

A HSCHCOH C6H6

AcO

+

AcO

AcO

I

SCN

S-C-NHAc

S-C-

!

11

278

S 285

NH2

286

coworker^'^^'^^^ reported the synthesis of various sugar thiocyanates by way of oxyphosphonium salts (290-292)as very reactive intermediates towards various nucleophiles, among them, thiocyanate ion. This approach starts from the unprotected methyl cu-D-hexosides 287-289, and proceeds by favored formation of oxyphosphonates 290-292 at the primary hydroxyl group. Thiocyanates 293-295 were obtained by treatment of 290-292 with

'0 :OoMe CH2OH

OH

R2

CH20i'(NMe2),CI-

R3

OMe

P(z+4:)I,

OH

R3

CH2SCN

DM NH,SCN F, 7- 10 h, 90"

,

R4

293 294 295

ZBIGNIEW J. WITCZAK

132

thiocyanate ion in N,N-dimethylformamide at 90- 100". Similarly, in the a,a-trehalose series, thiocyanate 296 and dithiocyanate 297 have been pre~ared.''~ CH2SCN

AcO 296 R'=OH 297 R ' = S C N

Gross and O r i e ~ ' 'reported ~ the synthesis of 4-S-acetyl-6-deoxy-4-thio-~altrose (302)and 4-S-acetyl-6-deoxy-4-thio-~-idose (303)by using, as starting materials, the corresponding thiocyanates prepared in 35% yield from ~ with thiothe 4-0-mesyl derivatives 298 and 299 by S N displacement

OBn 298 R' = H, R2 = OMS 299 R I = OMS, R~= H

300 301

OBn R~=SCN R' = H, R2= SCN

OAc 302 R' = SAC, R~ = H 303 R' = H, R2= SAC

cyanate ion under the standard conditions (in N,N-dimethylformamide for 5 h at 110"). However, simultaneous formation of the isomeric isothiocyanate in 5% yield was observed. Boigegrain and Gross'26 also reported an approach by way of thiocyanate to preparation of the 3,4,6-trideoxy-3,4acetyl-4-S-acetylepithio-a-D-allopyranoside derivative 309 and 1,2,3-tri-06-deoxy-4-thio-~-glucopyranose derivatives 315 and 316 by using the method previously applied by Dennis,"' as well as Dickerson."' The approach to 315 and 316 starts from the appropriate 3,4-anhydro sugars 310 and 311, and proceeds by way of thiocyanates 312 and 313. A similar ring-opening reaction of the 2,3-anhydromannoside 317 with ammonium thiocyanate in aqueous 2-methoxyethanol gave a preponderance of thiocyanate 318,formed by opening of 317 at C-3, as reported by Christensen and G o ~ d m a n . ' ~ ~ * ' ~ ~

MONOSACCHARIDE ISOTHIOCYANATES A N D THIOCYANATES

306

OH

308

133

OMS

1

MeOH, FeCI,

"O 'Q

OMe S

1

OBz

304

OMS

OH I

309

307

1. Zn.AcOH

2. Ac,O

HO

OR

A c s ~ O M e AcO

312 313

OAc 314

1

KSC"

OOMe Acsol CH3

0

OR

310 R = T s 311 R = A c

AcO

OAc

315 R' = H, R2 = OAc 316 R' = OAc, R2 = H

ZBIGNIEW J. WITCZAK

134

Evidence for structure 318 was provided by desulfurization of 318 with Raney nickel, with formation of the deoxy derivative 320. Additional confirmation of the trans-diaxial geometry of the thiocyanate was the formation of episulfide 321 from mesylate 319. Guthrie and Murphy131s132

ph-cfo CJMe \ - ph-c OCH2

Raney Ni

Ph-CH

Ho&oMe

OMe

NH,SCN

/ ,",,

CHZOH

320

318

0

317

kN

OCH2

OCHZ

'*"fQ

MsO

MeONa

0

OMe

OMe

S

NCS

321

319

later pointed out that epoxide 317, the precursor of 318, could be converted directly, although in poor yield, into episulfide 321 by thiocyanate ion. An interesting approach to the carbohydrate thiocyanates by ring opening of the corresponding epimine derivatives was reported by Gibbs and H 0 ~ g h . Treatment I~~ of 322 with potassium thiocyanate in N,N-dimethylformamide afforded methyl 4,6-0-benzylidene-2,3-dideoxy-3-(dimethylamino)-2-(thiocyanato)-a-~-altropyranoside(323) exclusively. The altro configuration of 323 was assigned from the 'H-n.m.r. spectrum on the basis

Ph

{DOMe -

___, KSCN Ph-CH DMF

c-Q (oMe 0 N+ Me'

'Me 322

Ct.Hz(NOp)oSO;

Raney Ni

0

NMe2 323

Ph-C(boMe

0 324

MONOSACCHARIDE ISOTHIOCYANATES AND THIOCYANATES

135

that trans ring-opening had occurred. Attempts to prepare a 2,3-dideoxy-3(dimethylamino) derivative by desulfurization of the thiocyanate 323 were unsuccessful, elimination occurring to give methyl 4,6-0-benzylidene-2,3dideoxy-a-~-eryythro-hex-2-enopyranoside (324). Similarly,formation of the unsaturated sugar derivative, methyl 2,3-dideoxy-a-~-erythro-hex-2enopyranoside (326), by treatment of methyl a-D-mannopyranoside 2,3carbonate (325) with thiocyanate ion has been observed.134An interesting CH2OH

CHZOH

325

326

mechanism for the formation of 326, similar to that previously reported, l3’- 32 has been proposed. The Goodman group rep~rted’~’ a synthetic approach to the preparation of S-P-D-galaCtOpyranOSyl-4-thi0-Dglucopyranose (330) (“thiolactose”) by using, as the starting material,

’

0

L””-v II

sCNl

p J

-0,

+o

SCN

2

)J+ S-CEN

methyl 2,3,6-tri-0-benzoyl-4-O-(methylsulfonyl)-~-~-galactopyranos~de (261) or methyl 2,3,6-t~-O-benzoyl-4-O-(trifluoromethylsulfonyl)-a-~galactopyranoside (327). Nucleophilic displacement of the 4-trifloxy group of 327 required only 12 h at 80°, and provided thiocyanate 262 in 85% yield, whereas the methylsulfonyloxy group at C-4 in 261 required 42 h at 140°, and provided the thiocyanate 262 in 68% yield, in contrast to previous b. S NDisplacement ~ of Sulfonyloxy Groups by Thiocyanate Ion in Pento~ of the 5-pfuranoses.-Schwarz and Yule136reported S N displacement tolylsulfonyloxy group in 2,3-O-isopropylidene-5-O-p-tolylsulfonyl-a-~xylofuranose (331) as a first step in the synthesis of 5-thio-~-xylopyranose (334). Owen and coworker^'^' applied S Ndisplacement ~ of the 5-p-tolylsul-

ZBIGNIEW J. WITCZAK

136

CH20Bz

CH20Bz KSCN

OBz 261 R ' = M s 321 R' = CF3SO2 CHZOH

328

OH

OBz 1

CH~OAC

H+,Ac,O, AcOH

CH20H

2. MeONa. MeOH

OAc 329

OH

330

Q3 =g>

CH~OTS

CH2SCN

0- CMel 331

CH2SH

=Qy

0- CMe2 332

/$

HOQOH

OH 334

0- CMe2 333

MONOSACCHARIDE ISOTHIOCYANATES A N D THIOCYANATES

137

fonyloxy group in 335 for confirmation of the resistance of the em-sulfonate group on C-3 in the furanose ring, as well as for comparison of the course of this displacement by thiobenzoate and thioacetate ions. The yield of thiocyanate 336 was not comparable to that reported earlier.'36

Ic;.

CH20Ts

CHZSCN

DMF,28 KScN h, 140"

'

Q

0- CMe2

0- CMe2

335

336

Tokuyama and c o ~ o r k e r s ' ~reported ~ ~ ' ~ ~ the displacement, with the thiocyanate ion, of the p-toluenesulfonate group in 2,3 :4,6-di-O-isopropylidene-1-0-p-tolylsulfonyl-cY-L-sorbofuranose (337) and 2,3-O-isopropylidene-l,6-di-O-p-tolylsulfonyl-a-~-sorbofuranose (341) in liquid ammonia, as well as in N,N-dimethylformamide. Treatment of 337 with potassium

CMe2

338

CMe2 337

340

339

Me2C KSCN P

DMF, 8 h, 140'

H0

HO 341

342

ZBIGNIEW J. WITCZAK

138

thiocyanate in N,N-dimethylformamide gives, instead of the thiocyanato sugar, the disulfide 339, which was considered to be formed by decomposition of the intermediate thiocyanate. It is noteworthy that similar S N ~ displacement of the methanesulfonyloxy group in octa-0-(methylsulfony1)sucrose produced 6,6'-dideoxy-1,2,3,4,3',4'-hexa-O-(methylsulfonyl)-6,6'di(thiocyanato)sucrose in 85% yield by selective, nucleophilic replacement of both of the primary methylsulfonyloxy groups by use of thiocyanate ion.'40 Nucleophilic displacement of p-toluenesulfonate groups at C-3 (usually highly resistant towards S N displacement) ~ in the furanose ring, with inversion of configuration, has been- reported by Defaye and Hilde~ h e i m . ' ~This ' displacement occurs much more readily than that of the CHlOH

0- CMel

NCS

0-CMe,

344

343

corresponding 5-0-tritylated compound 343 and 5-deoxy derivatives. It was suggested that intramolecular, electrophilic assistance is provided by the 5-hydroxyl group, as shown in 345, and that this would facilitate development of a negative charge on the sulfonate in the transition state. Treatment

.o\ o ,

I,'

H' \

Ar-Me

0- CMe,

345

of anhydro sugar 346 with potassium thiocyanate produced the stable, crystalline thiocyanate 332 in 12% yield. The fused, furanose episulfide 348

by by

CHI

CHZSCN

DM;:L8

0- CMel 346

0- CMe, 332

MONOSACCHARIDE ISOTHIOCYANATES AND THIOCYANATES

139

has been prepared by alkaline cleavage of thiocyanate 347 (synthesized by conversion of an epoxide into the 3-thiocyanate and subsequent to~ylation'~~). CHtOTr

CH,OTr

347

348

Goodman and reported the synthesis of thiocyanates as intermediates in the synthesis of the 3-thio-~-glucosederivative 354 and the 3-thio-~-allosederivative 355. The sequence started from the isomeric triflates 349 and 350. They found that the D-glum isomer 349 affords thiocyanate 351 in low yield, whereas the D - ~ Oisomer 350 gives thiocyanate 353 in 70% yield, when acetonitrile is used as the solvent. In the case of the D-gluco isomer 349,they also observed simultaneous formation of the partially deblocked derivative 352, as well as the unsaturated derivative 3deoxy- 1,2 :5,6-di-O-isopropylidene-cy.-~-erythro-hex-3-enofuranose (356), probably as a result of the presence of potassium triflate in the reaction mixture under these conditions. Notably, attempts to displace the sulfonyloxy group of 1,2 :5,6-di-0-isopropylidene-3-O-p-tolylsulfonyl-cy.-~-a~~ofuranose with the thiocyanate ion in N,N-dimethylformamide at high temperatures were unsuccessful, according to these IV. SPECTROSCOPIC PROPERTIES OF MONOSACCHARIDE ISOTHIOCYANATES The i.r. spectra of isothiocyanates and thiocyanates are more informative than 'H-n.m.r. and U.V. data. In contrast to thiocyanates, the isothiocyanates are distinguished by a strong, wide doublet band in the of 20201990 cm-' (see Table I). These can be used to distinguish isothiocyanates from thiocyanates, which have a sharp, medium-strong bandI4 at 21752100 cm-'. Ogura and TakahashP3 measured the c.d. spectra of a series of sugar isothiocyanates in acetonitrile solution. The spectra have a high 215 nm, and a somewhat less intense band at A,, intensity band at A,, 255 nm. 13 C-N.m.r. data for sugar isothiocyanates have been reported.33 Ogura and T a k a h a ~ hobserved i~~ the signal of the carbon atom of the isothiocyanate group in the range of 142.2-144.0p.p.m., whereas the C-1 signal was at 83.0-83.5 p.p.m. (see Table I).

ZBIGNIEW J. WITCZAK

140

349 R' = CF3S020,R2= H 350 R' = H, R2 = CF,SO,O ROCH2

NCS

/

OCH2

0- CMel

0-CMel

351 R=Me,C= 352 R = H

355

354

V. CONCLUSION It is rather obvious that sugar isothiocyanates (see Table 11) and thiocyanates (see Table 111) may now be considered to be not only classical, functional groups but also very good precursors for the synthesis of nucleoside analogs containing thio and deoxy sugars. The variety of methods for functionalization of sugar isothiocyanate molecules provides a number of attractive approaches to N-glycosyl heterocycles, a class of compounds of particular interest, which have been the subject of extensive investigation by many aUthors.54-69,80-85,90-~O0 In the near future, further developments concerning the utilization of new procedures or reagents and, probably, the discovery of new aspects of the reactivity of both isothiocyanates and thiocyanates may be expected.

MONOSACCHARIDE ISOTHIOCYANATES AND THIOCYANATES

141

TABLEI 1.r. and '3C-N.m.r.-spectral Data for Some Sugar I s o t h i ~ y a n a t e s ~ ~

Compound a-D-Arabinopyranosyl isothiocyanate 2,3,4-tri-O-acetylP- D-GlUCOpyraIIOSyl isothiocyanate 2,3,4,6-tetra-O-acetyl2,3,6-tri-0-acetyl-4-0-(2,3,4,6-tetra- 0acetyl-P- D-galactopyranosyl) 2,3,6-tri-0-acetyl-4-0-(2,3,4,6-tetra-O-acetyl-a-~glucopyranos y I) 2,3,6-tri-0-acetyl-4-0-(2,3,4,6-tetra-O-acetyl-~-~glucopyranosyl P-D-Ribofuranosyl isothiocyanate 2,3,5-tri-O-benzoyl-

2050

83.3

142.2

2100

83.5

144.1

1990

78.5

144.0

2025

83.0

144.2

2025

83.3

144.0

2000

88.5

144.4

TABLEI1 Isotbiocyaoates of Sugars

Compound

M.p. (degrees)

a-D-Arabinopyranosyl isothiocyanate 2,3,4-tri-O-acetylsyrup P-D-Galactopyranosyl isothiocyanate 2,3,4,6-tetra-O-acetylsyrup D-GlUCOnyl isothiocyanate 2,3,4,5,6-penta-O-acetyl132-135 a-D-GlUCOfUranOSe 6-deoxy-1,2 :3,5-di-O-isopropylidene6-isothiocyanato68 a-D-Glucopyranose 6-deoxy-6-isothiocyanatosyrup 1,3,4,6-tetra-O-acetyl-2-deoxy72-73 2-isothiocyanatoa-D-Glucopyranosyl isothiocyanate

[ a ] ~ Rotation (degrees) solvent

References

33,49,96 34,49 90

+45

MezCO

38

-

-

38

+73

DMF

39

4-249 +16.2

CHCI, CC4

37 30

3,4,6-tri-O-acetyI-2-deoxy-

2-thiocyanato2,3,4-tri- O-acetyl-6-bromo-6-deoxy-

94.5-96 164.5

(continued)

ZBIGNIEW J. WITCZAK

I42

TABLEI1 (continued)

M.p. (degrees)

Compound P-D-Glucopyranosyl isothiocyanate 2-acetarnido-3,4,6-trii-O-acetyl2-deoxy3,4,6-tri-O-acetyI-2-benzamido2-deoxy2,3,4,6-tetra-O-acetyI-

[a]D

(degrees)

References

161

+9.5

CHCI,

29

190 113-115

+38 +4.4

CHCI, CHCI,

32 7,8,33, 42,49,96

-

-

33,35,100

-

-

33

191-195

-

-

33

-

-

-

34,95

-

-60

CHCI,

36

-

+686

CHCI,

36

syrup

+375

CHCI,

20,21

68-69

-105

CHCI,

20,21

96-97

-

-

34,42

2,3,6-tri-0-acetyl-4- 0-(2,3,4,6-tetra-0acetyl-P- D-glucopyranosy1)157-159 2,3,6-tri-0-acetyl-4-0-(2,3,4,6-tetra-0acetyl-a-D-glucopyranosy1)120-123

2,3,6-tri-O-acetyl-4-0-(2,3,4,6-tetra-Oacetyl-P- D-glucopyranosyl)

Rotatlon solvent

2-acetamido-4-0-(2-acetamido3,4,6-tri-0-acetyl-2-deoxyp-~-glucopyranosyI)-3,6-di-OacetylHex-1-enitol, 3,4,6-tri-O-acetyI1,5-anhydro-2-deoxy-~-arabino2-isothiocyanatoHex-1-enitol, 4,6-di-O-acetyll,S-anhydr0-2-3-dideoxy-~ribo-3-isothiocyanatoHex-3-epopyranoside, ethyl D-threo2,3,4,6-tetradeoxy-2-isothiocyanato-6- 0-(methylsu1fonyl)Hex-3-enopyranoside, ethyl D-erythro2,3,4,6-tetradeoxy-2-isothiocyanato-6-O-(methylsulfonyl)P-D-Ribofuranosyl isothiocyanate 2,3,5-tri-O-benzoyl-

For these reasons, the field of carbohydrate isothiocyanates and thiocyanates will remain a rich area of investigation for many years to come. ACKNOWLEDGMENTS The author thanks Dr. James R. Daniel, Department of Food and Nutrition, h r d u e University, West Lafayette, Indiana 47907, for helpful discussions, and for reading the manuscript. Thanks are also due Professor Roy L. Whistler for his valuable advice and encouragement, and for making available the needed facilities.

M O N O S A C C H A R I D E ISOTHIOCYANATES AND THIOCYANATES

143

TABLE 111 Thiocynnates of Sugars ~

Compound

~~~

Map. (degrees)

~~~

(degrees)"

Allofuranose 5,6-di0acetyl-3-deoxy1,2-O-isopropylidene99-100 +79.6 3-thiocyanato-a-~3-deoxy-1,2-O-isopropylidene-3thiocyanato-a- D109-110 +66 3-deoxy-l,2 : 5,6-di0isopropylidene-3thiocyanato-a-D48-50 +63.9 Altropyranoside,methyl a - ~ 4,6-0benzylidene-3-deoxy-3-thiocyanato188-190 0 4,6-0benzylidene-3-deoxy-2-0(methylsulfonyl)-3-thiocyanatoSYNP 4,60benzylidene-2,3-dideoxy-3(dimethylamino)-2-thiocyanato99-101 +62 2,3-di-O-benzyl-4,6-dideoxy-4-thiocyanatoSYWP p-D-Fructofuranosyla-D-glucopyranoside, 6,6'-dideoxy-6,6'-dithiocyanato2,3,4,1',3',4'-hexa-O-acetyl169-171 +71 2,3,4,1',3',4'-hexa-O-benzoyl92-95 +39.9 1,2,3,4,3',4'-hexa-O-(methyIsulfonyl)177-180 +68.5 Galactopyranoside,methyl WD2,3-di0-acetyl-6-deoxy-6-thiocyanato-40p - tolylsulfonyl126-129 +167 2,3-di-O-acetyl-4,6-dideoxy-4,6-di(thiocyanato)- 183-185 +134 2,3,4-tri-O-acetyl-6-deoxy-6-thiocyanato2.3-di-O-benzoyl-4-deoxy-4-thiocyanato132-133 +89.4 2,3-di-0-benzoyl-4,6-dideoxy-4,6-di(thiocyanato)212-214 $93.5 a-D-Glucofuranose 5,6-di-O-acetyl-3-deoxy-l,2-0isopropylidene-3-thiocyanatoSYNP -29.4 3-deoxy-1,2-0-isopropylidene-3-thiocyanatosyrup -76.6 3-deoxy-1,2 : 5,6-di-O-isopropylidene-3thiocyanato43-44 -75.5 Glucofuranoside,methyl p-D3-deoxy-3-thiocyanato-2- O-p-tolylsulfonyl5-0-tritylGlucopyranose 1,3,4-tri-O-acetyl-6-deoxy-6-thiocyanato2-0-p-tolylsulfonyl-a-D1,2,3,4-tetra-O-acetyl-6-deoxy-6-

thiocyanato-a-D-

References

135 135 135 129-131 129,130 133 125 140 140 140

122 122 123 115 113,122 135 135 135

142

136

+137

109,110

11.7-1I8

+24

7,109,110

100-101

+144.9

1,3,4,6-tetra-O-acetyl-2-deoxy-2-

thiocyanato-a-D-

37 (continued)

ZBIGNIEW J. WITCZAK

144

TABLE111 (continued)

Compound Glucopyranose 1,3,4,6-tetra-O-acetyl-2-deoxy-2thiocyanato-p-D1,2,3,4-tetra-0-acetyl-6-O-pto1ylsulfonyl-p-DGlucopyranoside, methyl a - ~ 2,3-di-O-acetyI-4,6-dideoxy-4,6di(thiocyanat0)2,3-di-O-acetyl-6-deoxy-4-0(methylsulfonyl)-6-thiocyanato2,3,4-tri-O-acetyl-6-deoxy-6-thiocyanato2,3,6-tri-0-benzoyl-4-deoxy-4-thiocyanato4,6-dideoxy-4-thiocyanato-2-O-p-

tolylsulfonyl-3-O-(trimethylsilyl)Glucopyranoside, methyl p-D2,3-di-O-acetyl-6-deoxy-6-thiocyanatoa-D-Glucopyranosyl bromide 2,3,4-tri-O-acetyl-6-deoxy-6-thiocyanatoa-D-Glucopyranosyl chloride

3,4,6-tri-O-acetyl-2-deoxy-2-thiocyanatoa-D-Ghcopyranosyl isothiocyanate 3,4,6-tri-O-acetyl-2-deoxy-2-thiocyanatoP- D-Glucopyranosyl thiocyanate 2,3,4-tri-O-acetyl-6-brorno-6-deoxy2,3,4,6-tetra-O-acetyIGlucopyranoside, methyl p-D2,3,4-tri-O-acetyl-6-deoxy-6-thiocyanato3,4,6-tri-O-acetyl-2-deoxy-2-thiocyanatoGulopyranoside, methyl WD2-0-acetyl-4,6-dideoxy-4-thiocyanato2,3-di-O-acetyl-4,6-dideoxy-4-thiocyanato2,3-di-0-benzoyl-4,6-dideoxy-4-thiocyanato4.6-dideoxy-4-thiocyanato-2O-p-tolylsulfonylHex-1-enitol, 1,5-anhydro-~-arabino3,4,6-tri-O-acetyl-2-deoxy-2-thiocyanatoHex-2-enopyranoside, ethyl a - D - threo6-azido-2,3,4-trideoxy-4-thiocyanato2,3,4-trideoxy-6-0-(methylsulfonyl)4-thiocyanatoHex-2-enopyranoside, ethyl a-D-erythro2,3,4-trideoxy-6- 0-(methylsulfonyl)4-thiocyanato-

M.p. (degrees)

Iff ID (degrees)”

References

99-101

72.5

37

117-1 18

+27.9

8

144-145

+56

171.5- 172.6 101-103 194-194.5

+150 +150.8 +60.3

-

-

134-135

+15.6

8

160

+212.1

8

93-95

+120.6

37

+249

37

94.5-96

122 122 8 118-121,135 126- 128

164.5 132-133

-16.4 -20.9

30 8

134- 135 syrup

+15.6 +99.9

7 37

123-124

-

syrup

+20.2

-

-

126-128 125 126,127 126- 128 37 22, 23

91-92

-275

20.21

85-86

+115

20, 21 (continued)

MONOSACCHARIDE ISOTHIOCYANATES AND THIOCYANATES TABLEI11

(continued)

Compound Idopyranoside, methyl a - ~ 2,3-di-0-benzyl-4,6-dideoxy-4-thiocyanatoa-D-Mannopyranose 1,3,4,6-tetra-O-acetyl-2-deoxy-2-thiocyanatoMannopyranoside, methyl a-D3,4,6-tri-0-acetyl-2-deoxy-2-thiocyanato-

2,3,4-tri-O-acetyl-6-deoxy-6-thiocyanato-

M.p. (degrees)

lab (degrees)"

-

-

125

SYNP

+83.5

37

syrup

+79.3 +93

37 123

+1.3

37

+98.4

37

-

Mannopyranoside, methyl p-D3,4,6-tri-O-acetyl-2-deoxy-2-thiocyanato126-127 a-D-Mannopyranosyl chloride SYNP 3,4,6-tri-O-acetyl-2-deoxy-2-thiocyanatoa-D-Ribofuranose 3-deoxy-1,2-O-isopropylidene-3-thiocyanato- 101.5-192.5 a-L-Sorbofuranose l-deoxy-2,3 :4,6-di-O-isopropylidene-lSYNP thiocyanatoa,a-Trehalose 2,3,4,2',3',4'-hexa-O-acetyI164 6,6'-dideoxy6,6'-dithiocyanatoa-D-Xylofuranose 5-deoxy-1,2-0-isopropylidene-5-thiocyanatoa

Rotation solvent, CHC13.

145

+50

-10.6

References

141

138,139

+116

123

-30

137

This Page Intentionally Left Blank

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 44

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE BY BARRYv. MCCLEARY* A N D NORMANK. MATHESONt

* Biological and Chemical Research Institute, N.S. W. Department of Agriculture, Rydalmere 21 16, Australia

t Department of Agricultural Chemistry, The University of Sydney, N.S. W.2006, Australia

I. INTRODUCTION In an earlier article' on the application of enzymic techniques to the analysis of the structure of polysaccharides, the (Y-D- and P-D-glucans were discussed, as well as more-general aspects of the preparation and use of catabolic enzymes in such analyses. The present article describes enzymic contributions to knowledge of the structures of other polysaccharides, but an account of subsequent research on a- and P-D-glucans is also included. The properties and action patterns of glycosidases and polysaccharide depolymerases have been as also have the enzymes involved in biosynthetic pathway^.^.' An understanding of biosynthesis can provide insights into the structures of polysaccharides. Reviews concerning particular polysaccharides have appeared, and references to these will be given in relevant sections. Enzymes depolymerizing polysaccharides may have an endo or an em action pattern, and may hydrolyze, or cleave by elimination. Both the conformation of the polysaccharide and the active site of the enzyme need to be considered in the enzyme-glycan interaction. endo-Enzymes split, by a random type of depolymerization, glycosidic bonds situated internally in ( 1 ) J. J. Marshall, Adv. Carbohydr. Chem. Biochem., 30 (1974) 257-370. (2) H. M. Flowers and N. Sharon, Ado. Enzymol., 48 (1979) 29-95. (3) K. Nisizawa and Y. Hashimoto, in W. Pigman, D. Horton, and A. Herp (Eds.), The Carbohydrates, 2nd edn., Vol. 2A, Academic Press, New York, 1970, pp. 241-300. (4) R. F. H. Dekker and G . N. Richards, Adu. Carbohydr. Chem. Biochem., 32 (1976) 271-352. (5) N. K. Matheson and B. V. McCleary, in G . 0. Aspinall (Ed.), The Polysaccharides, Vol. 3, Academic Press, New York, 1985. pp. 1-105. (6) H. Nikaido and W. Z. Hassid, Adu. Carbohydr. Chem. Biochem., 26 (1971) 351-483, (7) D. W. James, J. Preiss, and A. D. Elbein, in Ref. 5, pp. 107-207.

147

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148

BARRY V. McCLEARY AND NORMAN K. MATHESON

the glycan chain, yielding a series of oligosaccharide fragments, some of which can be further cleaved. exo-Glycanases sequentially release monosaccharide or oligosaccharide repeating-units from one end of the glycan chain, most commonly the nonreducing end. Lyases cleave by elimination, producing an alkenic bond at the nonreducing end. endo-Hydrolases also catalyze transglycosylation, namely, the transfer of an oligosaccharide fragment from the enzyme-product complex to another oligosaccharide, instead of to water; this reaction should be considered in the interpretation of results. Many glycosidases that hydrolyze low-molecular-weight substrates also hydrolyze polysaccharides, releasing monosaccharide units from nonreducing termini. Some enzymes have been described as endo-glycosidases. Their substrates are the oligosaccharide chains of glycoproteins, and they usually hydrolyze at one point near the linkage to protein (see Section XI). In at least one case, endo-p-D-galactosidase (EC 3.2.1.103), the enzyme also hydrolyzes between repeating units in both keratan sulfate (see Section IX,4) and glycolipids (see Section XI). With some endo-glycosidases, suitable substrates are not available that would permit determination of whether multiple scission can occur. There appears to be some problem in the classification of these enzymes. They hydrolyze polymer chains containing multiple constituent sugars, but the enzymes from phages [that hydrolyze bacterial polysaccharides (see Section X)] are described as endo-glycanases. Sometimes, a division between an endo and exo pattern may not be clear; * ~ 4.2.2.5). exo-Maltotetraohyfor example, chondroitin AC l y a ~ e * (EC drolase" (EC 3.2.1.60) can, under some act in an endo manner. The behavior of this enzyme could be the result of a high rate of ex0 hydrolysis of exterior a-(1 + 4) chains of amylopectin, combined with a much lower rate of endo hydrolysis"" of the limit dextrin. Pullulanase (EC 3.2.1.41) (see Section XIII,2) hydrolyzes pullulan by an endo mechanism, but preferentially removes exterior chains from amylopectin. The hydrolysis products from the action of Basidiomycete exo-( 1 + 3)/3-D-glucanase (EC 3.2.1.58) on the laminaran of Eisenia bicyclis,l'b*l'c which has p-(1+6) linkages in the (1+3)-p-~-glucan chain, as well as single D-glucosyl branches linked p-( 1 + 6), indicated that this enzyme, which normally cleaves ( 1 + 3)-/3-~-glucansby an ex0 action, can

(8) (9) (10) (11) (lla) (llb) (llc)

T. Yamagata, H. Saito, 0.Habuchi, and S. Suzuki, J. Biol. Chem., 243 (1968) 1523-1535. H.-P. Ulrich, U. Klein, and K. von Figuta, Z. Pfiysiol. Chem., 360 (1979) 1457-1463. Y. Sakano, E. Kashiyama, and T. Kobayashi, Agric. Bid. Chem., 47 (1983) 1761-1768. J. Schmidt and M. John, Biochirn. Biopfiys. Acru, 566 (1979) 88-99. T. Nakakuki, K. Azuma, and K. Kainuma, Curbohydr. Rex, 128 (1984) 297-310. F. Nanjo, T. Usui, and T. Suzuki, Agric. Biol. Chem., 48 (1984) 1523-1532. R. Yamamoto and D. J. Nevins, Carbohydr. Res., 122 (1983) 217-226.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

149

act in an endo manner on the Eisenia D-glucan prior to ex0 cleavage of the fragments. A particular exo-glycanase or glycosidase may not have all the distinguishing characteristics of one of these classes of enzyme^.^ In one cellobiohydrolase (EC 3.2.1.91; from Trichoderma case a (1 + 4)-P-~-glucan viride)lId that released cellobiose from cellulose was found then to act further as a P-D-glucosidase (EC 3.2.1.21), hydrolyzing this disaccharide to D-glucose. Enzymes having the same name and Enzyme Commission (EC) number, but isolated from different sources, may show major differences in rates of reaction and action patterns. Some glycosidases can hydrolyze sugars having more than one configuration, for example p-D-gluco and P-galacto, but Many others have a strict requirement for one gro~p-configuration.'~-'~ show quantitative, and even qualitative, differences in their capacity to hydrolyze glycosidic bonds to different hydroxyl groups in the next sugar residue. Thus, the V,,,/Km value for the reaction of buckwheat (Y-Dglucosidase (EC3.2.1.20) with maltose was more than 500 times that for hydrolysis of is~maltose.'~ P-D-Glucosidase from almond emulsin hydrolyzes cellobiose, but has little action on 4-O-~-~-glucosyl-~-mannose.'~ Glycosidases do not hydrolyze a glycon that is substituted by another sugar. An enzyme preparation from Aspergillus niger, initially believed to be a P-D-glucosidase that hydrolyzed cellobiose substituted on 0 - 6 of both the D-glucose residue and the D-glucosyl group, releasing 6- O-a-~-xylosylD-glucose, has been to be a mixture of P-D-glucosidase and an exo-glycanase that releases a heterodisaccharide. exo-Glycanases differ in their ability to hydrolyze near a branch point. exo-( 1 + 4)-P-~-Mannanase could not cleave an unsubstituted D-mannosyl group attached to the nonreducing end of a D-mannose residue substituted by a D-galactosyl group," but exo-( 1 + 3)-P-~-glucanasecan bypass a D-glucosyl group that is

( l l d ) G. Beldman, M. F. Searle-Van Leeuwen, R. M. Rombouts, and F. G . J . Voragen, Eur. J. Biocbem., 146 (1985) 301-308. (12) D. E. Walker and B. Axelrod, Arch. Biocbem. Biopbyr, 187 (1978) 102-107. (13) M. A. Chinchetru, J . A. Cabezas, and P. Calvo, Comp. Biocbern. Pbysiol, B, 75 (1983) 719-728. (14) G . A. Levvy and S. M. Snaith, Adv. Enzymol, 36 (1972) 151-181. ( 1 5 ) S. Chiba, K. Kanaya, K. Hiromi, and T. Shimomura, Agric. Bid. Cbem., 43 (1979) 237-242. (16) B. V. McCleary and N . K. Matheson, Carbobydr. Rex, 119 (1983) 191-219. (17) T. Watanabe, K. Takahashi, and K. Matsuda, Agric. Biol. Cbem., 44 (1980) 791-797. (17a) Y. Kato, J. Matsushita, T. Kubodera, and K. Matsuda, J. Biocbem. (Tokyo),97 (1985) 801 -8 10. (18) B. V. McCleary, Carbobydr. Rex, 1 1 1 (1983) 297-310.

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BARRY V. McCLEARY AND NORMAN K. MATHESON

p-( 1+ 6 ) a b s t i t u t e d by another D-glucosyl group, releasing gentiobiose.” Different endo-glycanases need various lengths of uninterrupted, homoglycan chain in a branched polymer for hydrolysis to occur. All these types of differences in action pattern mean that the more precisely this has been determined for an enzyme from a particular source the more significant will be the results. Modern methods of protein purification, such as ion-exchange, gel, and affinity chromatography, allow the preparation of pure polypeptides, free from contaminating activities. For unequivocal results, the enzyme applied should consist of a single protein species having a single catalytic activity. The purity of enzymes used in the studies described in this article has varied from cell extracts having little or no description of possible extraneous activities to purified proteins characterized as single polypeptides by gel electrophoresis and isoelectric focusing. Enzymes may be used to detect major linkage-types in polysaccharides, and to purify specific polysaccharides from mixtures by selectively depolymerizing contaminants. They produce oligosaccharides in high yield, cleave polymers having acid-resistant linkages, and enable the isolation of oligosaccharides containing acid-sensitive bonds. Combined with fractionation, quantitative recovery, and characterization of fragments, details of glycan structure can be determined. The availability of size-exclusion gels on which oligosaccharides can be separated has allowed the isolation of fractions in reasonable quantities. These fractions from the enzymic cleavage of the polymers can then be examined by using further enzymic degradation, methylation analysis, n.m.r. spectroscopy, and mass spectrometry. With some polysaccharides, enzymes provide the only way at present available for studying aspects of fine structure. Many biosynthetic enzymes have also contributed to our knowledge of the structures of polysaccharides. The high specificity of reaction of glycosyltransferases limits the structure they can form and, in some cases, for example, the glycoproteins, a polymer having one biological activity has been converted into another, having a different activity, by a linkage-specific glycosyltransferase. 11. POLYSACCHARIDES HAVINGA (1 + 4)-@-D-GLUCANBACKBONE 1. Cellulose

Polysaccharides having a (1 + 4)-linked, @-D-glucan backbone include the homopolymer cellulose, plus others, such as xyloglucan and xanthan, (19) T. E. Nelson, J. Johnson, E. Jantzen, and S. Kirkwood, J. Biol. Chem., 244 (1969) 5912-5980.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

151

in which this backbone is substituted by neutral, or charged, branch units. On hydrolysis of swollen cellulose by cellulase [endo-( 1+ 4)-p-~-glucanase (EC 3.2.1.4)],'9a the major products are cellobiose and cellotriose, together with D-glucose and cellotetraose.2"-22The recovery of these in high yield is consistent with cellulose being composed of (1+ 4)-linked p-D-glucosyl residues. Owing to the highly insoluble nature of cellulose, such chemically modified substrates as acid-swollen or water-soluble 0-(carboxymethy1)cellulose are used as enzyme substrates. Substitution restricts the extent of hydrolysis, and this property has been used in studies on the location and distribution of chemical substituents in these polymers.23 2. Xyloglucans

Xyloglucans, which also contain lesser proportions of D-galactose, and may also contain L-fucose and L-arabinose, have been referred to as amyloids, because of their color reaction with iodine. The first thorough examination of a xyloglucan was of the polysaccharide from the seeds of Tamarindus i n d i ~ a It . ~ is ~ susceptible to hydrolysis by cellulase, and it yielded, as major components, hepta-, octa-, and nona-saccharide fractions, consistent with a (1+ 4)-p-~-glucanbackbone. These fractions were separated by paper chromatography, and partially characterized. Treatment of the polysaccharide with a preparation that contained p-D-galactosidase (EC 3.2.1.23) and p-D-glucosidase, as well as cellulase, yielded 6 - 0 - w ~ xylosyl-D-glucose (isoprimeverose), D-glucose, and D-galactose. It was concluded that the polymer consists of a main chain of p-(1+4)-linked D-glucosyl residues to which D-xylosyl groups are attached a - ( 1+ 6) to three out of every four main-chain residues. The combined yield of hepta-, octa-, and nona-saccharides, and their compositions, indicated that there was an average structural-unit made up of one unsubstituted and three substituted D-glucosyl residues. The configuration of the D-galactosidic linkage was established by using a preparation containing P- D-galactosidase. Treatment of an alkali-soluble xyloglucan from the seed of Annona muricuta with cellulase liberated tetra- and penta-saccharides, together with D-glucose, cellobiose, and a small proportion of a ~-xylosylcellobiose.~~ From the molar amounts of these products, it may be postulated that the (19a) (20) (21) (22)

T. M. Wood, Biochem. Soc. Trans., 13 (1985) 407-410. S. P. Shoemaker and R. D. Brown, Biochim. Biophys. Acru, 523 (1978) 133-146; 147-161. G. Okada, J. Biochem. (Tokyo), 80 (1976) 913-922. T. Kanda, K. Wakabayashi, and K. Nisizawa, J. Biochem. (Tokyo),79 (1976) 977-988;

989-995. (23) S. S. Bhattacharjee and A. S. Perlin, J. Polym. Sci., Purr C, 36 (1971) 509-521. (24) P. Kooiman, Red. Truu. Chim. Pup-Bus, 80 (1961) 849-865. (25) P. Kooiman, Phytochemisrry, 6 (1967) 1665-1673.

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BARRY V. McCLEARY AND NORMAN K. MATHESON

polymer consists of an average structural-unit (1) of cellotetraose substituted at 0 - 6 of one D-glucosyl residue by a disaccharide group composed of a D-xylosyl residue and a D-galactosyl group.

t

1 ff-D-Xyl 2

t

1 P-D-Gal 1

In the reported structures of products of cellulase digestion of xyloglucans from different sources, there has been considerable variation that may reflect different action-patterns of enzyme preparations as much as actual diff erences in the structures of the polysaccharides. On reaction of xyloglucan from the walls and culture medium of suspension-cultured, sycamore four major oligosaccharide products were isolated. Stuctures were proposed for the heptamer (2) and the nonamer (3). P-D-GlC-(1 +d)-P-D-GlC-(1+ 4)-P-D-GlC-(1+ 4)-D-GlC 6 6 6

t

1 ff-D-Xyl

t

t

1 Cr-D-XYl

1 ff-D-XY1

2

(26) W. D. Bauer, K. W. Talmadge, K. Keegstra, and P. Albersheim, Plant Physiol., 51 (1973) 174-187. (27) A. Darvill, M. McNeil, P. Albersheim, and D. P. Delmer, in N. E. Tolbert (Ed.), The Biochemistry of Plants, Vol. I, Academic Press, New York, 1980, pp. 91-162.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

153

Treatment of a hemicellulosic arabinoxyloglucan from the midrib of leaves of Nicotiana tabacum with cellulase gave a complex mixture of oligosaccharides.28 Separation and characterization of these provided the first convincing evidence for the attachment of cY-L-arabinofuranosyl groups to D-xylose residues. To obtain oligosaccharides having simpler structures, the arabinoxyloglucan was pretreated with mild acid (to remove Larabinose), before treatment with ~ e l l u l a s e Tri. ~ ~ and penta-saccharides were then released, and were characterized chemically and by n.m.r. spectroscopy as 4 and 5. Nine components were separated by gel chromatography

5

4

of the cellulase digest of intact arabinoxyl~glucan.~" The major fraction contained L-arabinose, D-xylose, and D-glucose in the ratios of 1 :2 : 3, and had the proposed structure 6. The absolute point of attachment of the @-D-GlC-(I+ 4)-P-D-GlC-(1+4)-D-GlC 6 6

t

t

1 Cr-D-XYl

'1 ff-D-Xyl

2

t

1 a-L-Ara 6

L-arabinofuranosyl group in 6, and the anomeric configuration of those in another fraction, defined as 7, were not determined. An alkali-insoluble @-D-GlC-(1 -+ 4)-P-D-GlC-( 1 -* 4)-D-GIC 6 6

t

1 ff-D-Xyl 2

t

1 ff-D-XYl 2

t

t

1

L-Ara

1

L-Ara

I (28) S. Eda and K. Katii, Agric. Biol. Chern., 42 (1978) 351-357. (29) M. Mori, S. Eda, and K. Katii, Agric. Biol. Chern., 43 (1979) 145-149. (30) M. Mori, S. Eda, and K. Katii, Carbohydr. Rex, 8 4 (1980) 125-135.

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BARRY V. McCLEARY AND NORMAN K. MATHESON

fraction from cotyledons of tora bean3’ contained L-arabinose, D-xylose, D-galactose, and D-glucose in the molar ratios of 1.0: 0.2: 0.1 : 1.2. Extensive digestion with a-L-arabinofuranosidase (EC 3.2.1.55) released most of the L-arabinosyl groups, leaving a galactoxyloglucan. Digestion of the xyloglucans from etiolated soybean, Vigna sesquipedalis, and mung-bean hypocotyls, and examination of the profiles of released oligosaccharides by gel chromatography, indicated that the structures of these are very similar, and are based on two oligosaccharide units, one of which consists of D-glucose and D-xylose, and the other, of D-glucose, D-xylose, D-galactose, and ~-fucose.~’ Oligosaccharides in the mung-bean xyloglucan digest were separated preparatively by gel c h r ~ m a t o g r a p h y , ~ ~ and identified as 2-5,8, and 9 by using methylation analysis, and hydrolysis p - ~ - G l c -1(+ 4)-D-GlC 6

p - ~ - G l c -1(+ 4)-D-GlC 6 6

t

t

t

1

1

(Y-D-XYl 2

1 a-D-Xy1

(Y-D-Xyl 9

t

1

L

t

1

(Y-L-FUC 8

by a-L-fucosidase (EC 3.2.1.51), p-D-galactosidase, and an Aspergillus oryzae enzyme preparation. This enzyme was able to hydrolyze a D-ghcoside substituted at the primary hydroxyl group by a D-xylosyl group by an ex0 mechani~rn,”~ and this explains the production of isoprimeverose in the as the cellulase preparation was hydrolysis of tamarind-seed xylogl~can,’~ not pure. The structure of a decasaccharide from a mung-bean hydrolyzate was later e s t a b l i ~ h e d ~ from ~ ” the action of the A. oryzae exo-enzyme to be 3, with a ~-galactosylgroup attached to the xylose unit on the penultimate D-glycosyl residue at the nonreducing end. Treatment with a-D-xylosidase, followed by methylation analysis, confirmed that the D-XYIOSYI group on the D-glucosyl residue at the nonreducing terminus was unsubstituted. Similar oligosaccharides were found in a digest of soy-bean x y l o g l ~ c a n . ~ ~ Soluble and cell-wall xyloglucans from etoliated mung-bean hypocotyls (31) (32) (33) (33a) (34)

K. Ohtani and A. Misaki, Agric. Bid. Chem., 44 (1980) 2029-2038. Y. Kato, N. Asano, and K. Matsuda, Plan! Cell Physiol., 18 (1977) 821-829. Y. Kato and K. Matsuda, Agric. Biol. Chem., 44 (1980) 1751-1758; 1759-1766. J. Matsuchita, Y. Kato, and K. Matsuda, Agric. Biol. Chem., 49 (1985) 1533-1534. T. Hayashi and K. Matsuda, 1. Biol. Chem., 256 (1981) 11,117-11,122.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

155

have been ~ o m p a r e d . ~Both ' gave very similar, oligosaccharide patterns on cellulase digestion, and the difference in solubility of the polysaccharides appeared to be due solely to a difference in molecular weight. Oligosaccharide 9 was also present in a cellulase digest of jojoba-seed xyloglucan." Incubation of 9 with an A. oryzae enzyme preparation gave isoprimeverose exclusively, and similar treatment of a trisaccharide product gave only D-glucose and isoprimeverose. Xyloglucans from cell walls of oat-coleoptile tissue,36immature barley,37 ~~ soybean rice endospe~m,~'Phaseolus c o c c i n e u ~ , suspension-cultured bamboo shoot? and elongating pea-tissue4'" have also been characterized by employing cellulase preparations. Rice-endosperm, cell-wall xyloglucan3*with cellulase gave, among the reaction products, an octa- and a penta-saccharide, for which structures 10 and 5 were respectively proposed. P-D-Gk-( 1 +4)-P-D-GlC-( 1 + 4)-P-D-GlC-( 1 + 4)-D-GlC 6 6 6

t

1 Cr-D-XYl 2

t

t

1

1

Cr-D-XyI

Cr-D-xyl

t

1

P-D-Gal 10

The anomeric configuration of the terminal D-galactosyl group was established with P-D-galactosidase. The xyloglucan of suspension-cultured ricecells released40bcellobiose, 2,4,5, and 2 with a (1 + 2)-P-~-galactosylgroup attached to a D-XYIOSYI residue, as well as 10a. P-D-GlC-( 1 + 4)-P-D-GlC-( 1 +4)-P-D-GIC-( 1+ 4)-D-GlC 1

1

t

t

6 a-D-Xyl

6 Cr-D-XYl 10a

Y. Kato and K. Matsuda, Agric. Biol. Chem., 45 (1981) 1-8. J. M. Labavitch and P. M. Ray, Phyrochemisrry, 17 (1978) 933-937. Y. Kato, K. Iki, and K. Matsuda, Agric. Biol. Chem., 45 (1981) 2745-2753. N. Shibuya and A. Misaki, Agric. Biol. Chem., 42 (1978) 2267-2274. M.A. O'Neill and R. R. Selvendran, Carbohydr. Res., 111 (1983) 239-255; 145 (1985) 45-58. (39a) T. Hayashi, Y. Kato, and K. Matsuda, Plant Cell Physiol., 21 (1980) 1405-1418. (40) Y.Kato, R. Shiozawa, S. Takeda, S. Ito, and K. Matsuda, Carbohydr. Res. 109 (1982) 233-248. (40a) T. Hayashi and G. Maclachlan, Plant Physiol., 75 (1984) 596-604. (40b) Y. Kato and K. Matsuda, Plant Cell Physiol., 26 (1985) 437-445.

(35) (36) (37) (38) (39)

156

BARRY V. McCLEARY AND NORMAN K. MATHESON

The proportions of these indicated that the polymer from the suspensioncultured cells was more highly branched than a seedling preparation.@" It also contained D-galactosyl groups, as did the endosperm cell-wall polysachar ride,^' which was in contrast to the seedling xyloglucan, which, on cellulase hydrolysis, produced only cellobiose, 4, 5, and 10a. Oligosaccharides 4 and 5 were present in the cellulase digests of xyloglucan from both immature-barley3' and b a m b o o - ~ h o ocell-walls. t~~ In both cases, treatment with A. oryzae enzyme preparation gave D-glucose and isoprimeverose. From the molar ratio of preponderant oligosaccharides and monosaccharides released on cellulase digestion of barley xyloglucan, the average structural unit can be described as 11.

11

The xyloglucan from cell walls of Phaseolus c o c c i n e ~ scontained ~~ Larabinose, L-fucose, D-galactose, D-xylose, and D-glucose in the ratios of 4 :6 :9 :34 :46. Treatment with a-L-fucosidase released -85% of the Lfucose, and this fact, together with the results of methylation analysis, showed that L-fucose was present solely as terminal, a-linked groups. Xyloglucan has been synthesized in uitro with a particulate-enzyme fraction from suspension-cultured, soybean cells.34The enzyme catalyzed the transfer of the D-glucosyl group from UDP-~-['~C]glucoseand of the D-xylosyl group from UDP-~-['~C]xylose into a ~yloglycan.~' On treatment of this xyloglucan with cellulase, oligosaccharides 2, 4, 5, 8, and 9, and a small proportion of a nonasaccharide were obtained.34 Treatment of these oligosaccharides with an A. oryzae preparation gave [ ''C]monosaccharides and a [14C]disaccharide having chromatographic and electrophoretic mobilities indistinguishable from those of isoprimeverose.

3. Xanthan Xanthan is an extracellular polysaccharide produced by Xanthomonas campestris and related species. The structure of xanthan was determined ~ h e m i c a l l y ~as* *a ~p-( ~ 1 + 4)-linked D-glucan to which are attached trisaccharide side-chains on each second D-glucosyl residue, as depicted in 12. (40c) (41) (42) (43)

Y. Kato, S. Ito, K. Iki, and K. Matsuda, Planr Cell Physiol., 23 (1982) 351-364. T. Hayashi and K. Matsuda, Plant Cell Physiol., 22 (1981) 517-523. P.-E. Jansson, L. Kenne, and B. Lindberg, Carbohydr. Res. 45 (1975) 275-282. L. D. Melton, L. Mindt, D. A. Rees, and G . R. Sanderson, Carbohydr. Rex, 46 (1976) 245-257.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

157

P-D-Man 1

.1

4 P-D-GIcA 1

3. 2

a-D-Man 6Ac 1

31 4)-P-D-Gk-(I

/

%,,

12

However, in the polysaccharides obtained from some mutant strains, there are deviations from this idealized structure.& Xanthan is relatively resistant to enzymic hydrolysis, but it has been cleaved by an enzyme preparation from a Bacillus sp. at moderate temperatures and in the presence of buffer salts, yielding mono- and oligo-sa~charides.~~ A partially purified, enzyme ~ r e p a r a t i o hydrolyzed n~~ deacetylated or depyruvated xanthan, and also xanthan from several wild-type and mutant strains of Xanthomonas. The release of reducing material varied little with xanthan preparations having differences in 0-acetyl and pyruvic acetal contents. Under similar conditions of incubation, cellulase acted only on xanthan from mutant strains that had defective side-chain formation. The observation4’ that the stability of the secondary-ordered structure of xanthan (and as a consequence, its susceptibility to hydrolysis by cellulase) is a function of temperature, pH, and ionic strength has allowed the application of enzymic techniques to structural analysis. Xanthan is hydrolyzed in salt-free, aqueous solutions at elevated temperatures; that is, hydrolysis occurs if the chain is unordered. It was proposed that, at lower temperatures and in the presence of salt, the small side-chains organize around the (1 + 4)-P-~-glucanbackbone and protect it from hydrolysis. The action of various cellulase preparations on solutions of commercial xanthans, and those from laboratory strains containing both pyruvate and acetate, pyruvate only, or acetate only, in the unordered state has been (44) (45) (46) (47)

C. Whitfield, I. W. Sutherland, and R. E. Cripps, J. Gen. Microbiol., 124 (1981) 385-392. S. M. Lesley, Can. J. Microbiol, 7 (1961) 815-825. 1. W. Sutherland, J. Appl. Bacieriol., 53 (1982) 385-393. M. Rinaudo and M. Milas, Int. J. Biol. MacrornoL, 2 (1980) 45-48.

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BARRY V. McCLEARY AND NORMAN K. MATHESON

Reaction products were separated by chromatography on BioGel P-30 and P-6 or P-2, and consisted of large fragments, two major oligosaccharides, and products of lower molecular weight (identified as D-glucose, cellobiose, and in some hydrolyzates, D-mannose). The large fragments contained D-glucose, D-mannose, and D-glucuronic acid in the expected 2 : 2 : 1 molar ratio, and apparently consisted of oligomers of the repeating unit of xanthan. Acetate and pyruvic acetal were present in nonstoichiometric proportions. The two oligosaccharides were also composed of D-glucose, D-mannose, and D-glucuronic acid in the molar ratios of 2: 2: 1. Hydrolysis with P-D-mannosidase (EC 3.2.1.25) confirmed the presence of a P-linked D-mannosyl group at the nonreducing terminus on the branch unit. This enzyme was not active against oligosaccharides carrying a terminal acetal on this residue. The penta- and deca-saccharides from the hydrolyzate of strain 1128 xanthan, which was not pyruvated, and a small amount of nonacetylated material from other strains, released Dmannose and a tetrasaccharide or an octasaccharide, in which the ratio of D-glucose :D-mannose :D-glucuronic acid was 2 : 1 : 1. It was concluded that the results indicated probable non-regularity of substitution of side chains, with -10% of the D-glucosyl residues that would be expected to carry side chains in a regular, repeating structure not being substituted. Some endoglucanase preparations contain a lyase that produces a tetrasaccharide having an unsaturated glycosyluronic group and pyruvic acetalated manno nose.^"^^ 111. POLYSACCHARIDES HAVINGA P-D-XYLANBACKBONE

Hemicellulosic xylans constitute a family of polysaccharides, based on an unbranched (1 + 4)-P-~-xylanbackbone, to which side chains of other sugar residues are attached. These are short, and may be single a-(1 + 2)linked 4-~-methyl-~-glucosyluronic groups, single a-(1 + 3)- or a-(1 + 2)linked L-arabinofuranosyl groups, or more-extended side-chains, in which L-arabinofuranosyl residues carry additional s u b s t i t ~ e n t s . Some ~ ~ * ~plant ~ xylans are partly acetylated. Seaweeds contain mixed-linkage ( 1 + 3), (1 + 4)-P-~-xylans,and the polymer from Rhodymenia palrnata has been studied in some detail.” Xylans and substituted xylans are susceptible to hydrolysis by endo-( 1 + 4)-P-~-xylanase(EC 3.2.1.8). Reaction of this enzyme with (1 + ~ ) - P - D (47a) (48) (49) (50) (51)

1. W. Sutherland, Carbohydr. Res., 131 (1984) 93-104.

I. W. Sutherland, personal communication. T. E. Timell, Adv. Carbohydr. Chem., 20 (1965) 409-483. K. C. B. Wilkie, Ado. Carbohydr. Chem. Biochem., 36 (1979) 215-264. H. Bjorndal, K.-E. Eriksson, P. J. Garegg, B. Lindberg, and B. Swan, Acra Chem. Scand., 19 (1965) 2309-2315.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

159

xylans, as well as arabino-, glucurono-, arabinoglucurono-, and arabino-4- 0methylglucuronoxylans, yields D-xylose, D-xylo-oligosaccharides, and a range of D-xylo-oligosaccharides containing side chains of other sugar residue^.^^^^^^*'^ Two endo-(1+ 4)-p-~-xylanaseswere purified from Irpex l a ~ t e u s , ~one ’ ~ of which showed no activity with 0-(carboxymethy1)cellulose, and the other, slight activity. The former hydrolyzed larch-wood xylan some 35% (releasing D-xylose, xylobiose, and xylotriose), and the latter hydrolyzed 19% and released xylobiose and xylotriose. Hydrolysis is affected by the degree of substitution. A rice-seed arabinoxylan was hydrolyzed much less5*than a corn-cob arabinoxylan that had a lower degree of main-chain substitution ( h a : Xyl = 1 :6.3). The isolation of a trisaccharide composed of L-arabinose and D-xylose in the ratio of 1 :2 on hydrolysis of wheat-straw xylan established that L-arabinosyl units are integral to the mo~ecule.’~-~~ The fine structure of wheat-flour arabinoxylan has been investigated by using an enzyme preparation having a high endo-( 1 + 4)-P-~-xylanase activity and essentially devoid of p-D-xylosidase (EC 3.2.1.37) and a-Larabinofurano~idase.~~ This preparation partially degraded arabinoxylan, a pentosan, giving substantial amounts of at least five oligosaccharides, and leaving 60% of the original substrate as a polymeric material having an average molecular weight of less than one-tenth of that of the original pentosan.Themajoro1igosaccharideswere (1 + 4)-p-~-xylo-bioseand-triose, with a significant proportion of 32-O-a-~-arabinofuranosylxylotriose (13),

indicating that many of the a-L-arabinofuranosyl branches were made up of a single group. Because there was only a trace of L-arabinose in the hydrolyzate, xylobiose and xylotriose represented unsubstituted, interbranch segments of the xylan main-chain. These oligosaccharides would have been derived from longer, unbranched segments in the polymer, as it appeared that, for cleavage to occur,s6 two unbranched D-xylosyl residues (51a) (52) (53) (54) (55) (56)

T. Kanda, Y. Amano, and K. Nisizawa, J. Biochem. (Tokyo), 98 (1985) 1545-1554. N . Shibuya, A. Misaki, and T. Iwasaki, Agiic. Biol. Chem., 47 (1983) 2223-2230. C. T. Bishop, J. Am. Chem. Soc., 78 (1956) 2840-2841. C. T. Bishop and D. R. Whitaker, Chem. Ind. (London), (1955) 119. H. R. Goldschrnid and A. S. Perlin, Can. 1. Chem., 41 (1963) 2272-2277. A. S. Perlin and E. T. Reese, Can. J. Biochem. Physiol., 41 (1963) 1842-1846.

160

BARRY V. McCLEARY AND NORMAN K. MATHESON

need to be in sequence. As the D-xylo-oligosaccharides constituted 13% of the xylan component, and the incidence of branching was high (2 out of 5 D-XYIOSYI units), it was concludeds5 that the branching was not uniform. The polysaccharide was considered to consist mainly of highly branched regions, in which isolated and paired L-arabinofuranosyl branches are separated by single D-xylosyl residues: at unequal intervals, averaging 20-25 main-chain units, this type of sequence is interrupted by unsubstituted regions that are at least 2 to 5 D-XYIOSYI residues long. Infrequently, a branch is separated from neighboring branches by at least two or more D-xylosyl units on each side. Enzymic hydrolysis of various arabinoxylans has produced a-Larabinofuranosyl-( 1 + 4)-p-~-xylobiose,branched at the reducing residues7 or at the nonreducing D-XYIOSYI end,s3*58*s9*59a as well as (1+4)P-D-xylotriose substituted on the red~cing,’~ or nonreducing D-XYIOSYI units. Hydrolysis of corn-cob arabinoxylanS9”gave 13 and the tetrasaccharide substituted 33 by an L-arabinofuranosyl group, as well as several oligosaccharides in which the branching Larabinofuranosyl residue was further substituted p-( 1 + 2) by a D-XYIOSYI group. These were tetrasaccharide 13a and two pentasaccharides having (1 + 4)-p-~-xylotriosesubstituted by the heterodisaccharide on either the nonreducing or central D-xylosyl units. P-D-XYl 1

J.

2

a-L-Araf 1

J. 3

/%D-XYl-(1 +4)-D-xyl 13a

Further information on the fine structure of arabinoxylan should be forthcoming when hydrolysis is performed with a purified (1+4)p-D-xylanase, such as that from Cryprococcus albidus,60-62whose subsite(57) S. Takenishi and Y. Tsujisaka, Agric. Bid. Chem., 37 (1973) 1385-1391. (58) G. 0. Aspinall, I. M. Cairncross, R. J. Sturgeon, and K. C. B. Wilkie, J. Chem. Soc., (1960) 3881-3885. (59) R. F. H. Dekker and G . N. Richards, Carbohydr. Res., 43 (1975) 335-344. (59a) I. Kusakabe, S. Ohgushi, T. Yasui, and T. Kobayashi, Agnc. Bid. Chem., 47 (1983) 2713-2723. (60) P. Biely, Z. KrPtkL, and M. VrSanskP, Eur. J. Biochem., 119 (1981) 559-564. (61) P. Biely, M. VrHanskP, and Z. KrPtkL, Eur. J. Biochem., 119 (1981) 565-571. (62) P. Biely and M. VrSanskB, Eur. J. Biochem., 129 (1983) 645-651.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

161

binding requirements have been determined. Fractionation, and characterization, of higher oligosaccharides in the hydrolyzate would also yield useful data. (1 + 4)-P-~-Xylanasehas also been employed in analysis of the distribugroups in tion of a-L-arabinofuranosyl and 4-O-methy~-~-glucosyluronic arabinoglucuronoxylans and 4-0-methylglucuronoxylans from woody tissue. With ( 1 + 4)-P-~-xylanasepreparations, the latter gave63 D-xylose, D-xylo-ohgosaccharides up to a degree of polymerization (d.p.) of 6, and D-xylo-oligosaca range of acidic 4-O-methyl-~-glucosyluronic-containing charides having the general structure 14, where n was 1 to 5. Similar (4MeO)-a-~-GlcA 1

3.

2 p-D-xyl-( 1

4)-p-D-xyl-( 1*4)-D-Xyl 14

structures have been separated after partial hydrolysis with acid.64 Aspen polysaccharide gave6’ 14 in which n was 1. The polysaccharide, which had been reduced by means of sodium borohydride, also released the equivalent tetrasaccharide in which 4-0-methyl-~-glucosyluronicacid was replaced by 4- 0-methyl-D-glucose, showing that some uronic acid residues were esterified in the native polymer. Hydrolysis of the arabino-4- O-methylglucuronoxylan from redwood led to a proposal for irregular distribution of both 4-0-methyl-~-glucosyluronicand L-arabinofuranosyl side-chain groups.66A series of dialyzable oligosaccharides and a nondialyzable residue (20%) were obtained. Included in the products were 14 (where n was 1 or 2) and 15, in which n was 0, 1, or 2. 3-O-a-~-Arabinofuranosyl-~-xylose, a-L-Ara 1

3. 1s

the aldobiouronic acid 2-O-(4-O-methyl-cu-~-glucosyluronic acid)-Dxylose, and the aldotriouronic acid homolog were not found. The binding of the (1 + 4)-P-~-xylanaseappeared to involve a region of polysaccharide backbone consisting of three D-xylosyl residues (16), in which 0 - 2 of residue (63) (64) (65) (66)

T. E. Timell, Sven. Puppersridn., 65 (1962) 435-447. J. Havlicek and 0. Samuelson, Carbohydr. Res., 22 (1972) 307-316. J. Comtat, J.-P. Joseleau, C. Bosso, and F. Barnoud, Carbohydr. Res., 38 (1974) 217-224. J. Comtat and J.-P. Joseleau, Carbohydr. Res., 95 (1981) 101-112.

162

BARRY V. McCLEARY AND NORMAN K. MATHESON

1

R R' 1 1

1

3.3.

R

3 2 3 + 4)-p-D-xyl-(1 +4)-p-D-xyl-(1 + 4)-p-D-xyl-( 1 + C B A 16

C, and 0 - 2 plus 0-3 of residue B, were unsubstituted; R is an a-Larabinofuranosyl group or hydrogen atom, and R' is a 4-O-methyl-a-~glucosyluronic group or a hydrogen atom. The main, neutral oligosacwere charides released from white-willow 4- 0-methylgl~curonoxylan~~ D-xylo-tetraose and -hexaose, together with a small proportion of xylosaccharides of higher d.p., and traces of xylobiose. Glucuronic acid derivatives (14), in which n was 1 and 3, were the major acidic compounds, with a small proportion in which n = 2. From the structures of these products, and from the absence of D-xylo-triose, -pentaose, and oligosaccharides having a high degree of substitution, it was considered that substitution was regular. Hydrolysis, with cellulase, of an extracellular, acidic arabinoxylan from suspension-cultured soybean-cells released xylo-oligosaccharides.67a Hydrolysis of extracted, maize-shoot cell-walls with a Basidiomycete e n ~ y m e - p r e p a r a t i o n ~released ~~ 0-(5-O-feruloyl-a-~-arabinofuranosyl)(1 + 3)-O-p-~-xylosyl-( 1 + 4)-~-xylose(15, where n = 0, with a ferulic ester group attached to 0 - 5 of L-arabinose. Hydrolysis, by a partially purified p-o-xylanase from Schizophyllum commune, of an acetylated xylan from birch gave a series of acetylated xylo-oligosaccharides. Several of these having the same d.p. differed in the number of their acetyl groups, consistent with a non-regular acetyl substitution. Acetyl esterase activity (EC 3.1.1.6) acting on acetylated xylan was detected in several fungal The extent of hydrolysis of D-xylans by p-D-xylosidase should denote the extent of substitution, but variation in the action patterns of enzymes may be reflected in the various results that have been obtained using enzymes from different sources. P-o-Xylosidase hydrolyzed a soluble D-xylan from Shirakamba wood68 to the extent of 39%, and rice-straw D-xylan released p-DD-xylose,69 but other D-xylans underwent no significant hydr~lysis.~' Xylosidase has been employed in partially determining the structures of (67) M. KubaEkovi, 3. Karicsonyi, L. Bilisics, and R. Toman, Carbohydr. Res., 76 (1979) 177-188. (67a) Y. Kato and K. Matsuda, Planf Cell Physiol., 26 (1985) 287-294. (67b) Y. Kato and D. J. Nevins, Carbohydr. Res., 137 (1985) 139-150. (67c) P. Biely, J. Puls, and H. Schneider, FEES Lerr., 186 (1985) 80-84. (68) M. Matsuo, T. Yasui, and T. Kobayashi, Agric. Biol. Chem., 41 (1977) 1601-1606. (69) K. Sumizu, M. Yoshikawa, and S. Tanaka, J. Biochem. (Tokyo), 50 (1961) 538-543. (70) S . Takenishi, Y. Tsujisaka, and J. Fukumoto, J. Biochem. (Tokyo),73 (1973) 335-343.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

163

tri- and tetra-heterosaccharides obtained by hydrolysis of rice-straw arabinoxylan with p - ~ - x y l a n a s ep-D-Xylosidase .~~ reacted with the borohydride-reduced oligosaccharides, releasing D-xylose as the only reducing sugar and, in conjunction with periodate-oxidation data, this indicated that and -triose. the original structures were 3'-a-~-arabinofuranosylxylo-biose The oligosaccharides remaining after hydrolysis were not characterized. a-L-Arabinofuranosidase gave essentially complete removal of Larabinosyl groups from wheat-flour arabinoxylan, whereupon a xylan was (compare Ref. 73), demonstrating the exterior location of these units, and confirming that the ring size was furanose. With the enzyme from Dichomitus squalens, oat-spelt arabinoxylan lost 32% of L-arabinosyl units, wheat-bran arabinoxylan only 4%, and wheat-straw glucuronoa r a b i n ~ x y l a n , ~42%. ~" Unlike the xylans already discussed, Rhodymenia palmata p-D-xylan contains mixed (1-3) and (1+4) linkages. On reaction with a cellulase that had p-D-xylanase a series of (1 + 4) and mixed-linkage xylosaccharides was produced that included (1 + 4)-P-xylo-biose, -triose, -tetraose, and a mixed-linkage trisaccharide, namely, 32-O-p-~-xylosylxylobiose (17). endo-(1 + 4)-P-~-Xylanasehas been shown62 to catalyze p- D-xyl 1

J. 3 p-D-XYl-(1 + 4)-D-xyl 17

transglycosylation, not only to 0 - 4 but also to 0-3. At very low concentrations of substrate, applied to eliminate transglycosylation, a number of mixed-linkage oligosaccharides were detected." These included 32-O-~-xylobiosylxylobiose (18), 33-O-/3-~-xylo~ylxylotriose (19), 32-0-pxylotriosylxylobiose (20), 3'-O-~-xylobiosylxylotriose (21), and 33-0-pxylotriosylxylotriose (22). The structures were determined chemically, and p-D-xyl-(1+4)-p-D-xyl 1

1 3 p-D-xyl-( 1 + 4)-D-xyl 18 (71) (72) (73) (73a) (74)

P-D-XYl 1

J.

3 p-D-xYl-(l +4)-p-D-xyl-(l+ 4)-D-xyl 19

H. Gremli and H. Neukom, Carbohydr. Res., 8 (1968) 110-112. K. Tagawa and A. Kaji, Carbohydr. Res., 1 1 (1969) 293-301. K. A. Andrewartha, D. R. Phillips, and B. A. Stone, Carbohydr. Rex, 77 (1979) 191-204. J.-M. Brillouet and J.-C. Moulin, Carbohydr. Res., 144 (1985) 113-126. D. J. Manners and J. P. Mitchell, Biochem. J., 89 (1963) 9 2 ~ - 9 3 ~ .

164

BARRY V. McCLEARY AND NORMAN K. MATHESON

by using P-D-xylosidase to interrelate the oligosaccharides by sequential hydrolysis from the nonreducing end. The array and proportions of oligosaccharides were considered to be consistent with a random arrangement of linkages.

Iv. POLYSACCHARIDES BASEDO N

A

(1 + 4)-P-D-MANNAN BACKBONE

The (1 + 4)-P-~-mannantype of polysaccharide includes mannan; glucomannan, in which a proportion of the (1 + 4)-linked P-D-mannosyl residues is replaced by similarly linked D-glucosyl residues; galactomannan, in which the backbone carries (1 + 6)-linked a-D-galactosyl groups; and galactoglucomannan, which combines the structural features of glucomannan and g a l a c t ~ m a n n a n . ~ ~Glucomannan -’~ and galactoglucomannan generally occur in vivo in a partially acetylated

(75) 1. C. M. Dea and A. Morrison, Ado. Carbohydr. Chem. Biochem., 31 (1975) 241-312. (76) P. M. Dey, Ado. Carbohydr. Chem. Biochem., 35 (1978) 341-376. (77) H. Meier and J. S . G. Reid, in F. A. Loewus and W. Tanner (Eds.) Encyclopedia of Plant Physiology, New Series, Vol. 13A, Springer-Verlag, New York, 1982, pp. 418-471. (78) A. M. Stephen, in G. 0. Aspinall (Ed.), Tne Polysaccharides, Vol. 2, Academic Press, New York, 1983, pp. 97-193. (79) H. Meier, Acta Chem. Scand., 15 (1961) 1381-1385. (80) T. Matsuo and T. Mizuno, Agric. Biol. Chem., 38 (1974) 465-466. (81) T. Katz, Tappi, 48 (1965) 34-41.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

165

1. (1 + 4)-P-~-Mannan

The essentially unbranched nature of the (1+ 4)-linked P-D-mannans isolated from ivory and other sources has been confirmed by treatment of these polymers with endo-( 1 + 4)-p-~-mannanase (EC 3.2.1.78). Reaction with P-D-mannans from such palm seeds as ivory Pheonix canariensis,86 bangalow palm,87 and Livistona australis" yielded (1 + 4)-linked p-D-manno-oligosaccharides and D-mannose, with only traces of other products. The small amounts of other oligosaccharides, present in some hydrolyzates, appear to be (1 + 6)-linked a-D-galactosyl-P~-mannosaccharides~~: most purified mannans contain some D-galactose, which varies" in proportion from 1 to 5 % . Smaller proportions of D-glucose may be present. Effective cleavage of highly insoluble P-D-mannans by P-D-mannanase is facilitated by treatment with the enzyme immediately after neutralization of an alkaline solution of the polymer.

2. Galactomannan Galactomannan consists of a (1+ 4)-linked p-D-mannan backbone, to which are attached single a-D-galactosyl stubs at 0 - 6 of certain of the D-mannosyl The extent of D-galactosyl substitution of the D-mannan backbone varies from almost complete substitution in the polymers from seeds of some of the Trifolieae to -20% in Sophorajuponica galactomannan (Gal: Man = 17:83), and this average ratio is species-specific. The general pattern of distribution of the D-galactosyl groups along the p-D-mannan backbone (fine structure) also appears to be species-specific. Galactomannans and oligosaccharide fragments are susceptible to hydrolysis by a number of enzymes, including a-~-galactosidas.e~~ (EC 3.2.1.22), p-D-mannanase, exo-p-D-mannanase?' p-D-mannosidase, and exo-P-Dmannan mannobiohydrolase (EC 3.2.1 Early applications of enzymic

(82) G. 0. Aspinall, E. L. Hirst, E. G. V. Percival, and I. R. Williamson, J. Chem. Soc., (1953) 3184-3188. (83) G. 0. Aspinall, R. B. Rashbrook, and G. Kessler, J. Chem. Soc., (1958) 215-221. (84) E. T. Reese and Y. Shibata, Can. J. Microbiol., 11 (1965) 167-183. (85) S. R. Lee, Ph.D. Thesis, University of Minnesota, Minneapolis, 1965; Chem. Absrr., 68 (1968) 111,694~. (86) S. Clermont-Beaugiraud and F. Percheron, Bull. Soc. Chirn. Biol., 50 (1968) 633-639. (87) B. V. McCleary, N. K. Matheson, and D. M. Small, Phytochemistry, 15 (1976) 1111-1 117. (88) B. V. McCleary, Carbohydr. Res., 71 (1979) 205-230. (89) B. V. McCleary, unpublished results. (90) P. M. Dey, Ado. Carbohydr. Chem. Biochem., 37 (1980) 283-372. (91) B. V. McCleary, Carbohydr. Rex, 101 (1982) 75-92. (92) T. Araki and M. Kitamikado, J. Biochem. (Tokyo), 91 (1982) 1181-1186.

166

BARRY V. McCLEARY AND NORMAN K. MATHESON

techniques to analysis of the structure of galactomannan employed seed The oligosaccharide products of reaction were separated, and characterized as (1 + 4)-P-~-mannobiose,p-D-mannotriose, and 6 - 0 - a - ~ galactosyl-D-mannose. The structures of these provided data on the linkage types in guar galactomannan. There is considerable interest in the nature of the distribution of Dgalactosyl groups along the mannan backbone of g a l a ~ t o m a n n a n .This ~~*~~ is due, in part, to the probable significance of this aspect of fine-structure in the interaction properties of mannan chain^.^^*^^ A range of techniques has been applied to the analysis of D-galactose distribution, with differing results. From an interpretation of periodate-oxidation and selective chemical depolymerization by p-elimination,lOOit was concluded that, in guar galactomannan, the D-galactosyl groups are arranged in small clusters of mostly two, and three or four neighboring units separated by sections of two, or three, contiguous, unsubstituted, D-mannosyl residues. Carob galactomannan was reported to consist of a mixture of long blocks of contiguous, unsubstituted D-mannosyl residues, as well as long blocks in which every second D-mannosyl residue is substituted with D-galactosyl groups, and shorter blocks in which there is a high density of D-galactosyl groups.99 It has been widely proposed that the determination of the finestructure should be amenable to enzymic study. For such methods to be unambiguous, highly purified, well characterized enzymes must be employed, and the degradation products be quantitatively separated into individual components and then characterized. Galactomannans have been treated with a-D-galactosidase, and the extent of removal of D-galactose has varied c ~ n s i d e r a b l y . ~With ~ ' ~ enzymes ~~-~~~ from lucerne'04 and guar seed,"' essentially all of the D-galactosyl groups (93) (94) (95) (96)

R. L. Whistler and J. Z. Stein, J. Am. Chem Soc., 73 (1951) 4187-4188. R. L. Whistler and C. G. Smith, J. Am. Chem. Soc., 74 (1952) 3795-3796. T. J. Painter, Lebensm. Wiss. Techno/., 15 (1982) 57-61. E. R. Morris, D. A. Rees, G . Young, M. D. Walkinshaw, and A. Darke, J. Mol. B i d ,

110 (1977) 1-16. (97) 1. C. M. Dea, E. R. Moms, D. A. Rees, E. J. Welsh, H. A. Barnes, and J. Price, Carbohydr. Res., 57 (1977) 249-272, (98) J. Hoffman, B. Lindberg, and T. J. Painter, Acra Chem. Scand., Ser. B., 29 (1975) 137; 30 (1976) 365-366. (99) T. J. Painter, J. J. GonzPlez, and P. C. Hemmer, Carbohydr. Res., 69 (1979) 217-226. (100) J. Hoffman and S. Svensson, Carbohydr. Rex, 65 (1978) 65-71. (101) P. A. Hui and H. Neukom, Tappi, 47 (1964) 39-42. (102) J. E. Courtois and P. Le Dizet, Carbohydr. Rex, 3 (1966) 141-151. (103) J. E. Courtois and P. Le Dizet, Bull. Soc. Chim. Biol., 52 (1970) 15-22. (104) B. V. McCleary, R. Amado, R. Waibel, and H. Neukom, Carbohydr. Res., 92 (1981) 269-285. (105) B. V. McCleary, Phytochemisrry, 22 (1983) 649-658.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

167

can be removed, leaving a ( 1 + 4)-P-~-mannanchain of high molecular weight, as indicated by viscometric behavior consistent with peripheral placement of the D-galactosyl groups. Evidence that a-D-galactosidases may preferentially remove D-galactosyl groups separated by one D-mannosyl residue has been obtained from the pattern of oligosaccharides produced on hydrolysis, by P-D-mannanase, of galactomannan pretreated with a-Dgalactosidase. The released oligosaccharides included high levels of 6'-0-aD-galactosyl-( 1 + 4)-P-~-rnannobiose(23), relative to that present in the a-D-Gd 1

.1 6 p-D-Man-(l+4)-~-Man 23

hydrolyzates of other galactomannans having similar D-galactose contents; this is consistent with the modified polymer (with the diminished D-galactose content) having a high proportion of sequences of -[Man-Man(Gal)],- in the chain.'06 The approximate, two-fold screw-axis of the (1 + 4)-P-~-mannanchain places the hydroxymethyl groups on neighboring D-mannosyl residues on opposite edges of the ribbon-like conformer, resulting in D-galactosyl groups that are separated by one D-mannosyl residue lying on the same edge of the ribbon, l637.107,108 and providing a stereochemical basis for their preferential removal by a-D-galactosidase. There was no e ~ i d e n c e ~ ~that . " ~ this enzyme sequentially removed D-galactosyl groups from adjacent Dmannosyl residues in a "zipper-like" fashion, producing completely unsubstituted, D-mannan segment^."^ (This is discussed later in this Section.) Also, the action of a-D-galactosidase on galactomannan was not affected by the fine-structural differences between different galactomannans. The extent of hydrolysis by P-D-mannanase is dependent on the Dgalactose content of the galactomannan, indicating that the D-galactosyl branch-units interfere with the association of the enzyme and the substrate. On hydrolysis of carob and Gleditsia ferox galactomannans, the nondialyzable fraction of the digest was progressively enriched in D-galactose.'Og When the reaction had ceased, this fraction was isolated in 10-20°/0 yield and the D-galactose content estimated to be 48%: It was concluded that the D-galactosyl groups were distributed in a partial, block-type structure. (106) B. V. McCleary, I. C. M. Dea, J. Windust, and D. Cooke, Carbohydr. Polym., 4 (1984) 253-270. (107) P. R. Sundararajan and V. S. R. Rao, Biopolymers, 9 (1970) 1239-1247. (108) B. A. Burton and D. A. Brant, Biopolyrners, 22 (1983) 1769-1792. (109) J. E. Courtois and P. Le Dizet, Bull. SOC.Chim. Eiol., 50 (1968) 1695-1710.

168

BARRY V. McCLEARY AND NORMAN K. MATHESON

In contrast, in a subsequent study,88the higher-molecular-weight fraction, released on hydrolysis of carob galactomannan, and recovered by ethanol precipitation, was only moderately enriched in D-galactose (36%). Examination, by t.1.c. and gel chromatography, of the oligosaccharides released by j3-D-mannanase hydrolysis of ten galactomannans indicated that the distribution of D-galactosyl groups in polysaccharides having medium to low D-galactosyl substitution is neither completely regular nor in blocks. Galactomannans from soybean and Leucaena leucocephala seed gave a higher proportion of 23 than expected from the D-galactose content of the polymer, and it was proposed that they contained a substantial proportion of the repeating unit -Man-Man(Gal)-; guar galactomannan, having a Gal :Man ratio similar to that of the L. leucocephala polysaccharide gave"' much less of 23. These results, together with studies employing chemical p r o ~ e d u r e s ~ ~ and - ' ~ ' n.m.r. spectroscopy,'" indicated that the distribution of D-galactosyl groups in guar and carob galactomannans is neither block-type nor in a uniform pattern, and is not statistically random. The distribution pattern is non-regular, and attempts have been made to refine this description by enzymic methods. In experimental'6.112and theoreti~al''~ analyses of the oligosaccharides released on hydrolysis of carob galactomannan by two P-D-mannanases having different action-patterns, the oligosaccharides up to d.p. 9 were separated by chromatography on Bio-Gel P-2 and by t.l.c., identified chemically and enzymically,'10~1'2 and their amounts measured. The only heterotrisaccharide released was shown to have structure 23 by methylation analysis and n.m.r. spectroscopy, and also by the production of one mol of D-mannose per mol, on hydrolysis by P-D-mannosidase. The only heterotetrasaccharide was shown to have the D-galactosyl group joined to the (reducing) D-mannose residue. It gave two mol of D-mannose per mol with j3-D-mannosidase, and a-D-galactosidase released P-D-(1 + 4)-~-mannotriose and one mol of D-galactose per mol. Characterization of higher oligomers was more complex, as, even after gel chromatography and t.l.c., fractions could consist of a mixture, and many of the oligosaccharides contained more than one D-galactosyl group. A pentasaccharide fraction released only ( 1 + 4)-p-~-mannotetraoseand one mol of D-galactose with a-D-galactosidase, but, after j3-D-mannosidase (110) B. V. McCleary, F. R. Taravel, and N. W. H. Cheetham, Curbohydr. Res., 104 (1982) 285-297. (111) H. Grasdalen and T. .I.Painter, Curbohydr. Res., 81 (1980) 59-66. (112) B. V. McCleary, E. Nurthen, F. R. Taravel, and J.-P. Joseleau, Curbohydr. Res., 118 (1983) 91-109. (113) B. V. McCleary, A. H. Clark, I. C. M. Dea, and D. A. Rees, Curbohydr. Res., 139 (1985) 237-260.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

169

hydrolysis, both 6-0-a-~-galactosyl-~-mannose and 63-O-a-~-galactosyl-pD-mannotriose were released, indicating that two pentasaccharides were present, with D-galactosyl groups joined either to the (reducing) D-mannose residue or the D-mannosyl residue penultimate from the nonreducing end in mannotetraose. Oligosaccharides of higher d.p. and having more than one D-galactosyl group attached were partially hydrolyzed by a-D-galactosidase, the products fractionated, and their structures then established by the use of p-D-mannosidase, p-D-mannanase, and a-D-galactosidase. The positions of hydrolysis of galactomannan, glucomannan, and p-Dmanno-oligosaccharides by A. niger and guar-seed p-D-mannanases were determined from the hydrolysis products, and a model defining the substrate sub-site binding requirements, in terms of polysaccharide conformation, was developed.'6 The patterns, and proportions, of oligosaccharides produced on hydrolysis of carob galactomannan fractions by the two p - ~ mannanases excluded a regularly repeating structure of one substituted and three unsubstituted D-mannosyl residues [ Man-Man-Man(Gal)]. This structure would have produced only mannobiose and 23,in equimolar amounts, instead of 24% and 16%, respectively, plus other oligosaccharides found experimentally. The nature of the oligosaccharides separated (up to d.p. 9), plus the Gal : Man ratio of fragments of d.p. >9 and <15 (which, together, quantitatively accounted for the polysaccharide) excluded block substitution or a high proportion of segments of mannan chain containing more than two adjacent, substituted D-mannosyl residues; that is, a structure with an average repeating unit [(Man),-( ManGal),,] where p > 2. Seventy-five percent of total galactose occurred as singlets or doublets in oligosaccharides up to d.p. 9: in the fraction of d.p.>9 (9.5% of the total), 45% of the D-mannosyl residues were unsubstituted. Comparison of experimental data with the theoretical, binomial distribution of D-galactosyl groups shows that the statistically random structure is also not possible. The theoretical percentage of neighboring pairs of D-galactosyl units for a binomial distribution in a polymer with a 1 9 9 1 D-galactose to D-mannose ratio is 17; the percentage found in oligosaccharides up to a d.p. of 9 was 28, and there was a possibility of even more occurring in the fraction of d.p. 10-14, as the percentage of substituted D-mannosyl units in this was still only 55%. The degree of nonregularity of substitution by D-galactosyl groups was defined'I3 in terms of a computer-simulated, chain-extending program, in which the probability of a given D-mannosyl residue's being substituted by a D-galactosyl group was dependent on the nature of substitution of the previous two residues, that is, a nearest-neighbor-second-nearest-neighbor model. The parameters were the experimentally determined, subsite bindingrequirements of the two enzymes, the amounts and structures of the oligosaccharides of d.p. 2 to 9 (or 7) released by the enzymes when hydrolysis

BARRY V. McCLEARY A N D NORMAN K. MATHESON

170

was essentially complete, the degree of P-D-mannanase hydrolysis, and the Gal : Man ratio of the polymer. Four probability factors were involved, P Plo, Pol, and PI]. The first integer indicates whether the designated as , nearest-neighbor is substituted (1) or unsubstituted (0), and the second integer refers to the second-nearest neighbor. These probabilities were optimized in turn, through a minimization of the sum of squared differences between the experimental data supplied and the corresponding computed values. For carob galactomannan, the best fit of data was obtained with high values for the probability factors Pooand Ploand low values for Pol and PI This indicates that, in carob galactomannan, the D-galactosyl groups are distributed non-regularly, with a higher proportion of couplets of Dmannosyl residues substituted by D-galactosyl groups than predicted for random substitution, and a lower proportion of substituted triplets. There was a low, predicted occurrence of regions in which every second Dmannosyl residue was substituted by D-galactosyl groups, and an extremely low prediction for small blocks of highly substituted regions. The presence, in the hydrolyzate of the hot-water-soluble, carob galactomannan fraction, of only 9.5% of oligosaccharides of d.p. >9 and <15, with a D-galactose content of 36%, provides further experimental confirmation of this model. Galactomannans from ten different, carob-seed varieties showed very similar patterns of released oligosaccharides on treatment with A. niger P-~-mannanase.'I~ Thus, not only is the D-galactose content species-specific but it would appear that so, also, is the distribution of D-galactosyl groups along the mannan chain. In agreement with this concept, samples of guar galactomannans from fifteen varieties also gave the same elution patterns in chromatography on Bio-Gel P-2 after hydrolysis with A. niger p-Dmannanase. Two of these are shown in Fig. 1 (a and b). The variety Pusa mosami had a 100-seed weight of 4.35 g, and the galactomannan contained 39.4% of D-galactose, whereas, for the variety MSSl Type 1, the corresponding values were 2.57 g and 38.3%. In contrast, quite different patterns of amounts of oligosaccharides were obtained on treatment, with P-D-mannanase, of galactomannans having similar Gal :Man ratios but from different species [carob and Caesalpinia pulcherima; Fig. 1 (c and d)]. Galactomannans from Caesalpinia spinosa, C. uesicaria, and Gleditsia triacanthos (honey locust), all having a Gal :Man ratio of -26: 74,also showed differences in patterns of amounts of oligosaccharide products, and thus, in fine-struct~re.'~ These fine-structural differences provide some insight into the different interaction-properties of galactomannans. The low level of trisaccharide 23 in the A. niger P-D-mannanase hydrolyzate,88*'06together with the known subsite binding-requirements of this enzyme,I6 demonstrate that guar galactomannan does not consist of the repeating unit -Man-Man(Ga1)-, as had

,.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

Elution

171

volume ImL)

FIG. 1.-Chromatographic Patterns, on Bio-Gel P-2, of Oligosaccharides Produced on Hydrolysis, by A. niger@-~-Mannanase, of Galactomannans from (a) Cyarnopsis tetragonolobus (guar) var. Pusa mosami, (b) Cyamopsis tetragonolobus var. MSSI-Type 1, (c) Carob, and (d) Caesalpinia pulcherima. [Numbers represent the d.p. of the oligosaccharides eluted.]

previously been proposed from a chemical study.'" Gel chromatography of the high-d.p. material, and determination of the Gal: Man ratios of the eluted fractions, gave no reaction products that had a D-galactose content approaching 50%, and thus, enzymic evidence shows that the original galactomannan cannot have a block-type distribution of D-galactosyl groups (compare, the results in Ref. 115). Rather, the structure is closer to that proposed from the results of &elimination, which indicated that the Dgalactosyl groups are arranged mainly in pairs and triplets.100 The amount of substitution, and tendencies in the pattern of substitution, of galactomannans can be related to possible effects in the biosynthesis.'16 (114) C. W. Baker and R. L. Whistler, Carbohydr. Res., 45 (1975) 237-243. (115) L. D. Hall and M. Yalpani, Carbohydr. Rer, 81 (1980) c l 0 - c l 2 . ( 1 16) N. K. Matheson, in D. R. Murray (Ed.), Seed Physiology, Vol. 1, Deoelopmenr, Academic Press, Sydney, 1984, pp. 167-208.

172

BARRY V. McCLEARY A N D NORMAN K. MATHESON

The enzymes involved are nucleoside diphosphohexosetransferases, probably GDP-D-mannosyltransferase (EC 2.4.1.32) and UDP-D-galactosyl1 -* transferase. For a structurally related polymer, (1 + 6)-a-~-xylosyl-( 4)-P-~-glucan,synthesis was shown to involve concurrent incorporation of D-xylose and D-glucose, and not substitution by D-xylose of a preformed (1 + 4)-/3-~-glucan The insolubility of (1 + 4)-P-~-glucanand mannan would make the latter mechanism unlikely. In the galactomannans of the Trifolieae, where almost all D-mannosyl residues are substituted, there would be no steric hindrance of the approaching UDP-D-galactosyltransferase-UDP-D-galactose enzyme-substrate complex by the existing substituents Ga* and Ga+ at the growing end of the polymer (see Fig. 2; M represents a D-mannosyl, and Gay a D-galactosyl, residue). The D-mannosyltransferase would also be near, providing a further interaction. In a species in which the galactomannan is less heavily substituted by D-galactosyl groups (-33-40%), when substituents Ga* or Ga+, or both, were present, these would sterically hinder the approach of the D-galactosyltransferase-substrate complex. In galactomannans having a D-galactose content greater than -4O%, hindrance by Ga+ would not be significant: in those having a D-galactose content of less than -33%, substituents farther back in the growing mannan-chain would have an effect. Like all enzymes, the conformation of D-galactosyltransferasesis genetically determined. Then, species differences in the amount of substitution would reflect differences in the susceptibility, to steric hindrance, of the enzyme in each species during substitution of the D-mannan chain. Both transferases are likely to be membrane-bound, which would intensify the steric effects. The probability factor in the transfer of a D-galactosyl group to a particular D-mannosyl residue would lead to non-regular substitution along the chain,

FIG.2.-A Chain.

Model for the Transfer of D-Galactosyl Substituents to an Extending D-Mannan

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

173

and to the variation in D-galactose: D-mannose ratios found in a population of molecules from a single species. The favored conformation of the ribbon-like structure of galactomannan, with a two-fold axis, places neighboring D-galactosyl groups on opposite sides of the chain. The new substituent to be added (see Fig. 2) would lie on the edge of the ribbon opposite to Ga*, and on the same side as Ga+; hence, in the substitution of galactomannan having a 33-40% content of D-galactose, the influence of the Ga' will still be significant, although it lies farther back along the growing chain than Ga*. Then, the relative degree of hindrance by Ga* and Ga+ would affect the pattern of D-galactosyl substitution. If hindrance by Ga* were significantly greater than by Ga+, the galactomannan would have a higher proportion of the sequence 24, and Ga

I

Ga

Ga

I

I

-M-M-M-M-M-M-M-M-

Ga

I

24

this may occur in L. leucocephala galactomannan. If hindrance by Ga+ were greater than by Ga*, there would be a preponderance of segments like 25, Ga

I

-M-M-M-M-

I

Ga

25

as is found in G. triacanthos galactomannan. Furthermore, considering a longer segment of the mannan chain, the production of a distribution like 26 would be favored, as the probability of substitution at M dand Me,and Ga

Ga

I

Ga

I

I

-M,-M~-Mj-Mi-M~-MM,-M~-MM,-M~-MM,-M~-MM,-

I

Ga

I

I

Ga

Ga

reducing end 26

at Mh and Mi, would tend to be lowered by the D-galactosyl groups on Mb and M,, and on Mf and M,, respectively. The galactomannan would then have a low degree of hydrolysis by A. niger P-D-mannanase relative to the amount of D-galactosyl substitution, as none of the D-mannosyl linkages in the segment Ma to Mh are susceptible to cleavage. The polymer would also release a high proportion of heptasaccharide 27 (relative to trisaccharide

174

BARRY V. McCLEARY AND NORMAN K. MATHESON cr-D-Gai 1

3. 6 P-D-Man-(1+ 4)-P-~-Man-( 1 + 4 ) - P - ~ - M a n -1(+ 4)-P-D-Man-(l+ 4 ) - ~ - M a n 6

t

1 a-D-Gal 21

23). (The reducing D-mannose residue in 27 and similar formulas is, of

course, always depicted at the right-hand end.) On the other hand, if, as already mentioned, hindrance by Ga* were greater than by Ga' (as would appear to occur in the synthesis of L. leucocephulu galactomannan), there would be a preference for segments like 24, leading to a high degree of hydrolysis by A. niger p-o-mannanase relative to the amount of D-galactosyl substitution, and release of a high proportion of trisaccharide 23 (relative to heptasaccharide).

TABLE1 Effect of Fine Structure on the Hydrolysis of Galsctomannan

Species

Gleditsiu rriucanthos Hot-watersoluble carob Cassia fistula Caesulpiniu vesicariu pufcherima spinosa a-D-Galactosidasemodified guar

Guar Leucaena leucocephala Mean

B

(YO)

D~~~ of hydrolysis

A" GalMan, Hepta

Degree of hydrolysis 48-D-galactose content

A XB

27

18

0.47

0.86

0.40

19 23 29 24 28

26 21 19 22 20

0.78 0.94 2.2 3.0 2.7

0.90 0.84 1.oo 0.92 1.oo

0.70 0.79 2.2 2.8 2.7

17 24 28 34 38 40

31 23 20 14

2.8 2.8 5.8 3.0 3.5 2.7

0.91 0.96 1.00 1.oo 0.50 1.25

2.5 2.7 5.8 3.0 1.75 3.4

2.6

0.93

2.4

cal

5

10

to 63~4-di-0-a-~-galactosylmannoRatio of amount of 61-O-a-~-galactosylmannobiose pentaose released.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

175

In Table I, the indices derived from the formula (amount of trisaccharide 23)/ (amount of heptasaccharide 27 released) x [degree of hydrolysis( O h )]/[48 - D-galactose content( Yo)]

have been calculated for twelve galactomannans. The number 48 was derived from a plot of the degree of hydrolysis(%), as y, against D-galactose content(%), as x, for 20 galactomannans having D-galactose contents of 17 to 48%. The equation of the linear regression was y = - 0 . 9 3 9 ~+ 44.7, with a correlation coefficient of 0.968. When x = 0, y = 47.6. The calculated indices can be expected to reflect the level of sequences like 24, relative to 26. The former would release higher amounts of 23, and undergo more hydrolysis per unit length of mannan chain. The higher the index, the more sequences like 24 would be present. It may be seen from Table I that there are differences between galactomannans from different species. Three from one genus, Cuesulpiniu, were similar. The change in this index with decreasing D-galactose content of guar galactomannan treated with a-D-galactosidase is consistent with nonrandom removal involving the release of substituents from one side of the (1 + 4)-P-~-mannan ribbon. G. triucunthos galactomannan represents a polymer having a preponderance of couplets of neighboring D-galactosyl groups, and L. leucocephalu galactomannan, a polymer having more than the average of expected sequences of alternately substituted and unsubstituted D-mannosyl residues. Where there is steric hindrance to substitution, any sections of block distribution would be most unlikely. A sequence of 9 neighboring D-galactosyl groups (as in 28) would require substitution to occur 7 times, with Ga

Ga

Ga

Ga

I I I I -M-M-M-M-M-M-M-M-M-M-M-MI I I I Ga

Ga

Ga

Ga

I Ga

28

both Ga* and Ga' already present. Three separated substitutions by three sets of three neighboring groups (as in 29) would require only three substitutions, with both Ga* and Ga+ already present. Ga

Ga

Ga

Ga

I I I I -M-M-M-M-M-M-M-M-M-M-M-MI I I Ga

Ga

Ga

Ga

I

I

Ga

29

In species with galactomannans having -20-30% substitution of the mannan chain by D-galactosyl groups, the degree of blocking by either Ga*

176

BARRY V. McCLEARY A N D NORMAN K. MATHESON

or Ga+ would be high; also, there would be significant hindrance to substitution by a D-galactosyl group on the D-mannosyl residue three (and, to a varying degree, four to six) back from the sugar being substituted, leading to an average ratio of one D-galactose to three or four D-mannose units. Once a sequence of three or more unsubstituted D-mannosyl residues has appeared during synthesis, in accordance with the average degree of substitution, the chance of substitution of the next D-mannosyl residue would be very high, as there would be limitations on the distance over which a D-galactosyl group could restrict substitution; the maximum distance over which steric hindrance can occur would be -6-7 residues, as all legume-seed galactomannans have a D-galactose content of 17%, or more. A relatively high amount of couplets of neighboring D-galactosyl groups could reflect a capacity for a repeat substitution on the opposite side of the o-mannan chain, once the steric hindrance involved in formation of the enzymesubstrate complex has been overcome. A low occurrence of single, unsubstituted D-mannosyl groups would be due to a very high steric hindrance associated with placement of a new D-galactosyl residue on the same side of the D-mannan chain as an existing D-galactosyl group. The appearance of two neighboring, unsubstituted D-mannosyl units could be diminished by the presence of existing D-galactosyl couplets in a number of the reactions. Then, there would be a D-galactosyl substituent on the D-mannan chain on the same side as, and separated by three D-mannosyl residues from, the newly substituted D-galactosyl group. A model, represented by 30, can be described for the structure of galactomannan from the available hydrolytic and theoretical evidence, and the

-

30

proposed pattern of bi~synthesis.'~*''*.''~.''~ The -(Man)- and -Man(Gal)units are non-regularly interposed, and not adjacent to themselves. The sum of blocks of unsubstituted D-mannosyl residues ( a + +p) is equal to the sum of the blocks of substituted D-mannosyl residues ( q + * .+z), and, for a population of molecules, the Gal: Man ratio can be calculated by use of Eq. 1.

-

q+* * * + w z

(a+.

*

+p)+(q+-

--Gal * *

wz)-Man

(1)

For hot-water-soluble, carob galactomannan, with 18% of D-galactose, the probability of the subscript of (M) being 4 to 7 is high, 1,3, or 8 is low, and > 8 is negligible. The probability of the subscript of (M.Ga) being 1

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

177

or 2 is very high, and >3, negligible. Based on these proposals, a possible distribution of the proportions of D-mannosyl residues in substituted and unsubstituted segments, compared with a statistically random distribution, is shown in Fig. 3, which illustrates the high frequency of substituted couplets, the diminished occurrence of single unsubstituted D-mannosyl residues, and the very low occurrence of long segments (greater than 7) of unsubstituted D-mannosyl residues. In the statistically random structure, p and q * z are defined by the binomial expression of the the series a fraction of unsubstituted and substituted D-mannosyl units. In hot-water-soluble, carob galactomannan, the relatively high level of couplets of neighboring D-galactosyl groups would reflect a capacity for a repeat substitution on the opposite side of the mannan chain, once the steric hindrance involved in formation of the enzyme-substrate complex has been

--

-

9

FIG. 3.-Fractions of the D-Mannan Chain of Hot-water-soluble Carob Galactomannan (18% Content of D-Galactose) that Occur as Unsubstituted and Substituted D-Mannosyl I

1

Segments. [Key; -M-, segments of singlets of substituted D-mannOSyl residues; > -M-, segments of doublets and triplets of substituted o-mannosyl residues; MI, segments of single, unsubstituted D-InannOSyl residues; M,, segments of two neighboring, unsubstituted, Dmannosyl residues, and so on; O, proposed distribution; and O, calculated from random distribution.]

BARRY V. McCLEARY A N D NORMAN K. MATHESON

178

overcome. The low occurrence of single, unsubstituted D-mannosyl residues would be due to the very high steric hindrance associated with placement of a new D-galactosyl residue on the same side of the mannan chain as an existing D-galactosyl group. The occurrence of two neighboring, unsubstituted D-mannosyl units would be diminished by the presence of existing D-galactosyl couplets in about half of the reactions. Then, there would be a D-galactosyl substituent on the mannan chain on the same side as, and separated by three D-mannosyl residues from, the newly substituting D-galactosyl group. A way of reconciling the observed proportions of mannobi-, tri-, and tetra-oses and heterosaccharides,'6*"3released by guar-seed P- D-mannanase (from hot-water-soluble carob galactomannan), with this model, would be if the enzyme preferentially hydrolyzes appropriate, unsubstituted Dmannosyl residues (as in 31); then, it would only be able to hydrolyze on the reducing side of a substituted D-mannose (as in 32) if there were a Ga

Ga

I

-M-M-M-M-M-M-

I

-M-M-M-M-M-

t

T

I

I

I

Ga

32

31

sufficiently long section of main chain, towards the nonreducing end, remaining after the previous split. Thus, heptasaccharides 33 and 34 are Ga

Ga

I

I

M-M-M-M-M--M

M-M-M-M-M--M

33

34

not hydrolyzed, but hydrolysis might occur were the segments part of a longer molecule, as in 35, when mannotriose would be released. Substitution Ga

Ga

I

I

-M-M-M-M-M-M-M-M--M La

I

35

by D-galactosyl groups could sufficiently distort the conformation of a segment of mannan chain as short as d.p. 6 to interfere with binding between substrate and the guar-seed, but not the A. niger, P-D-mannanase. 3. Glucomannan

D-Glucomannans have a D-glucose :D-mannose ratio ranging from 1 :3 (salep) to 2:3 (konjac), and this appears to be a constant for a particular species. The establishment of the structure of glucomannan as a polymer

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

179

of (1 + 4)-linked P-D-mannosyl and p-D-glucosyl residues has come, in part, from the isolation of a range of (1 + 4)-linked P-D-oligosaccharides from P ~16.84.87.1 -17-1 1% or ~ endo-( ~ 1+ 4 )~- P - ~ - g~l u c a n a~s e ’ ~ ~hydro~* ’ ~ ~ ~ lyzates. Oligosaccharides commonly detected in the p-D-mannanase hydrolyzates of glucomannan include P-D-mannobiose, P-D-GIc-( 1+ 4 ) - ~ - M a n , p-D-mannotriose, P-D-GIc-(1 + 4 ) - p - ~ - M a n -1(+ 4 ) - ~ - M a n ,/3-D-mannotetraose, p-D-mannopentaose, and tetra- and penta-saccharides having a D-glucosyl group at the nonreducing end. Other oligosaccharides, 1+ 4 ) - ~ - M a n , P-D-G~c-( 1+~ ) - P - D such as P-D-Man-( 1 + 4)-P-~-Glc-( Glc-( 1+ 4 ) - ~ - M a n , and cellobiose, have been r e p ~ r t e d . ~ ” - ’ ~ ~ Hydrolysis of konjac glucomannan produced hetero-oligosaccharides for 1+ 4 ) - P - ~ - M a n which the structures P-D-GIc-(1+ 4 ) - ~ - M a n ,P-D-G~c-( 1 + 4)(1 + 4 ) - ~ - M a n ,P-D-GIc-(1+ 4)-P-D-GlC-(1 + 4 ) - ~ - M a n ,P-D-G~c-( 1+ 4 ) - ~ - M a nwere proposed. Sequences were P-D-GIc-(1 + 4)-P-~-Man-( determined with almond e m ~ l s i n . ” ’ ~P-D-Mannosidase has found use in the characterization of these oligosaccharides, but almond emulsin P-Dglucosidase unexpectedly had a very limited action on D-glucosyl groups linked glycosidically to D-mannosel6 (see Section X,2). (1 --* 4)-P-~-Glucomannanhas been synthesized in vitro by a solubilized enzyme preparation, from Phaseolus aureus hypocotyls, which contained both D-mannosyltransferase and D-glucosyltransferase a ~ t i v i t i e s . ~ Both ~~”~~ GDP-D-mannose and GDP-D-glucose were required. In the presence of just GDP-D-mannose, a (1 + 4)-P-~-mannanof relatively low molecular weight was the only polymeric product, and, with GDP-D-glucose, only (1 + ~ ) - P - D glucan was formed. If both nucleoside glycosyl diphosphates were present, glucomannan was produced. The D-glucosyltransferase required the continual production of nonreducing, acceptor molecules that contained Dmannose, but the D-mannosyltransferase did not require the production of acceptors containing D-glucose. However, the reaction was severely inhibited by GDP-D-glucose, and these properties were considered to lead to (1 17) H. Shimahara, H . Suzuki, N. Sugiyama, and K. Nisizawa, Agric. Biol. Chem., 39 (1975)

293-299; 301-312. (118) 0. Perila and C. T. Bishop, Can. J. Chem., 39 (1961) 815-826. ( 1 19) K. Shimizu and M. Ishihara, Agnc. B i d . Chem., 47 (1983) 949-955. (119a) R. Takahashi, 1. Kusakabe, S. Kusama, Y. Sakurai, K. Murakami, A. Maekawa, and T. Suzuki, Agric. B i d . Chem., 48 (1984) 2943-2950. (120) H. 0. Bouveng, T. Iwasaki, B. Lindberg, and H. Meier, Acru Chem. Scand., 17 (1963) 1796- 1197. (121) K. Kat6, A. Takigawa, Y. Yamaguchi, and Y. Ueno, Agric. Biol. Chem., 40 (1976) 2495-2497. (122) A. D. Elbein, J. Biol. Chem., 244 (1969) 1608-1616. (123) J. S. Heller and C. L. Villemez, Biochem. J., 129 (1972) 645-655.

~

180

BARRY V. McCLEARY AND NORMAN K. MATHESON

the synthesis of glucomannan (with non-regular replacement by D-glucose), instead of two homopolymers. Evidence for the presence both of isolated and contiguous D-glucosyl residues in the main chain of various glucomannans has, from the nature of the oligosaccharide products, been obtained with either endo-( 1 + 4)P-D-glucanase or P-D-mannanase. The endo-( 1 + 4) -P-~-glucanasedigest of a lily glucomannan contained 4-O-P-~-mannosylcellobiose" [ P-D-Man(1 + 4)-P-~-Glc-( 1 + 4)-~-Glc],and the P-D-mannanase digest of konjac glucomannan contained cellobiose,117indicating that both glucomannans contained contiguous D-glucosyl residues. However, cellobiose was not a reaction product of hydrolysis of salep glucomannan by several P-Dmannanases, and, furthermore, none of the oligosaccharides of low d.p. appeared to contain contiguous D-glucosyl residues.I6 Both endo-(1 + 4)-P-~-glucanaseand p-D-mannanase have also been employed in the analysis of the fine-structure of glucomannan, and, from the structures and proportions of reaction products, various repeating sequences have been p r o p ~ s e d . " ~However, ~ ' ~ ~ these studies have not considered the extensive degree of possible transglycosylation catalyzed by both of these enzymes.16s20With glucomannan, these reactions are particularly significant, because some of the enzyme-binding sub-sites can accommodate either a D-glucosyl or a D-mannosyl unit. Although these reactions can also possibly occur with galactomannans, they are far less significant, because of the effect of D-galactosyl groups on substrate binding; in general, any products of transglycosylation that contain a D-galactosyl group would be expected to re-form the original oligosaccharides on further hydrolysis. The ratio of the amounts of oligosaccharides produced on P-D-mannanase hydrolysis of glucomannan is also dependent on the physical nature of the substrate,I6 and consequently, enzymic hydrolysis would appear to have less potential in studies on glucomannan fine-structure. 4. Galactoglucomannan

The application of P-D-mannanase and endo-( 1 + 4)-P-~-glucanaseto the structural analysis of galactoglucomannans has also been exploited. Characterization of reaction products provides information on the location of the D-galactosyl branch-units and on the distribution of D-glucosyl residues within the main chain. The enzymes should also find use in determining whether a polysaccharide is a single molecular species or a mixture. Preliminary studies on a galactoglucomannan from seeds of Cercis siliquastrum have been performed, and the isolation of a trisaccharide containing D-galactose, D-glucose, and D-mannose from the p-D-mannanase hydrolyzate of this polysaccharide confirmed that the sugar residues were all covalently linked in a single polysaccharide

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

181

An extracellular polysaccharide preparation from suspension-cultured cells of Nicotiana tabacum, judged to be homogeneous by several physicochemical criteria, contained'24 D-galactose, D-glucose, D-mannose, D-xylose, and L-arabinose in the ratios of 1.02: 1.00: 1.01 :0.07: 0.16. Partial hydrolysis with oxalic acid gave a polysaccharide containing only D-galactose, Dglucose, and D-mannose (0.47 : 1.00:0.78). On treatment with cellulase, two oligosaccharides were purified from the hydrolyzate, namely, p-D-Man(1 + 4 ) - ~ - G l cand 36. It was concluded that the polymer consisted of a a-D-Gal 1

3.

6 P-D-Man-( 1 + 4)-D-GlC 36

p-( 1+ 4)-linked main-chain of alternating D-glucosyl and D-mannosyl residues and that about two-thirds of the D-mannosyl residues carried an a-D-galactosyl group. A similar polysaccharide was found in the hemicellulosic fraction of the cell-wall material prepared from suspensioncultured, tobacco cells. Enzymic hydrolysis of this polysaccharide (before oxalic acid treatment) gave a complex elution-profile on Bio-Gel P-2, and only one of the oligosaccharide reaction-products could be purified. Extraction of the a-cellulose fraction'24aof the midrib of tobacco leaves with alkaline borate gave a galactoglucomannan (Gal 15 :Glc 27 :Man 56) containing a small proportion of arabinose and xylose (2%), indicating the possible presence of xyloglucan. Hydrolysis with p-D-mannanase gave ( 4)-P-~-Man-( 1 + 4 ) - ~ - M a nand , (1 + p - ~ - G l c -1(+ 4 ) - ~ - M a n~, - D - G I C1-+ 4)-P-~-mannobiose,as well as higher oligosaccharides containing all three sugars; structures for pentasaccharide 36a and the hexasaccharide having P-D-GIc-(1 -+ 4)-P-D-Man-( 1 + 4)-P-D-GlC-( 1 + 4 ) - ~ - M a n 6

t

1

O-D-Gal 36a

an additional P-D-(1 + 4)-linked D-mannose residue at the reducing end were proposed from methylation analysis. A hexasaccharide structure was proposed in which the D-galactosyl unit in 36a was further substituted (124) Y. Akiyarna, S. Eda, M. Mori, and K. Kat6, Phytochernisrry, 22 (1983) 1177-1180. (124a) S. Eda, Y. Akiyarna, K. Kat6, R. Takahashi, I. Kusakabe, A. Ishizu, and J. Nakano, Carbohydr. Res., 131 (1984) 105-118. (124b) S. Eda, Y.Akiyama, K. Kat6, A. Ishizu, and J. Nakano, Carbohydr. Res., 137 (1985) 173- 18 1.

182

BARRY V. McCLEARY A N D NORMAN K. MATHESON

(1 + 2)-p by a D-galactosyl group: a heptasaccharide homolog was also described, (1 + 2)-p-~-Galactosylsubstitution is found in xyloglucan. The sequences in the p-( 1 + 4) chains of the oligosaccharides were consistent with the action pattern of p-D-mannanase with the (1+4)-@D-mannan or -D-glucomannan chain,I6 and also indicated that D-galactosyl substitution occurs on D-mannosyl residues. , and the Hydrolysis with cellulase released @-D-Man-(1 + 4 ) - ~ - G l c36, pentasaccharide having the proposed structure 36b, as well as the hexasaccharide with an additional galactosyl group P-D-( 1 + 2)-linked to the (1 + 6)a-D-galactosyl unit. The isolation of these products having a substituent P-D-Man-(l+4)-P-D-GlC-(l + 4 ) - P - ~ - M a n - ( l + 4 ) - D - G k 6

t

1

a-D-Gal 36b

on the D-mannosyl residue penultimate to the reducing end indicated that the cellulase hydrolyzed at D-glucosyl residues, but that the pattern of binding was different from that of p-D-mannanase16 and lysozymes. V. PECTICPOLYSACCHARIDES In the pectic polysaccharides, the most common constituents are Dgalactosyluronic, D-galactosyl, L-arabinosyl, and, in some cases, D-apiosyl units. Lesser proportions of L-rhamnosyl and D-XYIOSYI, and traces of L-fucosyl, units are also present. The structural relationships of the pectic substances are complex, and fractions prepared from various sources have included rhamnogalacturonan, galacturonan, arabinan, galactan, arabinogalactan, arabinogalactorhamnogalacturonan, and apiogalacturonan. 27,78,125-127 Enzymes128have so far been of limited significance in the characterization of the individual components, other than to confirm aspects of structures already determined chemically. They have, however, been used in the degradation of plant cell-walls, in order to isolate pro top last^.'^^ (125) P. Albersheim, W. D. Bauer, K. Keegstra, and K. W. Talmadge, in F. Loewus (Ed.), Biogenesis of Plunr Cell Wall Polysacchurides, Academic Press, New York, 1973, pp. 117-147. (126) G. 0. Aspinall, in Ref. 125, pp. 95-115. (127) P. Albersheim, in J. B. Pridham (Ed.), Plunr Curbohydrute Chemistry, Academic Press, New York, 1974, pp. 145-164. (128) t. RexovP-BenkovP and 0. MarkoviE, Adu. Curbohydr. Chem. Biochem., 33 (1976) 323-385. (129) S. lshii and T. Yokotsuka, Agric. Biol. Chem., 35 (1971) 1157-1159.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

183

Also, fractionated enzymes have allowed the separation of polymer segments. Treatment of suspension-cultured, sycamore cells with endo-( 1 + 4)-a-~-galacturonanase(EC 3.2.1.15) gave acidic and neutral polymer fractions that were separated by gel and ion-exchange chromatography: -75% of the D-galacturonic acid of the cell wall was removed, releasing -16% of the cell wall as soluble p r ~ d u c t s . ' ~ ~ *A' ~released '" rhamnogalacturonan 1+ fraction was found to consist of the repeating unit + 4)-a-~-GalA-( 2)-a-~-Rha-( 1 +. Fragments of apple cell-walls treated with endo-( 1 + 4)-a-~-gaIacturonanase'~~ were either of high molecular weight, containing L-arabinose and L-rhamnose, or of low molecular weight, rich in D-galactose and a glycuronic acid. Endo-galacturonanase released 95% of the glycosyluronic residues from potato-tuber as soluble fractions of various molecular weights. Methylation analysis of the fraction of highest molecular weight showed that it contained, in decreasing proportions, (1 + 4)-linked galactose, (1 + 5)-linked arabinose, (1 + 4)-linked galacturonic acid, and ( 1,2,4)-linked rhamnose. The fraction of intermediate molecular weight contained (1,3,4)-linked galacturonic acid, 1- and (1,3)-, as well as (1,3,4)linked rhamnose, and branched arabinosyl and galactosyl residues. A tomato endo-galacturonanase, acting on isolated tomato-fruit cellgave, in 5% yield, a fraction of high molecular weight that contained 58% of galactose, 15% of arabinose, 4% of rhamnose, and 22% of galacturonic acid. I3C-N.m.r. spectroscopy indicated linkages of ( 1 + 4)-a-~-galactosyland (1 + 5)-a-~-arabinosylresidues. In studies employing'33 pectin lyase (EC 4.2.2.10) and pectate lyase (EC 4.2.2.2), >go% of the D-galactosyluronic residues of purified, apple pectic-substances were found to be free from neutral side-chains, and the neutral sugars were associated with fragments having higher molecular weight. From the gelchromatographic patterns, it was concluded that the neutral sugars were concentrated in blocks of more highly substituted ("hairy") regions, separated by unsubstituted ("smooth") regions containing D-galactosyluronic residues. When cherry-fruit pectin was subjected to chemical p-elimination, (130) P. D. English, A. Maglothin, K. Keegstra, and P. Albersheim, Planr Physiol., 49 (1972) 293-298. (131) K. W. Talrnadge, K. Keegstra, W. D. Bauer, and P. Albersheim, Planr PhysioL, 51 (1973) 158-173. (131a) J. M. Lau, M. McNeil, A. G . Darvill, and P. Albersheim, Carbohydr. Res., 137 (1985) 111-125. (132) M. Knee, A . H. Fielding, S. A. Archer, and F. Laborda, Phytochernistry, 14 (1975) 22 13-2222. (132a) S. Ishii, Phytochernistry, 20 (1981) 2329-2333. (132b) R. Pressey and D. S. Hirnmelbach, Carbohydr. Res., 127 (1984) 356-359. (133) J. A. D e Vries, F. M. Rombouts, A. G . J. Voragen, and W. Pilnik, Carbohydr. Polyrn., 2 (1982) 25-33.

184

BARRY V. McCLEARY AND NORMAN K. MATHESON

followed by hydrolysis by endo-( 1 -$4)-a-~-galacturonanase,and the reaction products were fractionated by gel c h r ~ m a t o g r a p h y ,the ' ~ ~results were interpreted as indicating that the neutral sugars occurred both as long and short side-chains on highly substituted regions which were interspersed with unsubstituted regions, a model similar to that proposed'33 for apple pecticsubstances. Pectin lyase and endo-( 1 + 4)-a-~-galacturonanasewere employed in the degradation of an acidic polysaccharide from soy sauce.135 On partial hydrolysis with acid, a degraded fraction was obtained which, unlike the original polysaccharide, was susceptible to hydrolysis. This degraded fraction contained D-galacturonic acid (83%), D-xylose (13%), and a trace of L-rhamnose, but was devoid of D-galactose and L-arabinose, although these had been present initially. The fraction of lower d.p., produced on hydrolysis of degraded polysaccharide by endo-( 1 -$ 4)-cu-~-galacturonanase,consisted of D-galacturonic acid, its a-(l+4)-linked dimer and trimer, and two heterosaccharides identified as 37 and 38. Their structures were determined

p-D-Xyl-( 1 + 3 ) - ~ - G a l A 31

38

by methylation analysis and by using p-D-xylosidase. These results are consistent with the degraded polymer's having a backbone of a-(1+ 4)linked D-galactosyluronic residues with D-xylosyl units attached through p-( 1+ 3) linkages. Pectic polysaccharides contain sections, rich in p-( 1 +4)-linked D-galactosyl residues, which are susceptible to hydrolysis by endo-( 1+ 4)-pD-galactanase (EC 3.2.1.89). (1 + 4)-p-~-Galacto-bioseand -triose were produced from soybean arabinogalactan on enzymic hydrolysis, indicating that the D-galactosyl residues are p-( 1+ 4)-linked.'36 This galactanase was unable to hydrolyze coff ee-bean arabinogalactan, which has p-D(1 + 3)-galactosyl linkages. Soybean arabinogalactan gave (1 + 4 ) - p - ~ galactobiose as the major product, with small proportions of D-galactose and heterosaccharides. The low proportion of heterosaccharides was unexpected, but a possible explanation has come from more-detailed studies (134) J.-F. Thibault, Phyrochemisfry, 22 (1983) 1567-1571. (135) T. Kikuchi and H. Sugimoto, Agric. Bid. Chem., 40 (1976) 87-92. (136) S. Emi and T. Yamamoto, Agric. Bid. Chem., 36 (1972) 1945-1954.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

185

with P - ~ - g a l a c t a n a s e ,when ' ~ ~ (1 + 4)-P-~-galacto-tetraose, with lesser proportions of -triose and -biose, plus another fraction (which was excluded on chromatography on Bio-Gel P-2 and contained 87% of L-arabinose, 6% of D-galactose, 4% of L-rhamnose, and 3% of D-glucose) were obtained. The results suggested that the L-arabinose in soybean arabinogalactan occurs as oligo-L-arabinosyl units having a d.p. of at least 10, rather than as monoor short oligo-substituents on a galactan backbone. The more-highly substituted fragments of apple pectic substance^'^^ (segments of rhamnogalacturonan carrying neutral side-chains) were treated Oligosaccharides of d.p. of -25, containing with ( 1 + 4)-P-~-galactanase.'~~" mainly L-arabinosyl plus D-galactosyl units (but not ~-xylosyl,D-glucosyl, or L-rhamnosyl units) were released, indicating the presence of arabinogalactan side-chains. The distribution of methoxyl groups in apple and citrus pectic subs t a n c e ~ has ' ~ ~ been ~ assessed by fractionation of degradation products released by pectin- and pectate-lyases. Apiogalacturonans from the cell wall of Lemna minor have a galacturonan backbone with side chains composed of D-apiose. The content of esterified D-galacturonic acid is low (1-3.5%), and the D-apiose content varies from 7.9 to 38.1 '/o. Apiogalacturonans of high D-apiose content were not degraded by a commercial pectinase preparati~n,'~'butthose of low D-apiose content were, indicating that both sugars are part of the same polymer. Removal of L-arabinose from pectic fractions on treatment with a-Larabinofuranosidase is consistent with an exterior positioning of at least some of these residues. Fifty percent of the L-arabinose in a sugar-beet arabinan was released by this enzyme, leaving a polymeric product71having L-arabinose : D-galactose :L-rhamnose ratios of 5 :3 : 1. Enzymes from other sources have, with different polysaccharide preparations, given values for the degree of hydrolysis of 90% (Ref. 72), 22% (Ref. 73a), and'39 38%. In the first, an essentially a-(1 + 5)-linked L-arabinan was obtained, indicating preferential splitting of a-(1 + 3) bonds. Apple cell-wall fragments lost -75% of their ~-arabinose'~' and this sugar was also released from grapejuice arabinan.'"" Incomplete hydrolysis has been suggested14' as being (137) J . M . Labavitch, L. E. Freeman, and P. Albersheim, J. Biol. Chem., 251 (1976) 5904-5910. (137a) J. A. de Vries, C. H. den Uijl, A. G. J. Voragen, F. M . Rombouts, and W. Pilnik, Carbohydr. Polyrn., 3 (1983) 193-205. (137b) J. A. de Vries, F. M. Rombouts, A. G. J. Voragen, and W. Pilnik, Carbohydr. Polym., 4 (1984) 89-101. (138) D. A. Hart and P. K. Kindel, Biochem. J., 116 (1970) 569-579. (139) A. Kaji, M. Sato, and Y. Tsutsui, Agric. Biol. Chem., 45 (1981) 925-931. (139a) J. C. Villetaz, R. Amado, and H. Neukom, Carbohydr. Polym., 1 (1981) 101-105. (140) M. Tanaka, A. Abe, and T. Uchida, Biochim. Biophys. Acta, 658 (1981) 337-386.

186

BARRY V. McCLEARY A N D NORMAN K. MATHESON

due to the presence of pyranoid rings, a-(1 + 2) linkages, and D-galactosyl units. An endogenous activity did not release all of the L-arabinosyl units from an arabinogalactan and a cell-wall polysaccharide fraction of lupin ~oty1edons.l~~ P-D-Galactosidase gave almost no hydrolysis of the cell-wall polysaccharide. A mixture of a-L-arabinofuranosidase and P-D-galactosidase, or P-D-galactosidase alone, with the partially acid-hydrolyzed polysaccharide, gave extensive, but still incomplete, hydrolysis of D-galactosyl units. The arabinans are highly branched polymers of a-L-arabinofuranosyl residues having a-(1-* 3) and a-(1 + 5 ) linkages. Beet arabinan was hydrolyzed to the extent of only 3% by endo-a-~-arabinofurananase,'~~ in agreement with the highly branched structure. On treatment with a-L-arabinof~ranosidase,'~ a polymer of a-(1 + 5)-linked L-arabinose could be precipitated from solution. This was hydrolyzed by endo-a-L-arabinofurananase to the extent of 23%, with release of a series of L-arabino-oligosaccharides initially and, on extended incubation, of L-arabinose and ( 1 + 5 ) a-~-arabinobiose,'~~ providing further evidence for the structure of the arabinan substrate. Partially debranched arabinan was hydrolyzed by endoa-L-arabinofurananase at 16 times the rate for native arabinan.'43 VI. AGAROSEA N D RELATED POLYSACCHAR~DES

Agarla consists of a spectrum of polysaccharides with three idealized extremes in structure, namely, neutral agarose, pyruvic acetalated agarose with little sulfation, and a sulfated ga1a~tan.l~'Agarose is made up of alternating, repeating, (1 + 4)-linked, 3,6-anhydro-a-~-galactosyl and (1 + 3)-linked P-D-galactosyl residues. '44,146,147 The D-galactose content in acid hydrolyzates can be estimated by oxidation with D-galactose oxidase (EC 1.1.3.9) followed by a 'H-n.m.r.-spectroscopic determinati~n.'~'The fraction termed agaropectin has some of the 3,6-anhydro-~-galactosyl residues replaced by 6- 0-sulfo-L-galactosyl resid~es,'~'and there can be partial replacement of D-galactosyl residues with the pyruvic acetal, namely, 4,6-0-( 1-carboxyethy1idene)-~-galactosyl residues. The terms agarose and (141) (142) (143) (144) (145) (146) (147) (148) (149)

N. K. Matheson and H. S . Saini, Carbohydr. Res. 57 (1977) 103-116. A. Kaji and T. Saheki, Biochim. Biophys. Acta, 410 (1975) 354-360. L. Weinstein and P. Albersheim, Plant Physiol., 63 (1979) 425-432. C. Araki and K. Arai, Bull. Chem. SOC.Jpn., 29 (1956) 339-345. M. Duckworth and W. Yaphe, Carbohydr. Res., 16 (1971) 189-197; 435-445. C. Araki and S. Hirase, Bull. Chem. SOC.Jpn., 33 (1960) 291-295. C. Araki and K. Arai, Bull. Chem. SOC.Jpn., 30 (1957) 287-293. J. N. C. Whyte and J. R. Englar, Carbohydr. Res., 57 (1977) 273-280. C. Araki, Proc. In?. Seaweed Symp., 5th, (1966) 3-17.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

187

agaropectin were introduced for the gelatinous (uncharged) and nongelatinous (charged) constituents of Japanese agar. The 3,6-anhydro-~galactosyl residues are derived from 6- 0-sulfo-L-galactosyl residues by enzymic conversion150at the polymer level (see Scheme 1). /

0

/

0

SCHEME1.-Conversion Residues.

of 6-O-Sulfo-a-~-galactosylinto 3,6-Anhydro-a-~-galactosyl

Major structural features of agarose and related polysaccharides were first determined by partial, acid hydrolysis and by using an agarase preparati~n.'--'~' On treatment of a Japanese agar with agarase, neoagarobiose and di(neoagarobiose) (39) were identified. Neoagarobiose had not previously been detected in partial, acid hydrolyzates, due to the susceptibility P-11-agarase

.1

+4)-a-~-AnGal-( 1+ 3)-p-~-Gal-( 1+ 4)-a-~-AnGal-( 1 + 3 ) - p - ~ - G a l -1(+ neoagarobiose

I

I agarobiose

t

I di(neoagarobiose) 39

of the a linkage to acid. The isolation of neoagarobiose and di(neoagarobiose), together with the knowledge that agarobiose was present in the partial, acid h y d r ~ l y z a t e , ' ~led ~ , 'to ~ ~the repeating unit accepted for agarose (39), and this has been confirmed with agarases from a range of bacteria. 145.1 51- 155 Essentially all enzymes that cleave agarose and related polysaccharides at the P-D-(1 + 4) linkage between the D-galactosyl and the (150) D. A. Rees, Biochem. J., 81 (1961) 347-352.

(151) W. Yaphe, Can. 1. MicrobioL, 3 (1957) 987-993. (152) M. Duckworth and J. R. Turvey, Biochem. J., 113 (1969) 139-142; 687-692; 693-696. (153) A. R. Sampietro and M. A. Vattuone de Sampietro, Biochim. Biophys. Acta, 244 (1971) 65-76. (154) M. A. Vattuone, E. A. de Flores, and A. R. Sampietro, Carbohydr. Res., 39 (1975) 164- 167. (155) M. Malmqvist, Carbohydr. Rex, 62 (1978) 337-348.

188

BARRY V. McCLEARY AND NORMAN K. MATHESON

3,6-anhydro-a-~-galactosylresidues are thus termed P-D-agarase (EC 3.2.1.81), but an enzyme active on the a - ~ 1+ ( 3) linkage between the 3,6-anhydro-a-~-galactosyl and the D-galactosyl residue (a-L-agarase) has also been reported.lS6Substitution of the disaccharide unit with an 0-sulfo or a pyruvic acetal group interferes with the reaction. Hydrolysis with P-D-agarase of three fractions from agar, representative of neutral agarose, pyruvic acetalated agarose with little sulfation, and sulfated galactan, gave both neutral and charged oligosa~charides.'~~ The ratios of these from the three fractions were 9 5 : 5 , 28:72, and 18:82, respectively. The neutral oligosaccharides obtained from all three fractions were tri(neoagarobiose), di(neoagarobiose), and neoagarobiose, and, in agarose, their ratios were 6 : 7 : 1. The charged oligosaccharides from sulfated galactans were separated by ion-exchange chromatography into those containing mainly 4,6-0- (1-carboxyethylidene)-~-galactosylresidues and those having a preponderance of sulfate groups. Two oligosaccharides containing the pyruvic acetal were characterized, and shown to be analogous to the hexa- and tetra-saccharide of the neutral series of oligosaccharides, but to contain a 4,6- 0-(1-carboxyethy1idene)-D-galactosylresidue in place of the penultimate D-galactosyl residue towards the nonreducing end of the oligosaccharides (40), indicating that the enzyme can hydrolyze near a Me

& g b o & oT I

HOzC-C

CHzOH

0

. P

OH

0

40

pyruvic acetal substituent. The high yield of oligosaccharides containing 4,6- 0-(1-carboxyethy1idene)-~-galactosyl residues, and yet free from sul-

fate, indicates that replacement of the D-galactosyl residues with 4,6-0-( 1carboxyethy1idene)-D-galactosyl residues occurs in those regions of the residues molecule where the replacement of the 3,6-anhydro-~-galactosyl by 6-O-sulfo-~-galactosylresidues is low. In a complementary manner, the portion of pyruvylated agarose that is resistant to enzymic attack has a greatly decreased content of pyruvic acetal, but is richer in sulfate. Treatment of the galactan sulfate fraction of agar with P-D-agarase yielded a series of oligosaccharides that could be fractionated on Sephadex G-25. The major (156) K. Young, K. C. Hong, M. Duckworth, and W. Yaphe, Roc. Inf. SeoweedSymp., 7rh, (1973) 469-472.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

189

reaction-products were sulfated oligosaccharides of high molecular weight, although pyruvylated hexa- and tetra-saccharides were present in smaller proportions. Porphyran, a related galactan, has a structure similar to that of agarose, except that alternation is between either D-galactosyl or 6-O-methyl-~or 6-0galactosyl on the one hand, and either 3,6-anhydro-~-galactosyl sulfo-L-galactosyl residues on the ~ t h e r . ' ~ ' -Native ' ~ ~ porphyran and porphyran treated with alkali (to remove most of the sulfate groups, with the residues) were both hydrolyzed by formation of 3,6-anhydro-~-galactosyl P-D-agarase more slowly than was a g a r 0 ~ e . With I ~ ~ native porphyran, there was only a 30% conversion of the polysaccharide into oligosaccharides, which included neoagarobiose, di( neoagarobiose), and a tetrasaccharide containing 6- O-methyl-~-galactosylresidues. From the relatively uniform ratio of D-galactosyl and 6-0-methyl-~-galactosylresidues throughout all of the reaction products, it was concluded that, within the alternating sequence of the D and L forms of galactosyl derivatives in porphyran, replacement of D-galactosyl by 6-O-methyl-~-galactosylresidues was not regular. Almost half of the alkali-treated porphyran was not degraded to a detectable degree, and there was an accumulation of tetra~accharide.'~'The polymer still contained some sulfate (1.8%, compared to 11.7% in the native polymer). Substitution by sulfate presents a serious hindrance to enzyme action: the methyl ether groups in porphyran lower the rate of hydrolysis. These effects account for the difference in hydrolysis between this polysaccharide and agarose. The arrangement of sulfate groups in the native porphyran was difficult to define, but sulfated oligosaccharides having a minimum d.p. of 8-10 and containing more than one sulfate group were produced. This mixture of sulfated oligosaccharides could be separated into six bands on ion-exchange chromatography, but each band was not a single molecular species. However, each gave a single band on electrophoresis, indicative of similar charge-to-size ratios of components within each fraction. None of the oligosaccharides isolated contained only one sulfate group, residues and all had, on average, two or more 3,6-anhydro-a-~-galactosyl per molecule. With another P-D-agarase,I6' 63-0-methyldi(neoagarobi0se)and 63,65-di0-methyltri(neoagarobiose), as well as two novel, monosulfated tetrasaccharides, namely, 41 and its 63-0-methylated derivative, were found in porphyran digests. Neutral oligosaccharides containing 6-0-methyl groups, (157) (158) (159) (160)

J. R. Turvey and T. P. Williams, Proc. Inr. Seaweed Symp., 4th, (1964) 370-373. N . S. Anderson and D. A. Rees, J. Chem. SOC.,(1965) 5880-5887. J. R. Turvey and J. Christison, Biochem. J., 105 (1967) 311-316; 317-321. L. M. Morrice, M. W. McLean, W. F. Long, and F. B. Williamson, Eur. J. Biochern., 133 (1983) 673-684; 137 (1983) 149-154.

190

BARRY V. McCLEARY AND NORMAN K. MATHESON

0 41

and sulfated oligosaccharides, were both terminated at their reducing ends by otherwise unsubstituted neoagarobiose. Characterization of the monosulfated tetrasaccharide allowed an interpretation of the I3C-n.m.r. spectra of the sulfated oligosaccharides of higher d.p. It was concluded that the sulfate residues occur in segments averaging 2.0-2.5 contiguous units. The relative amounts of neutral oligosaccharides were significantly different from those previously found'52 in a P-D-agarase hydrolyzate of porphyran. The variation was considered to reflect differences in the substrates studied, as well as in enzyme specificities. A second endo-enzyme fraction from the same source,16owhich is probably the same as P-D-di(neoagarobiose) hydrolase,I6' hydrolyzed porphyran to neutral oligosaccharides (24% ) which were mostly (>go% ) disaccharides (neoagarobiose and 6'- O-methylneoagarobiose in the ratio of 1:2). The degree of substitution in porphyran varies geographically and seasonally.162 Purified, extracellular P-D-agarase and cell-wall P-D-di(neoagarobiose) hydrolase have been employed in an analysis of the polysaccharides from several Graciluria spp., with the aim of providing an index for evaluating the gelling p r ~ p e r t i e s . ' ~ 'On " ~ ~hydrolysis by P-D-agarase, all of them gave the same pattern of neutral oligosaccharides, but the proportions differed. The four main neutral oligosaccharides were 6'-O-methylneoagarobiose, neoagarobiose, 63-0-methyldi(neoagarobiose), and di(neoagarobiose). The ratio of neutral to charged oligosaccharides also varied. Treatment with a mixture of P-D-agarase and P-D-di(neoagarobiose) hydrolase gave neoagarobiose and 6'-0-methylneoagarobiose as the only neutral products, and the ratio of these varied with the proportions of D-galactosyl and 6-0-methyl-~-galactosylresidues in the original polysaccharide. Although distinguished by their reaction products, there was no direct relationship between gelling ability and the nature of the oligosaccharide fragments. This was considered to be due to various arrangements of charged groups in the different polymers.

(161) D. Groleau and W. Yaphe, Can. J. Microbiol., 23 (1977) 672-679. (162) D. A. Rees and E. Conway, Biochem. J., 84 (1962) 411-416. (163) M. Duckworth, K. C. Hong, and W. Yaphe, Carbohydr. Res., 18 (1971) 1-9.

ENZYMIC ANALYSIS O F POLYSACCHARIDE STRUCTURE

191

VII. ALGINICACID Alginic is an unbranched polymer of 4-linked p-D-mannosyluronic and a-L-gulosyluronic residues, and the proportions of these two components is variable. The percentage of D-mannuronate in alginates of vegetative tissue of algae generally range^'^^.'^^ from 30-70%, but polymers containing >90% of D-mannosyluronic residues have been isolated from the receptacles of Fucus vesiculosus and Ascophyllum nodosum. 164~165Bacterial alginate contains O-acetyl groups. 167,168 GDP-D-mannuronic acid has been detected in Fucus gardneri, and incorporation into alginate by a particulate preparation was d e m ~ n s t r a t e d . ' ~ ~ a-L-Gulosyluronic residues are formed by epimerization of p-D-mannosyl2). uronic residues, after p o l y m e r i ~ a t i o n ' ~ ~(see - ' ~ Scheme ~

Ii /

0

SCHEME2.-Conversion of P-D-Mannosyhronic into a-L-Gulosyluronic Residues, and the Interconversion of the 4C, and the ' C , Conformers of the Latter.

Hydrolytic enzymes active with alginate have not been reported. Depolymerization occurs by elimination, releasing oligosaccharide fragments having an unsaturated glycosyluronic group (4-deoxy-~-erythro-hex-4enopyranosyluronate) at the nonreducing end.173Enzymes specific for either (164) A. Haug, in D. H. Northcote (Ed.), Plant Biochemistry, MTP Inr. Reu. Sci., Ser. One, 11 (1974) 51-88. (165) A. Haug, B. Larsen, and E. Baardseth, Proc. Int. Seaweed Symp., 6rh, (1969) 443-451. (166) F. G. Fischer and H. Dorfel, Z.Physiol. Chem., 302 (1955) 186-203. (167) A. Linker and R. S. Jones, J. Biol. Chem., 241 (1966) 3845-3851. (168) P. A. J. Gorin and J. F. T. Spencer, Can. J. Chem., 44 (1966) 993-998. (169) T.-Y. Lin and W. Z . Hassid, J. Biol. Chem., 241 (1966) 3283-3293; 5284-5297. (170) A. Haug and B. Larsen, Biochim. Biophys. Acta, 192 (1969) 557-559. (171) B. Larsen and H. Grasdalen, Carbohydr. Res., 92 (1981) 163-167. (172) D. F. Pindar and C. Bucke, Biochem. J., 152 (1975) 617-622. (173) J. R. Turvey, in D. J. Manners (Ed.), Biochemistry of Carbohydrates, MTP Int. Reu. Sci., Ser. Two, 16 (1978) 151-177.

192

BARRY V. McCLEARY AND NORMAN K. MATHESON

the a-L-gulosyluronic (L-guluronan lyase) or the P-D-mannosyluronic bonds (D-mannuronan lyase) (EC 4.2.2.3) have been identified.'74-'76In general, enzymic activities from algae and mollusks split the P-D-mannosyluronic linkage, whereas those of bacterial origin have a preference for cleaving the a-L-gulosyluronic bond. Enzymic cleavage of polymers containing glycosyluronic residues is particularly valuable, as the glycosyluronic linkage is resistant to acid hydrolysis, and uronic acids decompose in hot acid. Lyases have the advantage that they do not promote transglycosylation. The depolyrnerization of alginate by an elimination, rather than a hydrolytic mechanism, was d e r n o n ~ t r a t e d with ' ~ ~ an enzyme from abalone liver. The products included a disaccharide consisting of an unsaturated glycosyluronic group and a D-mannuronic acid residue. Two enzymes from the hepatopancreas of Dolabella auricula were both specific for the D-mannosyluronate linkage.'75 Reaction with oligoglycosiduronates, composed essentially of D-mannuronate, produced unsaturated di- (42), tri- (43), and higher oligosaccharides, where AXA is an unsaturated glycosyluronic group. Alginate is degraded to an extent proportional to the D-mannuronate content. AXA-( 1 + 4 ) - ~ - M a n A

AXA-( 1 + 4 ) - p - ~ - M a n A -1(+ 4 ) - ~ - M a n A

42

43

Alginates rich in L-guluronate and oligo-L-guluronan segments are rapidly lysed by L-guluronan 1 y a ~ e . The I ~ ~ end products of the action on oligoglycosiduronic segments composed entirely of 4-linked a-L-gulosyluronic residues are mainly176 44, 45, and 46. The same unsaturated group is produced from each glycosyluronic residue. AXA-( 1 + 4)-D-GuIA

AXA-( 1 + 4 ) - a - ~ - G u l A -1(+ 4 ) - ~ - G u l A

45

44

AXA-( 1 + 4)-a-L-GulA-(1 + 4)-a-L-GulA-(1 + 4 ) - ~ - G u l A 46

Cleavage of mixed uronic segments with L-guluronan lyase yielded the aforementioned oligouronic acids plus 47 and higher heterosaccharides, all with L-guluronate residues at the reducing end. Treatment of two alginates AXA-( 1 + 4 ) - p - ~ - M a n A -1( + 4 ) - ~ - G u l A

47

(174) I. Tsujino and T. Saito, Nature (London), 192 (1961) 970-971. (175) K. Nisizawa, S. Fujibayashi, and Y. Kashiwabara, J. Biochem. (Tokyo),64 (1968) 25-37. (176) J. Boyd and J. R. Turvey, Curbohydr. Res., 57 (1977) 163-171: 66 (1978) 187-194.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

193

with this enzyme yielded173blocks of P-D-mannosyluronic units with a d.p. of -25. The location of acetyl groups in bacterial alginates has been examined177 by using an L-guluronan lyase.I7* Incubation with Azotobacter uinelandii alginate yielded a fraction with which the acetyl groups were associated, and this was of higher molecular weight than the material free from acetyl groups. This higher-molecular-weight fraction was composed of D-mannosyluronic residues. If the alginate was treated with a phage D-mannuronan lyase, the products were almost all of low molecular weight, and many of the oligouronic acids were acetylated, establishing that the acetyl groups occur on the D-mannosyluronic residues. The exclusive location of the 0-acetyl groups on the D-mannosyluronic residues led to the suggestion that acyl groups may protect them from epimerization. This has been acting on native confirmed with a purified ~-mannuronan-C-5-epimerase and deacetylated polysaccharides; substitution was also found to protect neighboring D-mannosyluronic Multiple attack by the epimerase has been proposed.178b Partial, heterogeneous hydrolysis of alginate with acid, followed by specific dissolution or precipitation of fragments under different p H and salt conditions, or in the presence of particular cations,179gave fragments that were then analyzed electrophoretically, and characterized. This yielded fractions that were electrophoretically pure, but the sections rich in Lgulosyluronic residues still contained some D-mannosyluronic residues, and vice versa. Segments containing mainly D-mannosyluronic residues were further enriched in this component on continued hydrolysis with acid. Also, as there was only a moderate decrease in the d.p. of insoluble, resistant fragments, it was proposed that hydrolysis of these proceeds mainly from the chain ends, and that L-gulosyluronic units in the component rich in D-mannuronate are positioned terminally. The same proposal was made for the D-mannosyluronic units in segments consisting mainly of L-guluronate. From these observations, the average minimum lengths of sections of mainly D-mannosyluronic or of mainly L-gulosyluronic residues were estimated, and it was concluded that aliginate has a block type of structure, with three types of sections, one rich in L-gulosyluronic, one in D-mannosyluronic, and a third having essentially alternating sequences of L-gulosyluronic and D-mannosyluronic residues. (177) I. W. Davidson, I. W. Sutherland, and C. J. Lawson, J. Gen. Microbiol., 98 (1977) 603-606. (178) I. W. Davidson, I. W. Sutherland, and C. J. Lawson, Biochem. J., 159 (1976) 707-713. (1788) G. SkjPk-Braek, B. Larsen, and H. Grasdalen, Carbohydr. Res., 145 (1985) 169-174. (178b) B. Larsen, G. SkjHk-Braek, and T. J. Painter, Carbohydr. Res., 146 (1986) 342-345. (179) A. Haug, B. Larsen, and 0. Smidsrbd, Acm Chem. Scand., 21 (1967) 691-704.

194

BARRY V. McCLEARY AND NORMAN K. MATHESON

However, hydrolysis by L-guluronan lyase of an alginate fraction similar to the alternating fraction179gave, as the major reaction-products,”’ 45 and 47 in approximately equal amounts. From the significantly decreased levels of /3-D-mannosyluronic residues in the reaction products, it was concluded that more of the unsaturated uronic acid was derived from this residue, and that there was therefore a high frequency of -ManA-ManA-GulA- and -ManA-GulA-GulA- sequences in this fraction, indicating a significant deviation from an alternating structure. Another enzymic study181of the fine-structure of alginate employed two alginate lyases. L-Guluronan lyase from Klebsiella aerogenes specifically cleaved the linkage -GuIA~XA-in sequences of d.p. > 5 , where XA is either an a-L-gulosyluronic or a P-D-mannosyluronic residue, whereas the second lyase, from a Flavobacterium sp., appeared to cleave the -XAJManA- linkage specifically. An alginate from Ascophyllum nodosum was exhaustively digested, separately, by each of the enzymes, and the products fractionated by gel chromatography, and characterized by n.m.r. spectroscopy. The structures and proportions of the various oligomeric fractions showed that the Dmannosyluronic and the L-gulosyluronic residues in the native polymer are distributed less regularly than was previously envisaged.179Homopolymeric sequences ranged from 1 to 11 units in length, with all values represented. The distribution was not statistically random, as certain lengths, such as 6 for D-mannosyluronic residues (see Fig. 4), occurred more frequently than predicted. Digestion of alginate from Laminaria digitata with L-guluronan lyase also gave oligosaccharides containing blocks of D-mannosyluronic residues having d.p. values of 1 to 11, and the proportion with d.p. > 9 was less than 7 % of the total: oligosaccharides of d.p. 5-7 were the most abundant. Similar results were obtained with a number of other algal alginates. This provides a model alternative to the structure of algal alginate as being composed of 3 block types (poly-ManA, poly-GulA, and poly-alternatir~g).”~The three types merge into one another in a spectrum of structures. Long homopolymeric sections are rare; the major features are sequences of d.p. of 1-11, with d.p. values of -5-8 occurring more frequently than predicted for a random distribution.

-

(180) K. H. Min, S. F. Sasaki, Y. Kashiwabara, M. Umekawa, and K. Nisizawa, J. Biochem, (Tokyo), 81 (1977) 555-562. (181) A. J. Currie, Ph.D. Thesis, University of Wales, 1983; seen as, J. R. Turvey, personal communication.

ENZYMIC ANALYSIS O F POLYSACCHARIDE STRUCTURE

195

0.120,

V C

?!

L

3 r)

g 0.08-

0

x

r)

C 0,

3

V

0.04-

E LL

Ob

' 2' ' L' ' 6' ' 8 ' ' 10 ' Choin-length of ManA blocks

FIG. 4.-Frequency of Occurrence of Homopolymeric Sequences of D-Mannosyluronic Residues in Alginate from Ascophyllum nodosum. [Key: 0 , predicted values, based on a statistically random distribution of glycosyluronic residues; and 0, values determined experimentally.]

Examination, by n.m.r. spectroscopy, of the products formed by incubation of D-mannuronan C-5-epimerase with alginic acid containing 13% of L-guluronate indicated that reaction adjacent to an existing L-gulosyluronic residue was favored. The L-guluronate content increased"' to 59%. VIII. BACTERIAL PEPTIDOGLYCAN, CHITIN,A N D CHITOSAN In bacteria, the glycan strands of peptidoglycan usually consist of alternatand N-acetyling p-( 1 -P 4)-linked 2-acetamido-2-deoxy-~-glucosyl muramoyl(2-acetamido-2-deoxy-3-~-~-~actoyl-~-g~ucosy~) residues."' The cell-wall glycan of Micrococcus lysodeikticus is degraded by hen egg-white ly~ozyme'~'(EC 3.2.1.17) to di-, tetra-, and octa-saccharides. Lysozyme (muramidase) endo-hydrolyzes 2-acetamido-2-deoxy-~-~-glucosyl bonds in chitosaccharides and solubilized chitin substrates, but acts on the cell-wall peptidoglycans exclusively as an endo-N-acetylmuramidase, splitting only the glycosidic bond of N-acetylmuramoyl residues. Detailed X-ray

(182) J.-M. Ghuysen, Bacreriol. Reu., 32 (1968) 425-464. (183) D. M. Chipman and N. Sharon, Science, 165 (1969) 454-465.

196

BARRY V. McCLEARY AND NORMAN K. MATHESON

crystallographic, 184~185substrate and kinetic on this enzyme provided extensive information on the molecular architecture of the active site and the sub-site binding-requirements, allowing its confident use in structural studies of bacterial cell-wall peptidoglycans. Complementary to hydrolysis by l y s ~ z y m e ' an ~ ~endo-acting , N-acetyl-PD-glucosaminidase 190-192 degrades bacterial cell-wall peptidoglycan to the disaccharide N-acetyl-P-muramoyl-( 1+ 4)-2-acetamido-2-deoxy-~-glucose. The cell-wall peptidoglycan of Staphylococcus aureus has been characterized by employing these enzymes and a p e p t i d a ~ e . ' ~ ' -After ' ~ ~ solubilization of the wall by treatment with l y s o ~ y m e ,teichoic '~~ acids were removed by gel chromatography and electrophoresis, and the peptide substituents were detached from the glycan fragments by treatment with an N-actylmuramoylL-alanine amidase (EC 3.5.1.28). After removal of peptides, the carbohydrate fragments were separated chromatographically, and the disaccharides were shown to be 4-0-(2-acetamido-2-deoxy-~-~-glucosyl)-N-acetyl muramic acid (48) and 4-0-(2-acetamido-2-deoxy-~-~-glucosyl)-N-acetyl6-O-acetylmuramic acid (49). The p linkage was established in these disaccharides by use of the glycosidase N-acetyl-p -D-glucosaminidase (EC 3.2.1.30).'90*193 Reaction of the cell wall with peptidase released intact glycan.'" When the cell wall was incubated with peptidase and endo-p-N-acetylD-glucosaminidase, 2-acetamido-4-0- ( N-acetyl-~-muramoyl)-2-deoxy-~glucose and 2-acetamido-4- 0( N-acetyl-6-0-acetyl-~-muramoyl)-2-deoxyD-glucose were produced.'92 The results are consistent with a structure in which the glycan moiety is composed of unbranched chains of p-( 1 + 4)-linked 2-acetamido-2-deoxy-~-glucosyl residues, with each second residue substituted by a 3-O-~-lactoylgroup. About 50% of the N-acetylmuramoyl residues contained a 6- O-acetyl group, but the pattern of distribution of these is not yet known. On treatment of S. aureus peptidoglycan (184) C. C. F. Blake, L. N. Johnson, G. A. Mair, A. C. T. North, D. C. Phillips, and V. R. Sarma, Roc. R. SOC.London, Ser. B, 167 (1967) 378-388. (185) L. 0. Ford, L. N. Johnson, P. A. Machin, D. C. Phillips, and R. Tjian, J. Mol. Biol., 88 (1974) 349-371. (186) T. Imoto, L. N. Johnson, A. C. T. North, D. C. Phillips, and J. A. Rupley, in P. Boyer (Ed.), The Enzymes, 3rd edn., Vol. 7, Academic Press, New York, 1972, pp. 665-868. (187) J. A. Rupley, Roc. R. SOC.London, Ser. B, 167 (1967) 416-428. (188) D. M. Chipman, Biochemistry, 10 (1971) 1714-1722. (189) M. Leyh-Bouille, J.-M. Ghuysen, D. J. Tipper, and J. L. Strominger, Biochemistry, 5 (1966) 3079-3090. (190) T. Wadstrom and K. Hisatsune, Biochem. J., 120 (1970) 735-744. (191) D. J. Tipper, J. L. Strominger, and J. C. Ensign, Biochemistry, 6 (1967) 906-920. (192) D. J. Tipper and J. L. Strominger, Biochem. Biophys. Res. Commun., 22 (1966) 48-56. (193) D. J. Tipper, J.-M. Ghuysen, and J. L. Strominger, Biochemistry, 4 (1965) 468-473. (194) J.-M. Ghuysen and J. L. Strominger, Biochemisrry, 2 (1963) 1110-1119; 1119-1125.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

HNAc MeCH

HNAc MeCH

I

I

AcNH 48

COzH

197

AcNH

COZH

49

with either of the endo-hydrolases, free disaccharide is obtained only if an N-acetylmuramoyl-L-alanineamidase treatment is included, suggesting that essentially all of the N-acetylmuramoyl residues are substituted by peptide. Similar procedures have been employed in the structural analysis of the glycan moiety of M. lysodeikticus, which was found to have essentially the same fundamental repeat-structure as the S. aureus polymer, but with some difference^.'^' In all but a few strains, 0-acetyl substitution is absent, only some of the N-acetylmuramoyl residues are substituted by peptide (at least 40% are n ~ t ) , " ~and * ' ~a~small proportion of the muramoyl residues are not N-acetylated; splitting of the glycan with lysozyme is incomplete.'s2 The cell walls of several other bacteria have been treated with lysozyme, and the disaccharide fraction characteri~ed.'~'All peptidoglycans studied had the same fundamental repeat-structure. However, slight modifications to this structure can render the polysaccharide resistant to enzymic attack. The resistance of bacterial, cell-wall peptidoglycan to digestion by lysozyme and other enzymes lysing cell walls has been attributed to several factors, including the presence of 0-acetyl groups,198attachment of other polymers (such as teichoic acid), the occurrence of free amino groups (probably in Evidence the peptide portion), or a high degree of peptide cro~s-linking.'~~ that the mode of linkage of the cell-wall peptidoglycan in Micrococcus lysodeikticus and the external, antigenic polysaccharide is through a phosphoric diester linkage (Y to C-1 of the reducing-end D-glucose residue in the latter and 0 - 6 of muramic acid in the peptidoglycan was obtained by characterization of the residue from the action of lysozyme on cell-wall material.'99a (195) D. Mirelman and N. Sharon, J. Bid. Chem., 242 (1967) 3414-3427. (196) E. Muf~oz,J.-M. Ghuysen, M. Leyh-Bouille, J.-F. Petit, and R. Tinelli, Biochemistry, 5 (1966) 3091-3098. (197) D. Mirelman and N. Sharon, J. Biol. Chem., 243 (1968) 2279-2287. (198) W. Brumfitt, A. C. Wardlaw, and J. T. Park, Nature (London), 181 (1958) 1783-1784. (199) J. L. Strominger and J.-M. Ghuysen, Science, 156 (1967) 213-221. (199a) Nasir-ud-Din, M. Lhermitte, G . Lamblin, and R. W. Jeanloz, J. Biol. Chem., 260 (1985) 998 1-9987.

198

BARRY V. McCLEARY AND NORMAN K. MATHESON

The resistance of Bacillus cereus cell-wall peptidoglycan to lysozyme residues actionZmis due to the majority of the 2-amino-2-deoxy-~-glucosyl having free (nonsubstituted) amino groups. Polysaccharide and peptide components of the cell walls were converted into material susceptible to lysozyme by N-acetylation with acetic anhydride. The polysaccharides chitin and chitosan (N-deacetylated chitin)200a, which are structurally related to the glycan portion of bacterial cell-wall peptidoglycan, were initially characterized by chemical procedures, but almond emulsin enzymes proved useful in the establishment of the pglycosidic linkage in chitobiose.201The preparation cleaved P-linked 2acetamido-2-deoxy-~-glucosyl residues, but the a anomer was resistant. Degradation of chitin202and chitosan with (EC 3.2.1.14) and c h i t o s a n a ~ erespectively, ,~~~ together with isolation and characterization of the reaction products, confirmed the structure of chitin as a polysaccharide containing chains of 4-0-substituted 2-acetamido-2-deoxy-~-~-glucosyl residues, and chitosan as the N-deacetylated form of this polymer. Hydrolysis of chitin is affected by modification of the acetyl group.203

IX. GLYCOSAMINOGLYCANS The gl ycosaminoglycans characteristically have a repeating, disaccharideunit structure which is susceptible to endo-depolymerization. Except for hyaluronic acid, this repeating structure is masked by sulfation of hydroxyl groups (in heparin by N-deacetylation and sulfation), or by isomerization of P-D-glucosyluronic to a-L-idosyluronic residues, or by both.206-210 All except hyaluronic acid occur linked to protein as proteoglycans. Chondroitin sulfate and keratan sulfate, as proteoglycans, associate with protein and hyaluronic acid in a macromolecular complex.210*211 Chondroitin sulfates, (200) Y. Araki, T. Nakatani, K.Nakayama, and E. Ito, J. Bid. Chem., 247 (1972) 6312-6322. (200a) R. A. A. Muzzarelli, in Ref. 5, pp. 417-450.

(201) (202) (203) (204) (205) (206) (207) (208) (209) (210) (211)

L. Zechmeister and G. Toth, Forrschr. Chem. Org. Narursr., 2 (1939) 212-247. C. Jeuniaux, Merhods Enzymol., 8 (1966) 644-650. S. Hirano and Y. Yagi, Agric. Biol. Chem., 44 (1980) 963-964. Y. Tominaga and Y. Tsujisaka, Agric. Biol. Chem., 40 (1976) 2325-2333. A. Hedges and R. S. Wolfe, J. Bacteriol., 120 (1974) 844-853. R. W. Jeanloz, in W. Pigman, D. Horton, and A. Herp (Eds.), The Carbohydrates, Vol. 2B, Academic Press, New York, 1970, pp. 589-625. H. Muir and T. E. Hardingham'; in W. J. Whelan (Ed.), Biochemistry of Carbohydrates, MTP Int. Rev. Sci., Ser. One, 5 (1975) 153-222. L. Roden, in W. J. Lennarz (Ed.), The Biochemistry of Glycoproteins and Proteoglycans, Plenum Press, New York, 1980, pp. 267-371. L. Roden and M. I. Horowitz, in M. I. Horowitz and W. Pigman (Eds.), The Glycoconjugates, Vol. 2, Academic Press, New York, 1978, pp. 3-71. L.-A. Fransson, in Ref. 5, pp. 337-415. T. Hardingham, Biochem. Soc. Trans., 9 (1981) 489-497.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

199

dermatan sulfate, and keratan sulfate are released from the proteoglycan structure by proteolysis. Different proteinases degrade the protein section to various degrees. Selective cleavage with specific glycanases and glycan lyases can be used for removal of particular glycospminoglycans; thus, cartilage proteoglycan incubated with a chondroitinase leaves keratan sulfate attached to the protein core. 1. Chondroitin Sulfates

The repeating disaccharide unit of the main chain of chondroitin sulfate is 50, with 0-sulfo groups on the 4- or 6-hydroxyl groups of most 2-acetamido-2-deoxy-~-galactosyl residues. Fractions can be prepared +

4)-p-~-GlcA-( 1 + 3)-P-o-GalNAcSO4-(1 + 50

that have a high percentage of 4-sulfo (A) or of 6-sulfo (C) groups. Chondroitin sulfates can be hydrolyzed by testicular hyaluronoglucosaminidase212(hyaluronidase; EC 3.2.1.39, or lysed by chondroitin ABC lyase (EC 4.2.2.4) or chondroitin AC lyase,* to produce, in high yield, oligosaccharides having an even-numbered d.p. These have a strict, repeating sequence of alternating glycosyluronic and hexosaminyl residues. Lysis produces an oligosaccharide having a A4-unsaturated glycosyluronic group at the nonreducing end. Testicular hyaluronidase is specific for the Dglucosyluronic residue, and digestion with this enzyme gave tetrasaccharides that contained both 4- and 6-sulfated 2-acetamido-2-deoxy-~-galactosyl residues, showing that both types of substitution occur in a single polymer chain. Incubation conditions were chosen that did not favor transglycosylation.*I3This was confirmed by the isolation of related, unsaturated tetrasaccharides from reaction with chondroitin ABC lyase, which does not catalyze tra nsgl yc ~syl atio n ,~ and ~ ~by co-incubation with testicular hyaluronidase and an excess of P-D-ghcosiduronase (EC 3.2.1.31), the latter destroying the acceptor capability of the released o lig ~ s a c c h a rid e s .~Digestion ~~" of squid cartilage with chondroitin ABC lyase released a disaccharide additional to those with sulfate on either C-4 or C-6 of 2-acetamido-2-deoxy-~galactosyl residues: it contained21s two sulfate groups on a single 2acetamido-2-deoxy-~-galactosyl residue, on both C-4 and C-6. Cleavage (212) (213) (214) (214a)

M. Schmidt and A. Dmochowski, Biochim. Biophys. Acta, 83 (1964) 137-140. N. Seno, K. Anno, Y. Yaegashi, and T. Okuyama, Connect. Tissue Res., 3 (1975) 87-96. C. R. Faltynek and J. E. Silbert, J. Bid. Chem., 253 (1978) 7646-7649. W. Knudson, M. W. Gundlach, T. M. Schmid, and H. E. Conrad, Biochemistry, 23

(1984) 368-375. (215) S. Suzuki, H. Saito, T. Yamagata, K. Anno, N. Seno, Y. Kawai, and T. Fumhashi, J. Bid. Chem., 243 (1968) 1543-1550.

200

BARRY V. McCLEARY AND NORMAN K. MATHESON

products of chondroitin sulfate fractions from whale and shark cartilage with chondroitin AC and C lyases, indicated that 4-sulfated 2-acetamido-2deoxy-D-galactosyl residues were spaced along the polysaccharide chain in chondroitin C, which contained 95% of 6-sulfate linkages, and that these 4-sulfated residues did not occur consecutively in one region.216 D-Glucuronic acid was released by P-D-glucosiduronase (EC 3.2.1.31) from the tetrasaccharide produced by testicular hyaluronidase digestion, demonstrating the p linkage of the glycosyluronic residues.217The regularity of the main chain was revealed by limit digestion of proteoglycan with chondroitin AC and ABC lyases.2'8 The former depolymerized the carbohydrate portion to a residual, linkage tetrasaccharide, and the latter left a residual disaccharide joined to this core tetrasaccharide. Other catabolic enzymes that react are chondro-4- and -6-sulfatases (EC 3.1.6.9 and 3.1.6.10) that remove sulfate from disaccharide fragments, endo-P-Dglucosiduronase,218aand chondroitin C lyase,216which lyses chondroitin 6-sulfate. Aspects of the sulfation pattern in chondroitin A from whale cartilage and chondroitin C from shark cartilage have been determined after separation of hyaluronidase digests by gel and thick-paper chromatography and by liquid chromatography under elevated pressure.219On digestion with a bacterial chondroitinase, a hexasaccharide that had been reduced at the reducing end with borotritide released three distinctive, disaccharide fragments. Considering the sequence from the nonreducing end of the original oligosaccharide, the disaccharide units were released as a saturated disaccharide having a free reducing-end group, an unsaturated disaccharide with a free reducing-end group, and an unsaturated disaccharide with a tritiated 2-amino-2-deoxy-~-galactitolend-residue (51). Tetra- and hexa-saccharides were either only 4-sulfated or 6-sulfated from each source, but octa- and deca-saccharides contained both types of sulfation. A comparison of oligosaccharides released by chondroitinase AC from the chondroitin sulfates of three species of mollusks indicated differences in the patterns of s~lfation.~'~" Evidence for the structure of the main repeating-chain of chondroitin sulfate, as well as the sequence at the linkage region to protein, has come (216) Y. M. Michelacci and C. P. Dietrich, Biochim. Biophys. Acra, 451 (1976) 436-443. (217) R. Niemann and E. Buddecke, Z.Physiol. Chem., 363 (1982) 591-598. (218) V. C. Hascall, R. L. Riolo, J. Hayward, and C. C. Reynolds, J. Biol. Chem., 247 (1972) 4521-4528. (218a) K. Takagaki, T. Nakamura, M. Majima, and M. Endo, FEES Lerr.. 181 (1985) 271-274. (219) S. R. Delaney, H. E. Conrad, and J. H. Glaser, Anal. Biochem., 108 (1980) 25-34. (219a) H. B. Nader. T. M. P. C. Ferreira, J. F. Paiva, M. G . L. Medeiros, S. M. B. Jerhimo, V. M. P. Paiva, and C. P. Dietrich, J. Biol. Chem., 259 (1984) 1431-1435.

@-D-GIcA-(1 + 3)-p-~-GalNAcSo,-( 1 + 4)-p-~-GlcA-( 1 + 3)-p-~-GalNAcS0,-( 1 + 4 ) - p - ~ - G l c A1- + ( 3)-~-GalNAcS0,

I I

NaB’H,

p - ~ - G l c A -1(+ 3)-p-~-GalNAcS0,-(1+ 4)-p-~-GlcA-( 1 + 3)-/3-~-GalNAcS0,-(1 + 4)-p-~-GlcA-( 1 + ~)-D-G~INACSO,-O~H chondroitinae

f l - ~ - G l c A - ( l +3)-~-GalNAcS0,+AXA-(I + 3)-~-GalNAcS0,+AXA-(1 + 3)-o-GalNAcS0,-03H 51

AA

I

1 -&)-p-~-GlcA-( 1 + 3)-p-D-Gal-(1 + 3)-p-D-Gal-(1 + 4)-p-D-Xyl-ser +4)-p-~-GlcA-(1 + 3)-p-~-GalNAcSo,-(

I

AA 52

202

BARRY V. McCLEARY AND NORMAN K. MATHESON

both from enzymic degradative studies and from data acquired with biosynthetic enzymes, the nucleoside diphosphate glycosyltransferases that add a glycosyl group to the nonreducing end of a glycan. The structure of the main chain and linkage region can be represented as in 52, and each glycosidic linkage shown has a separate glycosyltransferase for its biosynthesis. Degradation of a proteoglycan from bovine nasal-septa by hyaluronidase, and proteolysis, gave a glycopeptide composed of D-glucosyluronic, 2acetamido-2-deoxy-~-glucosyl, D-galactosyl, and D-XYIOSYI residues.220Acid hydrolysis of this, or the original, polymeZ2’ released an aldobiouronic acid that was hydrolyzed by P-D-glucosiduronase, giving D-galactose as the neutral component. Neutral oligosaccharides obtained by partial hydrolysis with acid were shown by hydrolysis with P-D-galactosidase to contain /3 linkages and, in conjunction with chemical evidence, structure 52 was derived.222 Confirmation of sequence 52 followed from a study of the acceptor specificity of, and linkages formed by, the relevant glycosyltransferases. Purified UDP-D-xylose-protein D-xylosyltransferase (EC 2.4.2.26) added D-xylose to core protein from which carbohydrate had been removed by Smith degradati~n.’”-’’~ A chicken-cartilage homogenate then transferred D-galactose (by xylosylprotein 4-P-galactosyl transferase, EC 2.4.1.133) to D-Xylose and 3-O-~-xylosylform a P-D-Gal-( 1 + 4 )-~ -Xy llinkage.z26.227 serine also accepted. A second D-galactose was then linked (by EC 2.4.1.134) P-( 1+ 3), and P-D-Gal-(1 + 4 ) - ~ - X ywas l the smallest accept0r.2’~The same homogenate then transferred D-glucuronic acid from UDP-D-glucuronic acid (by EC 2.4.1.135) to the terminal D-galactosyl group and, although l react, the full sequence of Gal-Gal-Xyl-Ser P-D-Gal-(1 + 3 ) - ~ - G a could was better. This activity was separate from that which transferred Dglucuronic acid to the growing m ain -~ h ain .’~ Cell-free ~ preparations of embryonic-chicken cartilage transferred labelled sugar from UDP-Dglucuronic acid and UDP-2-acetamido-2-deoxy-~-glucoseto endogenous acceptor, and the polysaccharide formed had the composition of chondroitin (220) (221) (222) (223)

J. D. Gregory, T. C. Laurent, and L. RodCn, J. Biol. Cbem., 239 (1964) 3312-3320. L. RodCn and G. Armand, J. Biol. Chem., 241 (1966) 65-70. L. RodCn and R. Smith, J. Bid. Chem., 241 (1966) 5949-5954. T. A. Beyer, J. E. Sadler, J. 1. Rearick, J. C. Paulson, and R. L. Hill, Adu. Enzymol.,

52 (1981) 23-175. (224) J. R. Baker, L. RodCn, and A. C. Stoolmiller, J. Biol. Chem., 247 (1972) 3838-3847. (225) N. 9. Schwartz and A. Dorfman, Arch. Biochem. Biophys., 171 (1975) 136-144. (226) H. C. Robinson, A. Telser, and A. Dorfman, Roc. Narl. Acad. Sci. USA, 56 (1966) 1859- 1866. (227) T. Helting and L. Rodtn, J. Bid. Chem., 244 (1969) 2790-2798; 2799-2085.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

203

sulfate, and formed the same oligomers on hydrolysis with appropriate e n ~ y m e s . Desulfated ~ ~ ~ . ~ ~oligosaccharides ~ were effective acceptors, and, the higher the d.p. the faster the transfer. When the nonreducing, terminal sugar was a D-glucosyluronic group, only 2-acetamido-2-deoxy-~-galactose could be transferred and, when the nonreducing, terminal sugar was a 2-acetamido-2-deoxy-~-galactosyl group, only UDP-D-glucuronic acid reacted, consistent with the structure of a regularly repeating, disaccharide unit in the main chain. Separate enzymes have been defined for the formation of the different types of glycosidic linkage in chondroitin. Competition studies have shown that these are distinct enzymes; thus, there is a D-galactosyltransferase that forms the p-D-Gal-( 1 + 4 ) - ~ - X ylinkage l and another synthesizing the p-DGal-( 1 + 3 ) - ~ - G alinkage. l Although the D-xylosyltransferase can be extracted, the other activities were bound to the endoplasmic reticulum. Two N-acetyl-D-galactosaminyltransferases forming a p-( 1 + 4) linkage to Dglucosyluronic residues have been separated from calf a r t e r i a l - t i ~ s u e ~ ~ ~ ~ and, from their substrate specificities, it has been proposed that one is involved in synthesis of the carbohydrate-protein linkage-region and the other in main-chain elongation. In sulfation, substitution of polymeric chondroitin and chondroitin sulfates A and C by a hen-oviduct preparation has been described. The sulfate donor is adenylyl sulfate 3'-phosphate and a non-sulfated 2-acetamido-2deoxy-D-galactosyl residue positioned internally in the polymer chain can be s ~ b s t i t u t e d . ~Evidence ~' has been presented for another activity (from quail oviduct) that sulfates carbon atom 6 of a 2-acetamido-2-deoxy-~galactosyl 4-C-sulfate group at the nonreducing end of the chain, giving the 4,6-di-C-s~lfate.~~' 2. Hyaluronic Acid (Hyaluronan)

Enzymic studies of the structure of hyaluronic acid are consistent with a composition of alternating 2-acetamido-2-deoxy-~-glucosyl and Dglucosyluronic residues, both @linked. Hydrolysis with testicular hyaluronidase gave a series of oligosaccharides, up to a d.p. of 14, that were composed232of the repeating disaccharide unit + 4)-p-~-GlcA-( 1+. (228) J . E. Silbert, J. Bid. Chem., 239 (1964) 1310-1315. (229) A. Telser, H . C. Robinson, and A. Dorfman, Arch. Biochem. Biophys., 116 (1966) 458-465. (229a) K. Rohrmann, R. Niemann, and E. Buddecke, Eur. J. Biochem., 148 (1985) 463-469. (230) S. Suzuki and J . L. Strominger, J. Bid. Chem., 235 (1960) 257-266; 267-273; 274-276. (231) Y. Nakanishi, M. Shimizu, K. Otsu, S. Kato, M. Tsuji, and S. Suzuki, 1. Bid. Chem., 256 (1981) 5443-5449. (232) B. Weissmann, K. Meyer, P. Sampson, and A. Linker, 1.Bid. Chem., 208 (1954) 417-429.

204

BARRY V. McCLEARY AND NORMAN K. MATHESON

3)-p-~-GlcNAc-( 1 -* . The main product was the tetrasaccharide, transglycosylation o c c ~ r r e d , ’ ~ ~ and * ’ the ~ ~ enzyme showed specificity for D-glucosyluronic residues; the glycosidic linkage of L-idosyluronic residues in dermatan sulfate was not cleaved by this en~yme.’~’Bacterial hyaluronate lyase (EC 4.2.2.1) released the disaccharide having an unsaturated glycosyluronic g r o ~ p , ’ ~ ~and . ’ ~ ~leech hyaluronoglucosiduronase’3s (EC 3.2.1.36) gave mainly a tetrasaccharide with a D-glucuronic acid residue at the reducing end [P-D-G~cNAc-( 1-* 4)-/3-~-GlcA-( 1 + 3)-/3-~-GlcNAc-( 1 + 4)-~-GlcA]. Hyaluronate has been cleaved by testicular hyaluronidase into oligosaccharides that could be separated by gel chromatography into a homologous series ranging from d.p. 2 to 46, and leech hyaluronoglucosiduronase gave a similar result, with the products having the reverse sequence of monosaccharide residues.’39 The /3 linkage of the D-glucosyluronic residue followed from the release of D-glucuronicacid by P-D-glucosiduronase from oligosaccharides prepared by hyaluronidase digestion.’” The /3 linkage of the acetamido-2-deoxy-~-glucosylresidue was established by hydrolysis by N-acetyl-/3-D-hexosaminidase (EC 3.2.1.52) of oligosaccharides, derived from enzymic degradation of hyaluronic acid, that had a 2-acetamido-2deoxy-D-glucosyl group at the nonreducing t e r m i n ~ s . ’ ~ ~When - ’ ~ ~hyaluronate from rooster comb was digested with a mixture of /3-D-glucosiduronase and N-acetyl-P-D-hexosaminidase in a dialysis bag, there was a 99.6% conversion into monosaccharides and into oligosaccharides that were transferase products, consistent with the whole molecule’s being unbranched, and composed of equal parts of D-glucosyluronicand 2-acetamido-2-deoxyD-glucosyl residues and no significant proportion of other Biosynthetic studies on the formation of hyaluronic acid have yielded less information than have similar studies about the structure of chondroitin. Addition to small, well defined oligosaccharides has not been found. Using labelled nucleoside 5’-glycosyldiphosphates in a bacterial system, evidence has been obtained for the synthesis of hyaluronic acid of high molecular (233) (234) (235) (236) (237) (238) (239)

B. Weissman, J. Biol. Chem., 216 (1955) 783-794. P. Hoffman, K. Meyer, and A. Linker, J. Bid. Chem., 219 (1956) 653-663. L . - k Fransson, J. Biol. Chem., 243 (1968) 1504-1510. A. Linker, K. Meyer, and P. Hoffman, J. Bid. Chem., 219 (1956) 13-25. H. Greiling, H. W. Stuhlsatz, and T. Eberhard, 2.Physiol. Chem., 340 (1965) 243-248. A. Linker, K. Meyer, and P. Hoffman, J. Bid. Chem., 235 (1960) 924-927. M. K. Cowman, E. A. Balazs, C. W. Bergmann, and K. Meyer, Biochemistry, 20 (1981)

(240) (241) (242) (243) (244)

A. Linker, K. Meyer, and B. Weissmann, J. Bid. Chem., 213 (1955) 237-248. B. Weissmann, S. Hadjiioannou, and J. Tornheim, J. Biol. Chem., 239 (1964) 59-63. G. Bach and B. Geiger, Arch. Biochem. Biophys., 189 (1978) 37-43. T. M. Bearpark and J. L. Stirling, Biochem. 1,173 (1978) 997-1000. M. 0. Longas and K. Meyer, Biochem. J., 197 (1981) 275-282.

1379-1385.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

205

weight, and for the addition of single D-glucosyluronic groups from UDP-Dglucuronic acid, when it alone was incubated.245 3. Dermatan Sulfate

This structure resembles that of chondroitin sulfate, except that some of the P-D-glucosyluronic residues of the repeating disaccharide unit are replaced by a-L-idosyluronic residues. The proportion of D-glucosyluronic residues in the polymer varies according to the source, but there can be more L-idosyluronic than D-glucosyluronic residues. Sections containing P-D-glucosyluronic residues can be hydrolyzed by testicular h y a l ~ r o n i d a s eor , ~lysed ~ ~ by chondroitin ABC or AC 1 y a ~ e . ~ ~ ~ * ~ Lysis produces the same A4-unsaturated glycosyluronic group from both uronic acids. a-L-Idosyluronic sections can be lysed by chondroitin ABC lyase; and a chondroitin B lyase has been described that degraded only dermatan sulfate, indicating that a-L-idosyluronic residues are specifically a t t a ~ k e d . ~ ~ *Testicular ,’~~ h y a l ~ r o n i d a s epartly ~ ~ ~ hydrolyzed a highly purified preparation of dermatan sulfate from pig skin, to give fragments having D-glucosyluronic groups at newly formed, nonreducing termini, in agreement with the polymer’s containing D-glucosyluronic residues. Fractionation of the hydrolyzate separated a tetrasaccharide that contained both D-glucosyluronic and L-idosyluronic residues. This is consistent with the co-occurrence of both glycosyluronic residues in the polymer as L-idosyluronic residues do not participate in transglycosylation. Mixed sequences were also obtained on digestion of proteodermatan sulfate from bovine ~ ’ by chondroitin B lyase gave aorta with chondroitin AC l y a ~ e . ~Lysis oligosaccharides that were further degraded by chondroitin AC lyase, confirming previous conclusions that D-glucosyluronic residues are integral, and not a constituent of a contaminating polymer. The percentage of D-glucosyluronic and L-idosyluronic residues can be estimated from the for ’ ; example, extents of hydrolysis with chondroitin AC and ABC l y a ~ e s ~ ~ two dermatan sulfate fractions from rabbit corneal-stroma were shown to contain 36 and 42% of the uronic acids as ~-iduronate.”~Reaction of dermatan sulfate with hydrazine and nitrous acid gave disaccharides composed of uronic acid glycosidically linked to an anhydro sugar. These were (245) (246) (247) (247a) (248) (249) (250) (251)

A. C. Stoolmiller and A. Dorfman, J. Bid. Chem., 244 (1969) 236-246. L.-A. Fransson and L. Rodtn, J. Biol. Chem., 242 (1967) 4161-4169; 4170-4175. H. Saito, T. Yamagata, and S. Suzuki, J. Biol. Chem., 243 (1968) 1536-1542. K. Nagasawa, A. Ogamo, and K. Yoshida, Carbohydr. Res., 131 (1984) 315-323. Y. M. Michelacci and C. P. Dietrich, Biochem. J., 151 (1975) 121-129. N. Ototani and Z. Yosizawa, Carbohydr. Res., 70 (1979) 295-306. R. Kapoor, C. F. Phelps, L. Coster, and L.-A. Fransson, Biochem. J., 197 (1981) 259-268. J. D. Gregory, L. Coster, and S. P. Damle, J. Bid. Chem., 257 (1982) 6965-6970.

206

BARRY V. McCLEARY AND NORMAN K. MATHESON

characterized by glycosiduronase digestion, and a method of estimation of the ratio of L-iduronic to D-glucuronic acids was suggested, using these Dermatan sulfate contains 2-C-sulfated glycosyluronic residues, and the location of this sulfate on the a-L-idosyluronic residues was shownzs2by treatment of skin polysaccharide with hyaluronidase. Ion-exchange chromatography fractionated according to the sulfate content, and the degree of sulfation in the fractions was inversely proportional to the level of D-glucuronic acid. On treatment with chondroitin AC lyase, a highly sulfated fraction that contained 5% of D-glucuronic acid lost almost all of this, with little change in the average d.p. or degree of sulfation, indicating that the D-glucosyluronic residues were located terminally and were non-sulfated. Periodate oxidation, followed by acid hydrolysis, left L-idosyluronic units, consistent with sulfation of this acid. Oligosaccharide fragments, derived by enzymic hydrolysis of the polymer, were resistant to hydrolysis by chondrosulfatases, again locating the sulfation on L-idosyluronic residues.247An L-iduronate sulfatase has been isolated from human The distribution of regions containing D-glucosyluronic and L-idosyluronic residues was studied by sequential treatment with testicular hyaluronidase and P-D-glucosiduronase, and subsequent reaction with chondroitin AC l y a ~ e . ~D-Glucosyluronic '~ residues were judged to be predominantly in clusters, but isolated D-glucosyluronic and L-idosyluronic residues were also present. Examination of polysaccharide fractions revealed considerable heterogeneity. Selective periodate oxidation, followed by digestion of the Smith-degradation products with chondroitin AC lyase, or, alternatively, testicular hyaluronidase hydrolysis and periodate oxidation, followed by fractionation, and characterization, of the resultant oligosaccharides, led to further observations on the disposition of glycosyluronic residues and sulfate groups. Some 2-acetamido-2-deoxy-~-galactosyl residues are not s ~ l f a t e d , and ~~~ these *~~ appear ~ to be near to sulfated L-idosyluronic residues. Examinationzs6" of dermatan sulfates from nine sources with chodroitinases AC and B showed that they all differed in the proportion of A. S. B. Edge and R. G . Spiro, Arch. Biochem. Biophys., 240 (1985) 560-572. A. Malmstrom and L.-A. Fransson, Eur. J. Biochem., 18 (1971) 431-435. A. Wasteson and E. F. Neufeld, Methods Enzymol., 83 (1982) 573-578. L.-A. Fransson and A. Malmstrom, Eur. I. Biochem., 18 (1971) 422-430. L.-A. Fransson, L. Coster, A. Malmstrom, and I. Sjoberg, Biochem. J., 143 (1974) 369-378. (256) L.-A. Fransson, L. Coster, B. Havsmark, A. Malmstrom, and I. Sjoberg, Biochern. J., 143 (1974) 379-389. (256a) C. A. Poblaci6n and Y. M. Michelacci, Carbohydr. Res., 147 (1986) 87-100.

(251a) (252) (253) (254) (255)

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

207

6-sulfated disaccharide units and the relative amount and position of Dgluco- and L-ido-syluronic residues. Testicular hyaluronidase also released the glycopeptide fragments of the linkage region of pig-skin dermatan ~ u l f a t e , ~and ” analysis indicated that it was identical to that of chondroitin sulfate, and that a considerable proportion of the main-chain glycosyluronic residues near the linkage region were D-glucosyluronic. Depolymerization of aggregating chains of dermatan sulfate with testicular hyaluronidase gave larger amounts of hexa-, octa-, and deca-saccharides than did depolymerization of non-aggregating chains. Further degradation by chondroitin AC lyase gave tetrasaccharides having L-idosyluronic residues placed internally in the sequence, indicating that alternating sequences of D-glucosyluronic and L-idosyluronic residues were present in aggregating, but rare in non-aggregating, a-L-Idosyluronic residues are introduced after polymerization and before fation ion,^^^*^^^^ which is consistent with a non-regular distribution. Reaction occurs by epimerization of C-5, by a mechanism in which the initial step is abstraction of a hydrogen atom.260*261 Epimerization is linked with the presence of adenylyl sulfate 3 ’ - p h o ~ p h a t eindicating ,~~~ the co-occurrence of sulfation. Epimerization is an equilibrium reaction that favors the Dglucosyluronic configuration, but an -1doA-GalNAc4SO;- is not a substrate, and, hence, sulfation allows more L-idosyluronic residue formation.

4. Keratan Sulfate

Keratan sulfate, as well as showing structural affinities to the glycosaminoglycans, shares some characteristics of the glycoconjugates. The desulfated carbohydrate portion of the repeating unit of the main chain (53) is also found in glycoconjugates, and the linkage region to protein has similarities. + 3 ) - P - ~ - G a l -1(+ 4 ) - p - ~ - G l c N A c -1(+

53

Two types of keratan sulfate, corneal and skeletal, have been differentiated by the hydrolytic behavior of the linkage region. The former has an N(257) L.-A. Fransson, Biochim. Biophys. Acra, 156 (1968) 311-316. (258) L.-A. Fransson and L. Coster, Biochim. Biophys. Acra, 582 (1979) 132-144. (259) A. Malmstrom, L.-A. Fransson, M. Hook, and U. Lindahl, J. Biol. Chem., 250 (1975) 3419-3425. (259a) A. Malmstrom, J. Biol. Chem., 259 (1984) 161-165. (260) A. Malmstrom and L. Aberg, Biochem. J., 201 (1982) 489-493. (261) A. Malmstrom, Biochem. J., 198 (1981) 669-675.

208

BARRY V. McCLEARY AND NORMAN K. MATHESON

glucosaminyl linkage to L-asparagine, and the latter, an 0-glycosyl link residue to L-serine or L-threonine. from a 2-acetamido-2-deoxy-~-galactosyl The extent of sulfation varies with the source. Sulfate can be found on C-6 residues and also on C-6 of some Dof 2-acetamido-2-deoxy-~-glucosyl galactosyl residues. A P linkage both for the D-galactosyl and 2-acetamido-2-deoxy-~-glucosyl residues in the main chain was indicated by hydrolysis by P-D-galactosidase and N-acetyl-P-D-glucosaminidasefrom a Coccobacillus sp.262and from Aspergillus niger.263Keratan sulfate is hydrolyzed by an endo-P-D-galactosidase from Coccobacillus and Pseudomonas spp. and from Escherichia f r e ~ n d i i Flavobacterium ~~~, k e r a t o l y t i ~ u s and ~ ~ ~Bacteroides ~’~ f r a g i l i ~ . ’It~ ~ hydrolyses at D-galactosyl residues that are not sulfated. A mixture of oligosaccharides is produced, of which the smallest is P-D-G~cNAc~SO,. elution profile of this oligosaccharide mixture has been (1 + 3 ) - ~ - G a lThe found to vary with the source of the keratan sulfate, suggesting a use for the enzyme in studying differences of structure. Two proteokeratan sulfates were separated from corneal stroma and, after papain digestion, both reacted with E. freundii endo-P-D-galactosidase; one was transformed into fractions which were fully retarded in 6%-agarose gel chromatography and the other, into slightly larger fragments. The K,, values were2510.96 and 0.88. Skeletal keratan sulfate has been prepared from bovine nasal-cartilage by removal of chondroitin sulfate with chondroitin AC lyase, followed by proteolysis with papain265 (EC 3.4.22.2). The presence of terminal sialic was shown from its release on incubation of the skeletal polysaccharide from cartilage with neuraminidase266(EC 3.2.1.18). Enzymic digestion of corneal polysaccharide left D-mannosyl residues in the oligosaccharide-peptide fragment, indicating their location in the linkage r e g i ~ n . ~ The ~ ’ . ~structure ~~ in the linkage region of bovine-corneal proteokeratan sulfate has been determined with an oligosaccharide-peptide prepared by proteolysis, chemical desulfation, and digestion with A. niger P-D-galactosidase and N-acetyl-P-~-glucosaminidase.~~~ Reaction with (262) 0. Rosen, P. Hoffman, and K. Meyer, Fed. Roc., Fed. Am. SOC.Exp. BioL, 19 (1960) 147. (263) R. Keller, T. Stein, H. W. Stuhlsatz, H. Greiling, E. Ohst, E. Miiller, and H.-D. Scharf, Z. Physiol. Chem., 362 (1981) 327-336. (264) H. Nakagawa, T. Yamada, J.-L. Chien, A. Gardas, M. Kitamikado, S.-C. Li, and Y.-T. Li, 1. Biol. Chem., 255 (1980) 5955-5959. (264a) M. Kitamikado, M. Ito, and Y.-T. Li, J. Biol. Chem., 256 (1981) 3906-3909. (264b) P. Scudder, P. Hanfland, K. Uemura, and T. Feizi, J. B i d . Chem., 259 (1984) 6586-6592. (265) V. C. Hascall and R. L. Riolo, J. Biol. Chem., 247 (1972) 4529-4538. (266) N. Toda and N. Seno, Biochim. Biophys. Acra, 208 (1970) 227-235. (267) S. Hirano and K. Meyer, Biochem. Biophys. Res. Commun., 44 (1971) 1371-1375. (268) S. Hirano and K. Meyer, Connect. Tissue Rex, 2 (1973) 1-10.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

209

these two enzymes is consistent with /? linkages for both sugars in the main chain. The presence of a chitobiosyl unit linked to asparagine was estab(EC 3.2.1.96) lished by hydrolysis with endo-N-acetyl-p-D-glucosaminidase (see Section XI) and a terminal L-fucosyl group by hydrolysis269 with a-L-fucosidase. In conjunction with methylation analysis and the known specificity for the oligosaccharide chain of a particular endo-N-acetyl-p-D-glucosaminidase (D), a structure was proposed for the desulfated material, leading to 54 for the structure of the desulfated polymer. The same structure has + 3 ) - p - ~ - G a l - ( l +4 ) - p - ~ - G l c N A c - ( l k 2 ) - a - ~ - M a n 1

.1 3 P-D-Man-( 1 + 4)-p-~-GlcNAc-( 1 + 4)-~-GlcNAc-Asn 6 6

t

1

+ 3 ) - p - ~ - G a l - ( l +4)-P-~-GlcNAc-(lh2)-cu-~-Man

t

1 LPL-FUC

54

been derived by sequential glycosidase hydrolysis, combined with methylation analysis of a glycopeptide prepared by pronase and endo-P-D-galactosidase hydroly~is.~’~ This structure has also been deduced from chemical methods applied to a glycopeptide from monkey-corneal keratan sulfate, prepared by papain and endo-p-D-galactosidase hydroly~is.~~’ When bovine-corneal, peptido-keratan sulfate was degraded chemically, a tetrasaccharide fraction was obtained, and the sequence in this was determined from the hydrolytic pattern with E. coli P-D-galactosidase, Cunauuliu ensifomis a-D-mannosidase (EC 3.2.1.24), human-placental pD-mannosidase, and bovine-kidney a-~-fucosidase.~’~

5. Heparin, and Heparan Sulfate An understanding of the structures of the molecules of heparin and heparan sulfate has come, in part, from studies with degradative enzymes, but also with biosynthetic enzymes. Both polysaccharides are based on the disaccharide unit which, in the initial stage of biosynthesis, consists of (269) T. Stein, R. Keller, H. W.Stuhlsatz, H. Greiling, E. Ohst, E. Muller, and H.-D. Scharf, Z. Physiol. Chem., 363 (1982) 825-833. (270) H. Yamaguchi, J. Biochem. (Tokyo), 94 (1983) 207-213, 215-221; 95 (1984) 601-604. (271) B. Nilsson, K. Nakazawa, J. R. Hassell, D. A. Newsome, and V. C. Hascall, J. B i d . Chem., 258 (1983) 6056-6063. (272) A. Brekle and G. Mersmann, Biochim. Biophys. Acta, 675 (1981) 322-327.

210

BARRY V. McCLEARY AND NORMAN K. MATHESON

+ 4)-~-GlcA-( 1 + 4)-a-~-GlcNAc-( 1+. This polymer is subjected to partial

N-deacetylation, N-sulfation, epimerization of some D-glucosyluronic to L-idosyluronic residues, and sulfation at C-2 of L-idosyluronic and at C-6 of D-glucosaminyl N-sulfate units. Isolated preparations are a complex mixture of molecules having various degrees and patterns of modification. N-Sulfation 'leads to epimerization of a neighboring glucosyluronic unit. Heparan sulfate occurs as a proteoglycan (as a cell-surface component) and preparations from different sources may have a wide range of sulfation and e p i m e r i ~ a t i o n . ~ ' ~Heparin " . ~ ~ ~ " occurs intracellularly, being synthesized as a proteoglycan and, although similar modifications occur as to heparan sulfate, the final product is much more heavily sulfated (more than 80% of 2-amino-2-deoxy-~-glucosyl residues can be N-sulfated): some Dglucosyluronic residues are sulfated on C-2, 2-deoxy-2-(sulfoamino)-~glucosyl on C-3, and 2-acetamido-2-deoxy-~-glucosyl residues on C-6. Allowing for configurational change of the glycosyluronic residues, deacetylation of the amino sugar, and sulfation of both, and considering both sugars in a glycosidic linkage, at least 16 types of linkage can be present. Depolymerization occurs with induced enzymes from Hauobacterium heparinum. Two lyases, heparin lyase (heparinase; EC 4.2.2.7) and heparan sulfate lyase (heparitin lyase, heparitinase; EC 4.2.2.8) have been found, both of which release oligosaccharideshaving an unsaturated glycosyluronic group at the nonreducing end and an amino sugar residue at the reducing end.273-275 Action of heparin lyase requires regions having C,N-disulfated 2-amino-2-deoxy-~-g~ucosyl residues and L-idosyluronic residues, whereas heparan sulfate lyase acts in the absence of C,N-disulfated and in the presence of N-acetylated, N-sulfated, or N-acetylated- C-sulfated 2-amino2-deoxy-~-glucosylresidues, lysing at regions having D-glucosyluronic linkages. Purified forms of this enzyme show more s p e ~ i f i c i t y . ~Lysis ~~~-~~~~ produces the same unsaturated glycosyluronic group from either acid: glycosyluronic-specific lyases may exist. Early work on heparin and heparan sulfate was mainly concerned with isolation and identification of di- and tetra-saccharides. The main product of heparin lyase action on bovine-liver heparin the unsaturated, (272a) C. P. Dietrich, H. B. Nader, and A. H. Straw, Eiochem. Biophys. Res. Cornmun., 1 1 1 (1983) 865-871. (273) P. Hovingh and A. Linker, J. Eiol. Chem., 245 (1970) 6170-6175. (274) A. Linker and P. Hovingh, Fed. Proc., Fed. Am. Soc. Exp. EioL, 36 (1977) 43-46. (275) P. Hovingh and A. Linker, J. Eiol. Chern, 257 (1982) 9840-9844. (275a) M. E. Silva, C. P. Dietrich, and H. B. Nader, Biochim. Biophys. Ada, 437 (1976) 129-141. (275b) N. Ototani, M. Kikuchi, and Z. Yosizawa, Carbohydr. Res., 88 (1981) 291-303. (275c) I. Silverberg, B. Havsmark, and L.-A. Fransson, Carbohydr. Res., 137 (1985) 227-238. (276) A. S. Perlin, D. M. Mackie, and C. P. Dietrich, Carbohydr. Res., 18 (1971) 185-194.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

21 1

trisulfated disaccharide 55, indicating general aspects of the disaccharide repeating-unit. In another digest,277which yielded 85% of oligosaccharides, >30% of the 2-amino-2-deoxy-~-glucosyl residues bore two sulfate groups, AXA.2S04-( 1 + 4)-D-GlcNS046SO,

55

the second on (2-6, and at least 30% of the unsaturated glycosyluronic groups were nonsulfated. Heparin was degraded by heparin lyase, to afford27855 (52% ), a tetrasaccharide fraction (40% ), and lesser proportions of higher oligosaccharides. The tetrasaccharide fraction was converted by heparan sulfate lyase into the trisulfated disaccharide and into a disulfated disaccharide lacking a sulfate group on the acidic portion. Trisulfated disaccharide and tetrasaccharide were also detected as major products of both lung and mucosal heparin, and the relative proportions varied with the source.274The tetrasaccharide fraction contained both D-glucosyluronic and L-idosyluronic residues. When the products of digestion of whale heparin with heparin lyase were separated by ion-exchange and paper chromatography, 18 fractions were obtained. Among the oligosaccharide products were 55, AXA-( 1+ 4 ) - ~ GlcNSO,, AXA2S04-(1+ 4)-~-GlcNSo,,and AXA-( 1 + 4)-~-GlcNAc.The major fractions contained two, or three, sulfate groups per disaccharide unit. Structures for the A4-aldobiouronic acids were, in part, established by using a A4-hexosiduronase.279.280 Pig-mucosal heparin, digested with heparin lyase, gave 55: five tetrasaccharides, which all had a-~-GlcNS0,6S0, at the reducing end, and unsaturated glycosyluronic groups at the nonreducing terminus were also identified. The interior pairs of sugars were CY-D-G~CNS046S04with P-D-G~cAor a-~-IdoA2S0,, CX-D-G~CNAC with P-D-G~cA or a-L-IdoA, and a - ~ - G l c N S o ,with P-D-G~cA.The products of nitrous acid degradation were hydrolyzed by P-D-glucosiduronase, consistent with a P linkage for this acid.281An activity from mouse mastocytoma hydrolyzed heparin to a product that, on borotritide reduction, hydrolysis, and deamination, released tritiated L-gulonic acid, indicating that the enzyme was an

A. Linker and P. Hovingh, Biochemisfry, 1 1 (1972) 563-568. M. E. Silva and C. P. Dietrich, 1. Bid. Chem., 250 (1975) 6841-6846. N. Ototani, K. Nakamura, and Z. Yosizawa, J. Biochem. (Tokyo),75 (1974) 1283-1289. N. Ototani and Z. Yosizawa, J. Biochem. (Tokyo), 76 (1974) 545-551. Z. M. Merchant, Y. S. Kim, K. G. Rice, and R. J. Linhardt, Biochem. J., 229 (1985) 369-377. (281) T. Helting and U. Lindahl, J. Bid. Chem., 246 (1971) 5442-5447.

(277) (278) (279) (280) (280a)

212

BARRY V. McCLEARY AND NORMAN K. MATHESON

endo-/3-D-glucosiduronase282. This enzyme from platelet^:^^"'^^'^ hydro1 + 4)-cu~-GlcNSO, linkage with a requirement for lyzed a p-~-GlcA-( sulfamino but not ester sulfate. A similar activity from human placenta hydrolyzed heparan sulfate, and the amino sugar adjacent to the Dglucuronic acid at the reducing end of the fragments appeared to be residue.283endo-Glycosidases exclusively a 2-acetamido-2-deoxy-~-glucosyl have been detected in liver and platelets.282a*282b Degradation of heparan sulfate from lung with heparin lyase and heparan sulfate lyase gave five disaccharide fractions, and analysis and hydrolysis by glycosiduronase indicated that they were composed of AXA and 2-amino2-deoxy-~-glucosewith various extents of C- and N-sulfation and Na ~ e t y l a t i o n one ~ ~ ~was ; compound 55. Three sulfated tetrasaccharides were isolated from the products of heparin lyase action on beef-liver heparin.284a All had a sulfated, unsaturated glycosyluronic acid group at the nonreducing end. One contained an L-idosyluronic 2-sulfate residue and two 2-amino-2deoxy-D-glucosyl units substituted with sulfate on N-2 and C-6. In a second, L-idosyluronic 2-sulfate was replaced by a non-sulfated D-glucosyluronic residue. The third contained L-idosyluronic 2-sulfate, and the reducing 2-amino-2-deoxy-~-glucosylresidue was only mono-N-sulfated. The constitutions of bovine-lung and -kidney, as well as of porcine-kidney, heparan sulfates have been compared after quantitative digestion with a mixture of heparin lyase and heparan sulfate lyase, followed by separation of the unsaturated disaccharides by liquid chromatography under elevated pressure.285 Subsequent investigations on the structure of heparin concentrated on the isolation and structural determination of larger oligosaccharides, in order to determine the structural elements involved in anti-blood-clotting activity associated with the binding to antithrombin. A comparison of size distribution of oligosaccharides released by heparin lyase digestion of pigmucosal heparin with those calculated theoretially,^^'^ was consistent with a random distribution of cleavage sites within

(282) S. Ogren and U. Lindahl, J Biol. Chem., 250 (1975) 2690-2697. (282a) A. Oldberg, C.-H. Heldin, A. Wasteson, C. Busch, and M. Hook, Biochemistry, 19 (1980) 5755-5762. (282b) L. KjellCn, H. Pertoft, A. Oldberg, and M. Hook, J. Biol. Chem., 260 (1985) 8416-8422. (283) U. Klein and K. von Figura, 2.Physiol. Chem., 360 (1979) 1465-1471. (284) P. Hovingh and A. Linker, Carbohydr. Res., 37 (1974) 181-192. (284a) A. Linker and P. Hovingh, Carbohydr. Res., 127 (1984) 75-94. (285) N. Ototani, M. Kikuchi, and Z. Yosizawa, J. Biochem. (Tokyo), 94 (1983) 233-241. (285a) R. J. Lindhardt, Z. M. Merchant, K. G . Rice, Y. S. Kim, G . L. Fitzgerald, A. C. Grant, and R. Langer, Biochemistry, 24 (1985) 7805-7810.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

213

the polymer. N-Acetyl composition of the oligosaccharide fractions showed a random substitution by these groups relative to site cleaved. Oligosaccharide fragments have been made by deaminative cleavage or by heparin lyase digeStion.28~b,285c,286.286a,287,287a.287b Whale heparin, partially degraded with heparin lyase, was chromatographed on immobilized antithrombin, and an octasaccharide having high affinity was isolated.286Incubation of this with heparin lyase plus heparan sulfate lyase, heparin lyase alone, or heparan sulfate lyase alone, followed by separation and identification of disaccharide fragments by paper electrophoresis, led to the proposed structure, 56. Oligosaccharide fractions of AXA.2S04-(1 + 4)-a-D-GlcNS04-(1 + 4)-a-~-IdoA-( 1 + 4)-a-~-GlcNAc6SO,(1 + 4)-p-D-GICA-(1 + 4)-a-D-GlCNSO43SO4(1 + 4 ) - c ~ - ~ - I d o A 2 S O ~ -4)-D-GICNSO, (l+ 56

higher d.p. were separated and then digested further with heparan sulfate lyase and heparin lyase and the disaccharide products were fractionated.286a and AXA-( 1 + The proportions of a - ~ - I d o M S 0 , - 1( + ~)-D-GICNSO,~SO, 4)-~-GlcNS0,6S0, were higher, and of AXA-( 1+ 4 )-~ - Glc N S 0lower, , the more antithrombin activity was shown. Another structure was suggested for a fraction isolated similarly from porcine heparin. An octasaccharide that was prepared by partial deamination of porcine heparin, and which b c m d to a n t i t h r ~ m b i n , ~was ~ ~ ' converted ~~~" into a heptasaccharide by digestion with a-L-idosiduronase (EC 3.2.1.76) and was hydrolyzed to a pentasaccharide by an endo-P-D-glucosiduronase,indicating the positions, in the eight-sugar sequence, of a nonreducing, terminal a-L-idosyluronic group and a D-glucosyluronic residue. On nitrous acid cleavage, pig-mucosal heparin gave two octasaccharide fractions that bound with high affinity to human a n t i t h r ~ m b i n . " ~ One ~ of these (S) could be cleaved by heparin lyase, as well as heparan sulfate lyase, and the other (R) was not susceptible. Chemical degradation of the octasaccharide pro-

(285b) L. Thunberg, G. Backstrom, and U. Lindahl, Carbohydr. Res., 100 (1982) 393-410. ( 2 8 5 ~ )B. Casu, P. Oreste, G.Tom, G. Zoppetti, J. Choay, J.-C. Lormeau, M. Petitou, and P.Sinay, Biochem. J., 197 (1981) 599-609. (286) N. Ototani, M. Kikuchi, and Z. Yosizawa, Biochem. J., 205 (1982) 23-30. (286a) N. Ototani, C. Kodarna, M. Kikuchi, and Z. Yosizawa, J. Biochem. (Tokyo), 96 (1984) 1695- 1703. (287) U. Lindahl, L. Thunberg, G. Backstrorn, and J. Riesenfeld, Biochem. SOC.Trans., 9 (1981) 499-501. (287a) U. Lindahl, G. Backstrorn, and L. Thunberg, J. Biol. Chem., 258 (1983) 9826-9830. (287b) D. H.Atha, A. W. Stephens, A. Rimon, and R. D. Rosenberg, Biochemistry, 23 (1984) 5801-5812.

214

BARRY V. McCLEARY AND NORMAN K. MATHESON

duced, as the largest fraction, a tetrasaccharide that still showed antithrombin binding. Hydrolysis with a -L-idosiduronase, N-acetyl-a-D-glucosamine sulfatase (EC 3.1.6.14), N-acetyl-a-D-glucosaminidase (EC 3.2.1.50) and P -D-ghcosiduronase gave a sequence of ~-L-I~OA-~-D-GICNAC~SO~-P D-GICA-~-D-GLCNSO~~,~(SO~)~. Oligosaccharide R was not hydrolyzed by a-L-idosiduronase, showing a difference in structure from that of octasaccharide S. The 3-C-sulfated D-glucosaminyl-N-sulfate residue has been found only in active oligosaccharides. Reaction of human a -L-idosiduronase with glycosides of sulfated-23anhydrohexitols and anhydro-D-mannitol showed that sulfation enhanced catalysis. A model of substrate binding and a relationship to the disease termed mucopolysaccharidosis, which leads to incompletely degraded fragments of heparan and dermatan sulfates, was proposed.287’ A glycosaminoglycan isolated from lobsters was examined with heparin lyase and heparan sulfate l y a ~ e . ~It ~was ’ degraded much less extensively than beef-liver heparin by the former, and not degraded by the latter, indicating a structure intermediate between those of heparin and heparan sulfate. Heparan sulfates from three species of were found to be resistant to heparin lyase action. With heparan sulfate lyase, similar oligosaccharides in different proportions were obtained. Comparison with the products from bovine-pancreatic heparan sulfate showed the same oligosaccharides. Both heparan sulfate and heparin are derived from a proteoglycan that initially contains (1 + 3)-linked, alternating P-D-glucosyluronic and 2acetamido-2-deoxy-a-~-glucosyl residues. The structure of the linkage region to protein is similar to that in the chondroitin sulfates, and the ~ ~ ~main~~’ biosynthesis appears to follow a similar r e a c t i ~ n - p a t t e r n . ~The chain glycosyltransferases, N-acetyl-~-glucosarninyltransferase~’~-~’~ and ~-~-glucosy~uronotransferase,~~’*~’~ transfer substrate to oligosaccharide units containing the appropriate nonreducing, terminal sugar and linkage. The former did not transfer to a single, main-chain, disaccharide unit joined to the oligosaccharide at the linkage region, but did to a tetrasaccharide (287c) P. R. Clements, V. Muller, and J. J. Hopwood, Eur. J. Biochem., 152 (1985) 29-34. (288) E. E. Grebner, C. W. Hall, and E. F. Neufeld, Arch. Biochem. Biophys. 116 (1966) 391-398. (289) T. Helting, J. Biol. Chem., 246 (1971) 815-822. (290) T. Helting and U. Lindahl, Ac?a Chem. Scand., 26 (1972) 3515-3523. (291) W. T. Forsee, J. Belcher, and L. Rodin, Fed. Proc., Fed. Am. Soc. Exp. BioL, 37 (1978) 1777. (292) U. Lindahl, in G. 0. Aspinall (Ed.), Carbohydrate Chemistry, MTP Inr. Rev. Sci., Ser. TWO,7 (1976) 283-312.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

215

unit. If substrate was pretreated with P-D-glucosiduronase, to leave terminal a-L-idosyluronic groups, little or no transfer occurred. Reactions that complete the heparin molecule, such as, N-deacetylation, N-sulfation, epimerization of P-D-glucosyluronic to a-L-idosyluronic residues, 2-C-sulfation of a-L-idosyluronic, and C-sulfation of 2-amino-2deoxy-D-glucosyl residues, all occur on the polymerized molecule, and evidence has been obtained that these reactions occur in sequence.208*292-299 Five distinct components have been separated by DEAE-cellulose chromatography from reaction of a mastocytoma microsomal system with UDP-glucuronic acid, UDP-N-acetyl-D-glucosamine and adenylyl sulfate 3 ' - p h o ~ p h a t eand , ~ ~these ~ had the characteristics of products of the various reaction-stages. Only two were detected if adenylyl sulfate 3'-phosphate was omitted. The results have been interpreted as showing that biosynthesis follows the listed sequence of reactions. An N-acetyl-o-glucosaminyldeacetylase (EC 3.5.1.33), specific for polysaccharides having a heparin-like structure, has been detected in mouse-mastocytoma micro some^.^^^ The lack of reaction of UDP-N-acetyl-D-glucosaminyltransferase with oligosaccharides terminated at the nonreducing end with an a-L-idosyluronic group, and the absence of nucleoside 5'4 L-idosyluronic acid diphosphate) in tissue synthesizing heparin,208led to the exploration of an alternative mechanism of synthesis of the a-L-idosyluronic residues, resulting in the finding of C-5 D-glucosyluronic epimerase294-296*299 and the reaction with this enzyme was closely linked to 2-C-sulfation. The structures of sugars neighboring the D-glucosyluronic unit affect reaction.299aA D-glucosyluronic residue can be residue on the reducing side epimerized if the 2-amino-2-deoxy-~-glucosyl is N-acetylated and that on the nonreducing side is N-sulfated. The reverse arrangement is not reactive: the sequence D-GIcNAc-, L-IdoA is not found. Sulfate on C-2 of L-idosyluronic units or sulfation of C-6 of neighboring (293) U. Lindahl, M. Hook, G . Backstrom, I . Jacobsson, J. Riesenfeld, A. Malmstrom, L. Rodbn, and D. S. Feingold, Fed. Proc., Fed. Am. SOC.Exp. B i d , 36 (1977) 19-24. (294) M. Hook, U. Lindahl, G . Backstrom, A. Malmstrom, and L . - k Fransson, J. Biol. Chem., 249 (1974) 3908-3915. (295) 1. Jacobsson, G . Backstrom, M. Hook, U. Lindahl, D. S. Feingold, A. Malmstrom, and L. Rodbn, J. Bid. Chem., 254 (1979) 2975-2982. (296) A. Malmstrom, L. Rodbn, D. S. Feingold, 1. Jacobsson, G. Backstrom, and U. Lindahl, J. Bid. Chem., 255 (1980) 3878-3883. (297) M. Hook, U. Lindahl, A. Hallbn, and G . Backstrom, J. Bid. Chem., 250 (1975) 6065-607 1. (298) J . Riesenfeld, M. Hook, and U. Lindahl, J. Bid. Chem., 255 (1980) 922-928. (299) J. W. Jensen, L. Rodbn, 1. Jacobsson, U. Lindahl, H. Prihar, and D. S. Feingold, Carbohydr. Res., 117 (1983) 241-253. (299a) I. Jacobsson, U. Lindahl, J. W. Jensen, L. Rodbn, H. Prihar, and D. S. Feingold, J. Bid. Chem., 259 (1984) 1056-1063.

216

BARRY V. McCLEARY AND NORMAN K. MATHESON

2-amino-2-deoxy-~-glucosyl units prevents epimerization. These results are consistent with occurrence of epimerization after N-sulfation and prior to C-sulfation. 6. Proteoglycan Aggregate Proteinases and glycanases have both provided information about the proteoglycan section, made up of chondroitin and keratan sulfates covalently linked to protein, that, in combination with link-protein and hyaluronic acid, forms the cartilage proteoglycan aggregate.211Shorter oligosaccharide units are also attached. These are both 0-and N-linked,271 the former occurring along the whole protein chain, and the latter mainly in the region that binds to hyaluronate. N-Linked oligosaccharides have been isolated from papain digests of corneal, keratan sulfate proteoglycan.267-272 Proteoglycan aggregate, incubated with chondroitin sulfate ABC lyase plus trypsin, gave a keratan sulfate-rich peptide and a hyaluronate binding-region fragment. Digestion of disaggregated proteoglycan with papain gave single, chondroitin sulfate chains linked to peptide, but trypsin yielded peptide fragments having more than one hai in.^^^^.^^^^*^^^^ Treatment of cartilage from chicken embryo299C with chondroitinase AC and end0-P-Dgalactosidase, followed by pepsin and almond glycopeptide N-glycosidase (EC 3.2.2.18; see Section XI), released oligosaccharides containing Dmannosyl units. The binding region and link protein were prepared from the proteoglycan of pig-laryngeal cartilage299fby using digestion with chondroitinase ABC and trypsin. Purified binding-region interacted reversibly with hyaluronate, and this binding was shown to be stabilized by native link-protein. The isolated binding-region and link protein retained properties comparable with those involved in the structure and organization of proteoglycan aggregates. The results led to a model of a polypeptide chain with keratan sulfate and chondroitin sulfate chains attached, in which the keratan sulfate chains are seen as being primarily found towards one end of the peptide chain and next to the section of the polypeptide chain that binds to hyaluronic acid. After digestion with chondroitinase ABC, a dermatan sulfate proteoglycan from mouse c u l t u r e d - c e l l ~gave ~~~ two polypeptides, and papain digestion M. Luscombe and C. F. Phelps, Biochem. J., 103 (1967) 103-109. M. B. Mathews, Biochem. J., 125 (1971) 37-46. D. Heinegard and V. C. Hascall, Arch. Biochem. Biophys., 165 (1974) 427-441. N . Takahashi, H. Ishihara, S. Tejima, Y. Oike, K. Kimata, T. Shinomura, and S. Suzuki, Biochem. J., 229 (1985) 561-571. (2990 F. Bonnet, D. G . Dunham, and T. E. Hardingham, Biochem. J., 228 (1985) 77-85. (2990) J. R. Couchman, A. Woods, M. Hook, and J. E. Christner, J. Bid. Chem., 260 (1985) 13,755- 13,762. (299b) (299c) (299d) (299e)

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

217

of the proteoglycan indicated that most of the polysaccharide chains were clustered in a resistant segment. The location, in the polypeptide chain, of the particular serine to which dermatan sulfate is attached in bovine-skin proteodermatan was established by hydrolysis with cathepsin C (EC 3.4.14.1). Proteoheparan sulfates from fibroblasts299i were digested by thrombin, to yield two major fragments. The larger contained heparan sulfate chains and oligosaccharides. This was cleaved by trypsin into fragments containing heparan sulfate and those containing oligosaccharide chains. The D-xylosyl residues in bovine-lung heparan sulfate have been found to occur as the 2-phosphoric e ~ t e r . ~ ~ ~ j

X. BACTERIALPOLYSACCHARIDES Bacterial, extracellular polysaccharides mostly consist of heterosaccharide repeating-units, and they can be partially hydrolyzed to oligosaccharide fragments by endo-glycanases from infecting bacteriophages. Phages produce enzymes that specifically hydrolyze or lyse one type of linkage in these heteropolysaccharides, releasing oligosaccharides that are the repeating unit or multiples of it. The glycosidic bond hydrolyzed may differ from that preferentially split by acid, and high yields of these oligomeric products are obtained. Other substituent groups, such as acetal and ester, that may be sensitive to acid hydrolysis, remain on the fragments. 1. Klebsiella exo-Polysaccharides A survey of the enzymes from bacteriophages infecting Klebsiella spp. indicated that most of them hydrolyzed a P-D-glycosidiclinkage in a glycosyl residue that was itself linked3" at OH-3. The reducing-end residue released was not a glycuronic acid. An enzyme from a phage that infects one strain of Klebsiella can be effective in the hydrolysis of the polysaccharide from another strain. An activity acting on serotype K5 lysed the polymer, yielding a trisaccharide that contained an unsaturated glycosyluronic For example, the native, capsular polysaccharide from Klebsiella aerogenes type K54 incubated with a bacteriophage-induced enzyme, gave an (299h) R. K. Chopra, C. H. Pearson, G . A. Pringle, D. S. Fackre, and P. G . Scott, Biochem. J., 232 (1985) 277-279. (2991) L.-A. Fransson, L. Coster, 1. Carlstedt, and A. Malmstrom, Biochem. J., 231 (1985) 683-687. (299j) L.-A. Fransson, I. Silverberg, and I. Carlstedt, J. Biol. Chem., 260 (1985) 14,722-14,726. (300) D. Rieger-Hug and S. Stirrn, Virology, 113 (1981) 363-378. (300a) J. E. G. van Dam, H. van Halbeek, J. P. Kamerling, J. F. G . Vliegenthart, H. Snippe, M. Jansze, and J. M. N. Willers, Carbohydr. Rex, 142 (1985) 338-343.

218

BARRY V. McCLEARY AND NORMAN K. MATHESON a-D-GlcA-( 1+ 3)-a-~-Fuc-(1 + 3)-D-Glc 4

t

1 P-D-GIC 57

esterified tetrasaccharide (57) plus an octasaccharide that contained this structure as a repeating unit.301*302 In Table 11, oligosaccharide products of enzymic degradation of some Klebsiella polysaccharides are shown. The bacteriophage preparation hydrolyzing serotype K26 also acted as a p-Dgalactosidase, hydrolyzing the terminal glycosyl group, and producing some d i s a c ~ h a r i d e . ~Modifications ~~" to substituents on constituent sugars may, or may not, affect the hydrolysis reaction, and the effects of a number of these modifications are also listed. Depolymerization of K. aerogenes type 63 polysaccharide with a bacteriophage gave306a trisaccharide (58). Treatment with a-D-galactosidase released D-galactose, leaving an aldobiouronic acid, showing that the D-galactosyl group was nonreducing and terminal. a-D-Gal-(1+ 3)-a-~-GalA-(1 + 3 ) - ~ - F u c 58

Examination of the n.m.r. spectra of the hexasaccharide derived by partial, enzymic depolymerization of the serotype K18 polysaccharide, and of the parent polymer, indicated306"similar solution conformations, despite the large difference in d.p. Serotypes K21 and K32 polysaccharides have both been degraded to oligosaccharides having the 1-carboxyethylidene group intact, giving 59 and 60,respectively. Owing to the extreme acid-lability of this substituent in some structures, phage depolymerization may provide the only method of obtaining an intact repeating-unit from these polymers.307 (301) G. G. S. Dutton and E. H. Memfield, Carbohydr. Res., 105 (1982) 189-203. (302) A. Dell, G. G. S. Dutton, P.-E. Jansson, B. Lindberg, U. Lindquist, and I. W. Sutherland, Carbohydr. Res., 122 (1983) 340-343. (302a) J. L. Di Fabio, D. N. Karunaratne, and G. 0.S. Dutton, Carbohydr. Res., 144 (1986) 251-261. (303) U. Elsasser-Beile and S. Stirm, Carbohydr. Res., 88 (1981) 315-322. (304) H. Niemann, H. Beilhan, and S. Stirm, Carbohydr. Res., 60 (1978) 353-366. (305) H. Thurow, H. Niemann, and S. Stirm, Carbohydr. Res., 41 (1975) 257-271. (305a) G. G. S. Dutton, J. L. DiFabio, D. M. Leek, E. H. Memfield, J. R. Nunn, and A. M. Stephen, Carbohydr. Res., 97 (1981) 127-138. (305b) G. G. S. Dutton and D. N. Karunaratne, Carbohydr. Res., 138 (1985) 277-291. (305c) J. L. DiFabio, G. G. S. Dutton, and H. Parolis, Carbohydr. Res.. 126 (1984) 261-269. (306) G. G. S. Dutton and E. H. Memifield, Carbohydr. Res., 103 (1982) 107-128. (306a) G. G. S. Dutton, A. V. Savage, and M. (R.) Vignon, Can. J. Chem., 58 (1980) 2588-2591. (307) G. G. S. Dutton, K. L. Mackie, A: V. Savage, D. Rieger-Hug, and S. Stirm, Carbohydr. Res., 84 (1980) 161-170.

TABLEI1 Oligosaccbarides Released by Phage Hydrolysis of Kle6siella Polysaccbarides Oligosaccharide released

serotype 1"

Bond hydrolyzed

References

C02H

Me 'C'

I\

3 2 B-D-G~cA-( 1+ ~)-o-L-Fuc-(1+ 3)-D-GlC

+ 3)-B-D-GlC-(1 14)-B-D-GlCA-( + 1 +

303

a-D-GlcA 1

2b

1

3 /3-D-Man-(l+4)-cr-D-Glc-(l+ 3 ) - ~ - G l c

1

+ 3)-B-D-Glc-(1 4)-B-~-Man-( + 1 +

B-AXA-(1+ 4)-p-~-Glc2Ac-( 1+ 3 ) - ~ - M a n 6 4

5

+ 3)-B-~-Man-(l +4)-B)-D-GIcA-l( 1 +

6 4

C

6" (native)

\C02H

&D-Man-( 1 + 4)-cr-~-GlcA-( 1+ ~)-(Y-L-Fuc-( 1+ 3 ) - ~ - G l c 6 4

300a

\I

\/

Me'

304

'C Me'

'C02H

+ 3)-B-D-Glc-(1 + 13)-/3-~-Man-(1+

303

\/ C

Me' 6, esterified and then carboxyl-reduced

\CO,H no reaction

303

(continued)

TABLEI1

(continued)

OligosPccharide released

Serotype 6, depyruvated

Bond hydrolyzed

303

no reaction

11, native, or alkali-treated

1+ 3)-a-~-Gal-( 1+ 3)-D-Gk p-~-GlcA-( 4

References

1

+ 3)-p-D-Gk-( 1+ 3)-p-D-GkA-( 1+

305

t

1 a-D-Gal C Me/ h)

s

‘CO,H

.1

+ 3)-B-D-Gk-(l+ 3)-p-D-GICA-(1+

11, Smith-degraded (side chain removed) 11, esterified and then reduced 13

no reaction

Me

305

305

CO,H ‘C/

I\

4 5 P-D-Gd 1

1 4 CY-D-G~CA 1

t

3 p-D-Man-(1 + 4)-a-D-GlC-(l+ 3)-D-GlC

304

17

22b

26

a-~-Gl~A-(l+3)-a-~Man-(l+2)-a-~-Man-(l+ 3)-D-Gal

+ 3)-B-D-Gal-(l +2)-m-D-GlCA-( 1 1+

302a

4

h) h) r

t

1 a-D-Gk 6

t

1 B-D-GlC 4

t

1 B-D-Gal 4 6

\I C

Me’

‘CO,H (continued)

TABLEI1 Oligosaccharide released

Bond hydrolyzed

a-L-Rha-(1 + 3)-a-~-Rha-( 1+ 2)-a-~-Rha-( 1+3 ) - ~ - G a l

+ 3)-p-~-Gal-( 1 +3)-a-L-Rha-( 1+

p-~-Glc-( 1+ 3 ) - ~ - G a l 4

+ 3)-p-~-Gal-( 1 + 4)-p-D-GlG(1 +

arotype

36

(continued)

Me \ /

References

COzH

C

I\

6 4 B-D-GlC 1

1

4 P-D-GICA 1

N N

1 L

37b

1

1

305a 304

t

1 ~-D-GIC 6

t

1

p-Ad

305b

46

a-D-GlcA-(1+ 3)-a-~-Man-( 1-D 3)-a-D-Gal-(1+ 3)-D-Gal 4

+ 3)-/3-~-Gal-( 1 +3)-a-D-GlcA-(l+ 1

305a

f

i

p - D- M a n 4 X = 3 6

54

Me CO,H

+ 3)-D-GlC a-D-GlcA-(l + 3)-a-~-Fuc2Ac-(l 4

+ 3)-p-D-Gk-( 1

1 4)-a-~-GlcA-( + 1 -D

301 302

t

1 P-D-GIC N w N

a-D-GalA-(1+ 2)-a-D-Man-(1-D 3)-D-Gal 4

51"

13)-a-~-GalA-( + 3)p-~-Gal-( 1 -D 1 +

303

t

1

a-mMan 60

p-Dac 1

B-D-GIC 1

1

1

2

1

+ 3)-B-D-GlC-(l -D~)-~-D-GICA-( 1+

305a.305~

2 p-~-GlcA-( 1+ 3)-a-D-Gal-(1 + 3)-a-~-Man-( 1+ 3)-D-GIC 4

t

1 a-D-GlC (continued)

TABLEI1 (continued) Oligosaccbaride released

serotype

63

74

a-D-Gal-( 1 + 3)-a-~-GalMAc-(l+3)-~-Fuc

Me

\ /

Bond hydrolyzed

1

+ 3 ) - a - ~ - Fuo ( +3)-a-~-Gal-( l 1 -i

COzH

References 305a 306 305a

c

P-D-Gal 1

1 4 a-D-GlcA 1

1 3 a-D-Man-(1 + 2)-a-D-Man-(l+ 3)-D-Gal

1

+ 3)-/3-~-Gal-( 1+ 2)-a-~-Man-( 1+

acid. a Hydrolyzed by type 6 bacteriophage activity. Hydrolyzed by type 13 bacteriophage activity. ' 4-Deoxy-~-~-threo-hex-4-enopyranuronic 4-0-(D-l-Carboxyethyl)-P-D-glucuronic acid.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

225

a - ~ - G l c A -1(+ 3)-a-D-Man-(l+ 2)-a-D-Man-( 1 + 3)-D-Gal 4

t

1

59

Information about the structure of capsular polysaccharides has also been derived from the pattern of biosynthesis of these compounds. The early stages of synthesis involve the prior construction of an oligosaccharide-lipid whose structure ultimately contains the repeating, oligosaccharide unit of the polymer. Further reaction then leads to a polysaccharide composed of this oligosaccharide unit in a regularly repeating pattern.”’ This is in contrast to the non-regular structures of a number of plant polysaccharides (see Sections 11-V). 2. Extracellular Polysaccharides of Other Genera

The extracellular polysaccharides (succinoglycans) from Alcaligenes faecalis var. myxogenes 1OC3, Rhizobium meliloti, Agrobacterium rhizogenes, A. radiobacter, and A. tumefaciens have been degraded successively with succinoglycan depolymerase (an extracellular P-D-glycanase from Flauobacterium spp.) and then endo-(1 + 6)-P-~-glucanase(EC 3.2.1.75) (an intracellular e n ~ y m e ) . ’The ~ former enzyme released an octasaccharide repeating-unit from Alcaligenes faecalis, and the latter hydrolase converted the desuccinylated product into two tetrasaccharides. One of these, when depyruvylated, was hydrolyzed by almond P-D-glucosidase, to give gentiobiose as the sole disaccharide product. In conjunction with methylation analysis of the pyruvated and depyruvated compounds, these results led to structure 61 for this oligosaccharide. Methylation analysis and borohydride reduction gave structure 62 for the other oligosaccharide. Methylation (308) H. Nikaido, Adu. Enzyrnol., 31 (1968) 77-124. (309) M. Hisamatsu, J. Abe, A. Amemura, and T. Harada, Agric. Biol. Chem., 44 (1980) 1049- 1055.

226

BARRY V. McCLEARY A N D NORMAN K. MATHESON p - ~ - G l ~ - (3)-P-D-GlC-( l+ 1 + 3)-P-D-GIC-(1 + 6)-D-GIC 6 4

\I C

Me'

'CO,H 61

P-D-GIc-(1 + 4)-P-D-GlC-( 1 + 4)-P-D-GIC-(1 + 3 ) - ~ - G a i 62

analysis of the octasaccharide showed the presence of two (1 + 3)-, two (1 + 4)-, and two p-( 1+ 6)-linked D-glycosyl residues and one nonreducing (terminal) D-glucosyl group substituted with pyruvate, indicating structure 63;hydrolysis by endo-( 1+ 6)-p-~-glucanaseoccurs at the arrow. When the original polysaccharide was depyruvated and desuccinylated, and then digested with almond p-D-glucosidase, methylation analysis of the product indicated that the glycosidase had removed two of the p(1 + 3)-linked and one of the p-( 1+ 6)-linked D-glucosyl residues, and that one p - ( l + 6)-linked D-glucosyl group remained as a branch. This incomplete release of the D-glucan side-chain suggests that almond p-Dglucosidase may be an exo-D-glucanase. These results led to a proposal of structure 64 for the desuccinylated succinoglycan, in which hydrolysis by the extracellular p-D-glycanase takes place at the arrow marked with a, and by the intracellular (1 + 6)-p-D-glUCanaSe at the arrow marked b. When treated with succinoglycan depolymerase followed by (1-* 6)-P-~-glucanase,the exocellular polysaccharides from Rhizobiurn rneliloti U27, Agrobacteriurn radiobacter, and Alcaligenes faecalis var. rnyxogenes gave the same two tetrasaccharide fractions, as judged by paper chr~matography,~" confirming that these are all identical, apart from their acylation. The structures of the polysaccharides of R. trifolii AHU 1134, R. phaseoli AHU 1133, and R. lupini KLU, when similarly examined, were shown to differ from 64, in that the terminal sugar in the branch chains was a D-galactosyl group. The penultimate D-glucosyl residue of the branch chains and half of the terminal D-galactosyl groups were pyruvylated at 0 - 4 and 0-6 of these sugars, and there were -2 mol of acyl units per mol of repeating unit.3" When the extracellular, acidic polysaccharide from Rhizobiurn rneliloti I F 0 13336 was hydrolyzed with extracellular p-D-glycanase and then intracellular endo-(1 +6)-p-~-glucanase, two tetrasaccharides were (310) T. Harada, A. Amemura, P.-E. Jansson, and B. Lindberg, Carbohydr. Res., 77 (1979) 285-288. (311) A. Amemura and T. Harada, Carbohydr. Rex, 112 (1983) 85-93.

C

Me'

\CO,H

63

64

228

BARRY V. McCLEARY A N D NORMAN K. MATHESON

released, one of which was 62, but the second was quite different: from chemical evidence, structure 65 was proposed, with the ribosyluronic residue having a furanose ring.312Methylation analysis of the octasaccharide, and enzymic susceptibility, indicated that the D-glucosyl group was p-( 1 + 6)linked to the side chains. a-D-RibfA-( 1 -* 4)-a-D-GICA-(1 -* 4)-P-D-GIC-(1 + 6)-D-GIC 65

R. trifolii 4s polysaccharide was hydrolyzed with a phage-induced depolymerase into a heptasaccharide and its dimer, having the same ratio of components (D-glucose :D-glucuronic acid :pyruvic acid :acetyl = 5 :2 :1 : 2) as the native polymer.313 p-D-Glucosiduronase released 1.4 mol of Dglucuronic acid from the deacetylated heptasaccharide, and then, after depyruvation, D-glucose (2 mol) was released by almond p-D-glucosidase. From these results, combined with methylation, before and after enzymic hydrolysis, and n.m.r. spectroscopic data, repeating unit 66 was proposed, having a backbone structure different from those previously described. -*

4)-P-D-GICA-(1 + 4)-p-D-GICA-(1 -* 4)-P-D-GlC-(1 -*4)-a-D-GIC-(1 -* 6

t

1 P-D-GIC 4

f

1

r

1

Me’-‘CO,H

66

The extracellular polysaccharides of Rhizobium meliloti 201 have been examined by using enzymic degradation and chemical procedure^.^'^ A mixture of polysaccharides produced by the bacterium, when incubated with a bacterial enzyme that hydrolyzed one of these, gave oligosaccharides that could be separated by DEAE-cellulose chromatography. The major fraction was a pentasaccharide, for which methylation analysis and Smith (312) A. Amemura, M. Hisamatsu, S. K. Ghai, and T. Harada, Carbohydr. Res., 91 (1981) 59-65. (313) A. Amemura, T. Harada, M. Abe, and S. Higashi, Carbohydr. Res., 115 (1983) 165-174. (314) N. Yu, M. Hisamatsu, A. Amemura, and T. Harada, Agric. Biol. Chem., 47 (1983) 49 1-498.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

229

a - D - M a n - ( l + 4 ) - a - ~ - G l c A - ( l3+) - a - ~ - M a n - ( l 3+) - ~ - G l c 4

t

1 P-D-GlC

67

degradation, combined with its susceptibility to hydrolysis by jack-bean a-D-mannosidase, indicated structure 67. An extracellular polysaccharide from strains of Rhizobium japonicum was chemically degraded to a tetrasaccharide in high yield with lithium in ethylenediamine.315 Sequential glycosidase hydrolysis with Aspergillus niger a-D-galactosidase, jack-bean a-D-mannosidase, and a-D-glucosidase, in combination with methylation analysis, indicated structure 68 for this tetrasaccharide. Combined with structural analysis of a hydrogen fluoride degradation product (a tetrasaccharide containing glycosyluronic residues), this allowed a pentasaccharide repeating unit to be proposed for the polymer. a-D-Man-( 1 + 3)-a-D-GlC-(1 + 3)-D-GlC 6

t

1 a-~-Gal4Me 68

Reaction of the secreted polysaccharides of Rhizobium trifolii NA30, R. trifolii LPRS, R. leguminosarum LPRl, R. phaseoli LPR49, and a nonnodulating strain formed from R. trifolii LPRS with a bacteriophage enzyme released the same octasaccharide 68a from all.31Sa AXA-( 1 + 4)-P-D-GICA-(1 + 4)-P-D-GlCA-(1 + 4)-D-GIC 6

t

1 P-D-GIC 4

t

1 P-D-GlC 4

t '4 P-D-G~c:C= 36

Me COzH

t '4 P-D-Gal :C= 6

Me CO,H

68a

(315) A. J. Mort and W.D. Bauer, J. Bid. Chem., 257 (1982) 1870-1875. (315a) M. McNeil, J. Darvill, A. G. Darvill, P. Albersheim, R. van Veen, P. Hooykaas, R. Schilperoort, and A. Dell, Carbohydr. Res., 146 (1986) 307-326.

230

BARRY V. McCLEARY AND NORMAN K. MATHESON

The unsaturated glycosyluronic group was derived from a - ~ - G l c A -1(+ 4)-. A bacteriophage-induced enzyme hydrolyzed315bthe capsular polysaccharide of Acinobacter calcoaceticus BD4, releasing heptasaccharide 68b. a-L-Rha 1

3.

4 a-~-GlcA 1

5.

2 a-L-Rha-(1 + 3 ) - a - ~ - M a n -1(+ 3)-a-~-Rha-( 1 + 3)-a-~-Rha-( 1 + 3)-P-o-Glc

68b

The enzyme hydrolyzed the p-D-glucosyl-(1+ 3)-L rhamnosyl linkage that joins the heptasaccharide repeating units. A disaccharide obtained by partial, acidic hydrolysis, and composed of a D-glucosyluronic and a Dmannosyl unit was hydrolyzed by p-D-glucosiduronase.

3. Lipopolysaccharides The 0-antigen polysaccharides of Klebsiella serotype 0 5 and Escherichia coli 0 8 were prepared by mild hydrolysis of the lipopolysaccharides. A bacteriophage enzyme hydrolyzed both giving the trisaccharide P-D-Man-(l+ 2)-a-~-Man-( 1+ 2 ) - ~ - M a n . In structural of a lipopolysaccharide of Serratia marcexens CDC 1783-57 (014:H9), which consists of D-glucose, D-galactose, and 2-acetamido-2-deoxy-~-glucose in the ratios of 1:1:2, Smith degradation gave, as a major product, an oligosaccharide of a D-galactosyl and 2acetamido-2-deoxy-~-glucosyl residue joined to glycerol. Treatment with coff ee-bean or Aspergillus niger a -D-galactosidase and then jack-bean N acetyl-P-D-glucosaminidase, in conjunction with methylation analysis of the original polysaccharide, showing that the structure was 68c. a-D-Gal-(l+ 3)-p-~-GlcNAc-( 1 + 1)-L-glycerol 68C

Deamination of the polymer yielded 2,5-anhydromannitol and a trisaccharide composed of equimolar amounts of D-glucose, D-galactose, and 2,5-anhydromannitol. Yeast a-D-glucosidase released D-glucose from this (315b) N. Kaplan, E. Rosenberg, B. Jann, and K. J a m , Eur. 1. Biochem., 152 (1985) 453-458. (315c) P.-E.Jansson, J. Lonngren, G. Widrnalrn, K. Leontein, K. Slettengren, S. B. Svenson, G . Wrangsell, A. Dell, and P. R. Tiller, Carbohydr. Rex, 145 (1985) 59-66. (315d) C. J. Brigden and S. G. Wilkinson, Carbohydr. Res., 145 (1985) 81-87.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

23 1

trisaccharide, indicating a nonreducing (terminal) position for this sugar. Combined with methylation analysis and n.m.r. spectroscopy, structure 68d was proposed for a tetrasaccharide repeating unit.

The' linkage of the 0-D-galactosyl group in the lipopolysaccharide of Salmonella typhimurium was shown, by the application of D-galactose oxidase, to be to 0-3 of a D-glucosyl residue. Oxidation of the nonreducing (terminal) D-galactosyl group by this enzyme, followed by oxidation with bromine, producing a D-galactosyluronic group, strengthened the bond between it and the D-glucosyl residue sufficiently for the aldobiouronic acid to be isolated on acid hydrolysis, and allowed the (1 -* 3) linkage to be e~tablished.~'~' Xanthan, the (1 + 4)-P-~-glucan-basedpolymer from Xanthomonas spp. has been discussed in Section II,3. XI. GLYCOCONJUGATES Information on the structures of the glycan portion of glycoproteins has been obtained ( a ) by using specific glycosidases that sequentially remove glycosyl groups from the nonreducing ends of the oligosaccharide chains; (b) from the characteristics of specific, endogenous, degradative enzymes that modify asparagine-linked chains prior to further extension; ( c ) by using endo-glycosidases that split at, or near, the linkage of carbohydrate to protein, and whose reactivities are affected by the structure of the oligosaccharide chain; and ( d ) by employing biosynthetic enzymes, whose reactivities are controlled by both the terminal sugar being substituted, as well as by other glycosyl residues in the oligosaccharide. Sequences of glycosyl units, and the anomeric linkages in oligosaccharide fragments, have been determined by using a series of specific glycosidases to remove sugar groups sequentially from nonreducing chain-ends. This approach has been r e ~ i e w e d . ~ ' ~The . ~ ' ' oligosaccharide remaining after each (315e) S. M. Rosen, M. J. Osborn, and B. L. Horecker, J. Biol. Chem., 239 (1964) 3196-3200. (316) Y.-T. Li and S.-C. Li, in M. I. Horowitz and W. Pigman (Eds.), The Glycoconjugares, Vol. 1, Academic Press, New York, 1977, pp. 51-67. (317) R. Kornfeld and S. Kornfeld, in Ref. 208, pp. 1-34.

232

BARRY V. McCLEARY A N D NORMAN K. MATHESON

treatment can be separated by gel chromatography from the released monosaccharide, and the proportions of the latter determined; paper chromatography has also been used. Alternatively, the proportion of released monosaccharide can be estimated in the incubation mixture by using enzymes specific for the sugar; for example, by reaction with Dgalactose dehydrogenase (EC 1.1.1.48) linked to reduction with nicotinamide adenine dinucleotide. As the oligosaccharides of glycoproteins are branched, and also may contain, in any one chain, more than one sugar in a sequence susceptible to hydrolysis by a single glycosidase, quantitation on hydrolysis is essential. Sequential, glycosidase hydrolysis can be combined with Smith degradation to provide information about linkage types and branching and, in conjunction with methylation analysis, complete structures have been determined. Some earlier examples of sequential hydrolysis are of oligosaccharides from pineapple-stem bromelain,3'8 ovalb ~ m i n ; ' ~Phaseolus uulgaris lectin receptor-site from human erythrocyte^,^^' human yG-myeloma proteinsP2*and r i b o n ~ c l e a s eSelected . ~ ~ ~ examples of complete sequence-determination of N-asparaginyl-linked glycan chains are those of ~ v a l b u m i n , ~ 'IgE ~ . ~i m ~m ~~ . ~n o~g~l o b u l i npulmonary ,~~~ glyc o p r ~ t e i n Rous-sarcoma ,~~~ virus, and cell-membrane glyc~proteins.~~' A comparison of a-D-galactosidase hydrolyses of thyroglobulin from different mammalian sources showed a species-dependent occurrence of terminal a-D-galactosyl units ranging from o to 1 1 per The differences in the rates of hydrolysis of various linkage types by a particular glycosidase can be used to provide information about this aspect of structure. Jack-bean a-D-mannosidase cleaves a-(1 + 2) and a-(1+ 6) linkages much faster than a-(1+ 3). Oligosaccharides, obtained by endo-Nacetyl-/3-D-glucosaminidase hydrolysis of ovalbumin, were subjected to acetolysis, which selectively cleaved the a-(1+ 6) bonds. A tetrasaccharide isolated after this treatment was then incubated with jack-bean CY-D(318) (319) (320) (321) (322) (323) (324) (325) (326) (327) (327a)

Y. Yasuda, N . Takahashi, and T. Murachi, Biochemisrry, 9 (1970) 25-32. C.-C. Huang, H. E. Mayer, and R. Montgomery, Curbohydr. Res., 13 (1970) 127-137. R. Kornfeld and S. Kornfeld, J. Biol. Chem., 245 (1970) 2536-2545. R. Kornfeld, J. Keller, J. Baenziger, and S. Kornfeld, J. Biol. Chem., 246 (1971) 3259-3268. T. Sukeno, A. L. Tarentino, T. H. Plummer, and F. Maley, Biochemistry, 11 (1972) 1493-1501. T. Tai, K. Yamashita, M. Ogata-Arakawa, N. Koide, T. Muramatsu, S. Iwashita, Y. Inoue, and A. Kobata, J. Bid. Chem., 250 (1975) 8569-8575. T. Tai, K. Yamashita, S. Ito, and A. Kobata, J. Biol. Chem., 252 (1977) 6687-6694. J. I. Rearick, A. Kulczycki, and S. Kornfeld, Arch. Biochem. Biophys., 220 (1983) 95-105. S. C. Sahu and W. S. Lynn, Carbohydr. Res., 90 (1981) 251-260. L. A. Hunt, Biochem. J., 209 (1983) 659-667. R. G . Spiro and V. D. Bhoyroo, J. Bid. Chem., 259 (1984) 9858-9866.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

233

mannosidase, which gave rapid hydrolysis of one D-mannosyl group, followed by slow hydrolysis of a second, consistent323with structure 69. a-D-Man-(1 + 2 ) - a - ~ - M a n -1 (-* 3)-P-D-Man-(1 + 4 ) - ~ - G l c N A c o l 69

An a-L-fucosidase that specifically hydrolyzes L-fucosyl groups a-(1+ 3) -linked to 2-acetamido-2-deoxy-~-glucosyl residues, being unable to hydrolyze either a-(1 + 6) or a-(1 + 2) bonds, released most of the L-fucose from asialo-orosomucoid, and about half from lactoferrin, but produced no L-fucose from a2-macroglobulin, suggesting the absence of (1 + 3)-a-~-fucosyllinkages in this g l y c o p r ~ t e i nVarious . ~ ~ ~ viral and bacterial sialidases show specificity for either the a-(2-3) or the a-(2+6) linkages. Viral enzymes gave a very low rate of hydrolysis of a - ( 2 + 6 ) linkages, but, with bacterial enzymes, the preference was mixed. It also appeared that both the structure of the core oligosaccharide and of the protein affected the rate of hydrolysis.329 Glycosidases from different sources, hydrolyzing the same sugar and anomeric linkages, but with differing specificities for the position of linkage to the next sugar, can also be used in conjunction, to determine a sequence of a-D-mannosyl residues. The glycopeptide obtained by pronase digestion of lima-bean l e ~ t i n ~ has ~ 'been examined with jack-bean a-D-mannosidase (EC 3.2.1.77), which hydrolyzes all a linkages, Aspergillus niger a-Dmannosidase, which hydrolyzes only a-(1 + 2) bonds, and Arthrobacter exo-a-D-mannanase, that requires a sequence of a-linked D-mannosyl residues for effective action. The jack-bean enzyme released three mol of D-mannose, the A. niger enzyme, one, and the Arthrobacter enzyme, one. The Arthrobacter digest released two more residues if incubated with jackbean a-D-mannosidase, and one more if then treated with P-D-mannosidase. A partial structure (70) was proposed, consistent with that generally detected for this region of glycoproteins. a-D-Man 1

33 p-D-Man-(l+ 6

t

1 a-D-Man-(1+ 2)-a-D-Man 70

(328) M. J. Imber, L. R. Glasgow, and S. V. Pizzo, J. Biol. Chem., 257 (1982) 8205-8210. (329) A. P. Corfield, H. Higa, J. C. Paulson, and R. Schauer, Bioehim. Biophys. Acta, 744 (1983) 121-126. (330) A. Misaki and I. J. Goldstein, J. Biol. Chem., 252 (1977) 6995-6999.

234

BARRY V. McCLEARY AND NORMAN K. MATHESON

Glycosidases can differentiate between anomeric linkages. The presence in potato lectin of nonreducing (terminal) a-L-arabinofuranosyl bonds in oligosaccharide chains composed of L-arabinofuranosyl residues, when the d.p. was greater than three, was established with a-~-arabinofuranosidase.~~’ The inner linkages were p, and the few terminal D-galactosyl groups (3%) could be removed with a-D-galactosidase. For determination of the sequence of the lipid-linked precursor of N-mannosylasparaginyl-containingchains in vesicular-stomatitis virus G protein, glycosidase sequencing was adapted to radioactively labelled material,332owing to the small amounts of material available. D-Galactose oxidase identifies nonreducing (terminal) D-galactosyl groups by selective oxidation.332a Aspects of the structure of glycoproteins having asparaginyl N-linked chains have been determined from studies of the part of their biosynthesis that involves glycosidases (or possibly exo-glycanases). This has been Initially, the (Gl~)~(Man),(GlcNAc), section of the dolichyl diphosphate derivative of this compound is transferred to an L-asparagine residue in the polypeptide chain. Then, three D-glucosyl units and up to six of the D-mannosyl residues are sequentially removed by a-D-glucosidases and a-D-mannosidases. This is called “processing,” and some of the enzymes may be exo-glycanases. At least two a-D-glucosidases are involVed.334-340a The first releases the terminal a - ( l + 2)-linked D-glucosyl group; examples are a-D-glucosidase from hen-oviduct micro~ornes~~’ and from Saccharomyces cerevisiae extracts336;the second, which removes the two inner a-(1-* 3)-linked D-glucosyl units includes an a-D-ghcosidase from the endoplasmic reticulum of rat liver.339A mutant line of mouse-lymphoma cells, deficient in one of the a-D-glucosidases, produced mostly highmannose oligosaccharide side-chains having the structure (Glc),( Man),(GlcNAc),. In the presence of castanospermine, an inhibitor (331) D. Ashford, N. N. Desai, A. K. Allen, A. Neuberger, M. A. O’Neill, and R. R. Selvendran, Biochem J., 201 (1982) 199-208. (332) E. Li, I. Tabas, and S. Komfeld, J. Bid. Chem.. 253 (1978) 7762-7770. (332a) G . Avigad, Arch. Biochem. Biophys., 239 (1985) 531-537. (333) S. C. Hubbard and R. J. Ivatt, Annu Rev. Biochem., 50 (1981) 555-583. (334) L. S. Grinna and P. W. Robbins, J. Biol. Chem., 255 (1980) 2255-2258. (335) W. W. Chen and W. J. Lennan, J. Biol. Chem., 253 (1978) 5780-5785. (336) R. D. Kilker, B. Saunier, J. S. Tkacz, and A. Herscovics, 1. Bid. Chem., 256 (1981) 5299-5303. (337) J. J. Elting, W. W. Chen, and W. J. Lennan, J. Biol. Chem., 255 (1980) 2325-2331. (338) D. M. Burns and 0. Touster, J. Biol. Chem., 257 (1982) 9991-10,000. (339) R. A. Ugalde, R. J. Staneloni, and L. F. Leloir, Eur. 1. Biochem., 113 (1980) 97-103. (340) J. M. Michael and S. Kornfeld, Arch. Biochem. Biophys., 199 (1980) 249-258. (340a) H. Hettkamp, G . Legler, and E. Bause, Eur. J. Biochem., 142 (1984) 85-90.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

235

of a-D-glucosidase, the parent cells, produced mostly (Glc),( Man),( GlcNAc),, whereas, in the absence of inhibitor, normal, highmannose oligosaccharide chains were obtained.340b At least two a - ~ mannosidase activities are involved in processing the D-mannose-containing ~ e g m e n t . ~ The ~ ' , ~first, ~ ' ~which may be composed of two activities, removes up to four D-mannosyl units. An enzyme, purified from rat-liver Golgi bodies,342converted 71 into 72, releasing four D-mannosyl residues, and a-D-Man-( 1+ 2 ) - a - ~ - M a n -1(+ 2 ) - a - ~ - M a n 1

1 a-D-Man-( 1 + 2 ) - a - ~ - M a n 1

3 I B-o-Man-( 1 + . 4 ) - p - ~ - G l c N AAsn ~~ 6 I

a-D-Man 6

t

1 a-D-Man-(l+2)-a-D-Man 71

a-D-Man 1

1 3 I p-~-Man-(1+4)-p-~-GlcNA~Asn

a-D-Man

I

1

\ 3 1 /"6 a-D-Man 6

t

1 a-D-Man 72

thus showing a specificity for a - ( 1 + 2) linkages. Another a-D-mannosidase, also found in the rat-liver Golgi complex,343converts 73 into 74. The product of hydrolysis (72) by the first a-D-mannosidase must first be substituted with a 2-acetamido-2-deoxy-~-glucosyl group before further hydrolysis of (340b) (341) (341a) (342) (343)

G. Palamarczyk and A. D. Elbein, Biochem. J., 227 (1985) 795-804. W. T. Forsee and J. S. Schutzbach, J. Biol. Chem., 256 (1981) 6577-6582. B. Winchester, Biochem. Soc. Trans., 12 (1984) 522-524. I. Tabas and S. Kornfeld, J. Biol. Chem., 254 (1979) 11,655-11,663. D. R. P. Tulsiani, S. C. Hubbard, P. W.Robbins, and 0. Touster, J. Biol. Chem., 257 (1982) 3660-3668.

236

BARRY V. McCLEARY AND NORMAN K. MATHESON P-D-GIcNAc-(I + 2 ) - a - ~ - M a n 1

5.

a-D-Man 6

t

1 a-D-Man 13

P-D-GIcNAc-(1 + 2 ) - a - ~ - M a n 1

.1 3 I P-D-Man-(1+4)-P-~-GlcNAc+ Asn 6 I

t

1 a-D-Man 14

two or more D-mannosyl units can occur. Further substitution of these hydrolysis products by appropriate glycosyltransferases then leads to the glycoprotein structures. The a-D-glucosidase, and one of the a-D-mannosidases, that process the outer edge of their respective substrates, do not hydrolyze the nitrophenyl a-D-glycosides of D-glucose and D-mannose, whereas those that hydrolyze the inner section do. This property, the specificity for one linkage type and the incomplete hydrolysis of the chain, suggests that the former pair of enzymes may be exo-glycanases rather than glycosidases. An important aspect of the processing is that it is ordered. Hydrolysis by a-D-glucosidase precedes a-D-mannosidase action, and the release of D-mannosyl groups from different branches is not random, but proceeds in a definite sequence. A group of enzymes designated as endo-glycosidases hydrolyze at, or near, the linkage of carbohydrate to peptide. They have been useful, not only in releasing oligosaccharide chains from glycoproteins for further structural studies, but also, because their specificities, as governed by component glycosyl residues in the oligosaccharide chain that are distant from the site of hydrolysis, allow conclusions about structure to be made. Reviews of these enzymes have The different types of linkages (344) A. Kobata, A n d Biochem., 100 (1979) 1-14. (345) P. H. Atkinson and J. Hakimi, in Ref. 208, pp. 191-239.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

237

between oligosaccharide and protein346have distinctive hydrolases that act on them. The endo- N-acetyl-P-D-glucosaminidases [mannosyl-glycoprotein-( 1 + 4)-acetamidodeoxy-~-~-glucohydrolase] that have been isolated fall broadly into two classes. The first, which includes endo-N-acetyl-P-o' . ~from ~ ~ Clostridium glucosaminidase D from Diplococcus p n e ~ r n o n i a e ? ~C, perf ring en^,^^' and F-I from fig latex,350hydrolyze tri-D-mannosyl derivatives (75) and glycopepof di-(2-acetamido-2-deoxy-~-glucosyl)-~-asparag~ne tides much more readily than hexa-D-mannosyl derivatives. The a-(1+ 3)-linked D-mannosyl group should be present, and not further substituted a-D-Man 1

J. 3 P-D-Man-(1 + 4)-p-~-GlcNAc-( 1 + 4)-~-GlcNAc-,Asn 6

t

1

a-D-Man 75

at 0-2, and other glycosyl groups (D-galactosyl or 2-acetamido-2-deoxy-~glucosyl) can be joined to the D-mannosyl residue that is a-(l+ 6) -linked.3519351a Hydrolysis is very limited if the number of D-mannosyl residue residues exceeds five; and the first 2-acetamido-2-deoxy-~-glucosyl linked to L-asparagine can be substituted with an L-fucosyl group. Members of the second group of endo-N-acetyl-D-glucosaminidases readily hydrolyze oligosaccharide chains having a higher number of Dmannosyl residues. Several of these enzymes have been described, such as CIIfrom C. p e r f n ' n g e n ~ H , ~from ~ ~ . ~Streptornyces ~~ g r i s e ~ s , ~Aspergil~ ~ * ~ ~ ~ * ~ ~ lus o r y ~ a e ?a ~Flavobacteriurn ~~ ~ p . F-I1 , ~from ~ fig ~ latex,350 ~ and enzymes (346) (347) (348) (349) (350) (351) (351a) (352) (353) (353a) (353b)

A. B. Zinn, J. J. Plantner, and D. M. Carlson, in Ref. 316, pp. 69-85. N. Koide and T. Muramatsu, J. Biol. Chem., 249 (1974) 4897-4904. A. L. Tarantino and F. Maley, Biochem. Biophys. Res. Commun., 67 (1975) 455-462. S. Ito, T. Muramatsu, and A. Kobata, Arch. Biochem. Biophys., 171 (1975) 78-86. S.-C. Li, M. Asakawa, Y. Hirabayashi, and Y.-T. Li, Biochim. Biophys. Acta, 660 (1981) 278-283. S. Ito, T. Muramatsu, and A. Kobata, Biochem. Biophys. Res. Commun., 63 (1975) 938-944. T. Mizouchi, J. Amano, and A. Kobata, J. Biochem. (Tokyo), 95 (1984) 1209-1213. A. L. Tarentino, T. H. Plummer, and F. Maley, J. B i d . Chem., 249 (1974) 818-824. T. Tai, K. Yamashita, and A. Kobata, Biochem. Biophys. Res. Commun., 78 (1977) 434-441. J. Hitomi, Y. Murakami, F. Saitoh, N. Shigemitsu, and H. Yamaguchi, J. Biochem. ( T o k y o ) , 98 (1985) 527-533. K. Yamamoto, S. Kadowaki, K. Takegawa, H. Kumagai, and T. Tochikura, Agric. Biol. Chem., 50 (1986) 421-429.

238

BARRY V. McCLEARY AND NORMAN K. MATHESON

from Sporotricum dirnorpho~porum~~~ and rat liver.355 Differences in specificity have been reported for these activities from different sources, with respect to ( a ) further substitution of D-mannosyl residues by other and D-galactose); (6) substitution sugars (2-acetamido-2-deoxy-~-glucose residue next to L-asparagine by of the 2-acetamido-2-deoxy-~-glucosyl L-fucose; and ( c ) the smallest oligosaccharide chain that can be hydrolyzed. The differing specificities of these enzymes have enabled observations about glycoprotein structures to be made. The presence of a D-mannosyl residue P-linked to chitobiose, in contrast to the a linkages of all of the other D-mannosyl residues in ~ v a l b u m i nwas , ~ ~shown ~ by the initial preparation of Man-GlcNAc-GlcNAc-Asn by proteolysis and glycosidase hydrolysis. Reaction of this product with an endo-N-acetyl-D-glucosaminidase released D-GlcNAc-Asn and P-D-Man-( 1 + 4)-~-GlcNAc;the latter was clearly differentiated from a-D-Man-(1 + 4)-~-GlcNAc.Using an endo- Nacetyl-P-D-glucosaminidase from D. p n e ~ m o n i u edifferences ,~~~ have been detected in the population of oligosaccharide chains released by hydrolysis of glycopeptide chains from growing and non-growing, human-diploid cells. The glycopeptide mixture obtained by proteolysis of ovalbumin was 20% hydrolyzed by D. pneumoniae endo- N-acetyl-P-D-glucosaminidase,consistent with heterogeneity of the oligosaccharide chains.347 The neutral oligosaccharide chains of some glycoproteins (ribonuclease B and invertase) could be released by endo- N-acetyLj3-D-gIucosaminidaseH, whereas structures containing only acidic chains terminated by sialic acid (transferrin, fibrinogen, and a -acid glycoprotein) were resistant. Where the glycoprotein contained both types of chains (thyroglobulin and immunoglobulin M), only the neutral chains were released.352Two of the glycopeptide fractions (IV and V), obtained from ovalbumin by proteolysis and ion-exchange chromatography, were shown to be heterogeneous in the oligosaccharide section by electrophoresis in borate of the oligosaccharides obtained by endo-N-acetyl-P-D-glucosaminidasetreatment of each fraction.323 The differing behavior of fraction 111 on hydrolysis with endo-N-acetyl-P-Dglucosaminidase C , , or H showed that this fraction was also a mixture; C , , gave only 75% hydrolysis, whereas H hydrolyzed the substrate com~ l e t e l yAn . ~ examination ~~ of the membrane glycoproteins of BHK21 cells and Rous-sarcoma using digestion with endo-N-acetyl-P-Dglucosaminidase and glycosidases, followed by separation of the products (354) S. Bouquelet, G. Strecker, J. Montreuil, and G . Spik, Biochimie, 62 (1980) 43-49. (355) Y. Tachibana, K. Yamashita, and A. Kobata, Arch. Biochem. Biophys., 214 (1982) t 199-210. (356) A. L. Tarentino, T. H. Plummer, and F. Maley, J. Biol. Chem., 247 (1972) 2629-2631. (357) T. Muramatsu, P. H. Atkinson, S. G . Nathenson, and C. Ceccarini, J. Mol. BioL, 80 (1973) 781-799.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

239

by gel chromatography, indicated that asparagine-linked, acidic oligosaccharide chains all contained a core of two a-linked D-mannosyl residues and a third D-mannosyl residue joined p to a 2-acetamido-2-deoxy-~glucosyl residue, as in 76. (cr-D-Man),-P-D-Man-(1 + 4 ) - p - ~ - G k N A c -

76

An enzyme that cleaves the linkage between 2-acetamido-2-deoxy-~glucose and L-asparagine (glycopeptide-N-glycosidase, EC 3.2.2.18), releasing the intact oligosaccharide chain, has been isolated from almond seeds and jack-bean mea1.358-360 Both preparations show a broad spectrum for substrates; complex chains, and chains containing high levels of D-mannosyl residues are hydrolyzed, the former the more readily, and protein conformation affected the rate of oligosaccharide removal. Oligosaccharide chains can be substituted with sialic acid. Almond extract has been separated into three enzyme fractions by DEAE-cellulose c h r ~ m a t o g r a p h y .One ~ ~ ' of them preferred glycopeptides having shorter peptide chains. Another hydrolyzed glycoprotein having intact protein chains. Glycopeptide-N-glycosidase activity has been detected in an endo-N-acetyl-/3-glucosaminidase prepar, ~it was ~ ~able ~ to cleave short glyation from a Huvobacterium ~ p . and coprotein oligosaccharide On treatment with almond-seed enzyme, stem-bromelain glycopeptide quantitatively released peptide free from glycosyl units, and two oligosaccharides, which were linked to Lasparagine in the original molecule and which contained a sequence of two 2-acetamido-2-deoxy-~-glucosyl units at the reducing end. Their structures were determined,362by methylation analysis and by hydrolysis with (Y-Dmannosidase, to be 77 and 78. Pepsin digestion of ovalbumin gave fractions having all of the carbohydrate in two closely similar g l y ~ o p e p t i d e sAlmond . ~ ~ ~ enzyme quantitatively released both high-mannose and hybrid-type oligosaccharides in the same ratio from both glycopeptides, indicating that both types of oligosaccharides, (358) N. Takahashi, Biochem. Biophys. Res. Commun., 76 (1977) 1194-1201. (359) K. Sugiyama, H . Ishihara, S. Tejima, and N. Takahashi, Biochem. Biophys. Res. Commun., 112 (1983) 155-160. (360) A. L. Tarentino and T. H. Plummer, J. Bid. Chem., 257 (1982) 10,776-10,780. (361) N. Takahashi and H. Nishibe, Biochirn. BiOphyS. Ada, 657 (1981) 457-467. (361a) T. H. Plummer, J. H. Elder, S. Alexander, A. W. Phelan, and A. L. Tarentino, J. Biol. Chem., 259 (1984) 10,700-10,704. (361b) F. K. Chu, J. Biol. Chem., 261 (1986) 172-177. (362) H. Ishihara, N. Takahashi, S. Oguri, and S. Tejima, J. Biol. Chem., 254 (1979) 10,71510,719. (363) H. Ishihara, N. Takahashi, J. Ito, E. Takeuchi, and S. Tejima, Biochim. Biophys. Acra, 669 (1981) 216-221.

240

BARRY V. McCLEARY AND NORMAN K. MATHESON

2 3 P-D-Man-(1 + 4)-P-o-GlcNAc-( 1 -+ 4)-~-GlcNAc 6

t

1 a-D-Man-(l+6)-a-D-Man

11

WL-FUC 1

P-D-XYl 1

.1

J.

2 3 P-D-Man-(1+ 4)-P-~-GlcNAc-(l+4)-D-GlcNAc 6

t

1 a-D-Man 18

high-mannose and complex, are attached to the same L-asparaginyl unit in ovalbumin. Digestion of desialylated fibrinogen removed 40% of the total, neutral sugars, with equivalent release from both the P- and y-polypeptide chains. No significant differences in clotting ability appeared.364Sequential digestion of the released oligosaccharide with glycosidases gave the tentative sequence (Gal),-( GlcNAc)*-(Man),-GlcNAc-GlcNAc. endo-N-Acetyl-a-D-galactosaminidase (EC 3.2.1.97) from D. pneumoniae365.366 hydrolyzes the 0-glycosyl bond between the 2-acetamido-2deoxy-D-galactosyl residue and L-serine, or L-threonine, and has been found to release the disaccharide &,-Gal-( 1 + 3)a-GalNAc from a number of desialylated glycoproteins, including asialo-fetuin glycopeptide fraction C, human melanoma, human-bronchial and ovine- and porcine-submaxillary mucin, mouse melanoma, and fetuin glycopeptide, as well as antifreeze glycoprotein. The pattern of hydrolytic products indicated an exclusive specificity for the oligosaccharide sequence. endo-P-D-Galactosidases (EC 3.2.1.102) have been isolated from D. pneumoniae, and one released trisaccharides, as shown in 79 and 80, from type 2 chains in A and B blood-group mucins, re~pectively.3~’ Type 1 compounds, or D-glucosyl in which the linkage to 2-acetamido-2-deoxy-~-glucosyl (364) H. Nishibe and N. Takahashi, Biochim. Biophys. Acta, 661 (1981) 274-279. (365) Y. Endo and A. Kobata, J. Biochem. (Tokyo), 80 (1976) 1-8. (366) J. Urnernoto, V. P. Bhavanandan, and E. A. Davidson, J. Bid. Chem., 252 (1977) 8609-8614. (367) S. Takasaki and A. Kobata, J. Biol. Chem., 251 (1976) 3603-3609.

ENZYMIC ANALYSIS O F POLYSACCHARIDE STRUCTURE

24 1

a-L-FUC 1

.1 2 . 1 CY-D-GICNAC-(I + 3 ) - P - ~ - G a l -1(+ 4 ) - p - ~ - G l c N A c 19

(I-L-FUC 1

.1 2 4 c cr-D-Gal-(l+ 3 ) - P - ~ - G a l 1- (--t 4 ) - p - ~ - G l c N A(Gk)80

residues is (1 +3), and compounds having the H structure, lacking the terminal, nonreducing 2-acetamido-2-deoxy-~-glucosyl or D-galactosyl residue, were not hydrolyzed. endo-p-D-Galactosidase, isolated from Escherichia f r e ~ n d i i , ’ ~ which ~ , ’ ~ ~also ~ hydrolyzes the main chain of keratan sulfate (see Section IX,4), released oligosaccharide chains from glycolipids having the general structure 81, hydrolyzing at the arrow. A similar activity

.1

P-r,-Gal-( 1 + 4(3))-P-~-GlcNAc-( I + 3 ) - P - ~ - G a l - ( l 4)-~-Glc--lipid 3 --f

t

1

a-L-FUC 81

from D. p n e u r n ~ n i u e ~could ~ ’ ~ not hydrolyze keratan sulfate. Methylation analysis of A’ glycolipid, one of the branched variants of blood-group A-active glycolipid, suggested368either structure 82 or 83, compounds susO-L-FUC 1

.1

2 a-D-GalNAc-(1 + 3 ) - P - ~ - G a l 1- (+ 4)-p-~-GlcNAc 1

J. 3 p-D-Gal-( 1 + 4)-D-Glc-Cer 6

~-L-Fuc 1

t

.1

2 1 a-D-GalNAc-(I+ 3 ) - P - ~ - G a l1(+ 4 ) - P - ~ - G l c N A c - ( l + 3 ) - P - ~ - G a l -1( 4)-/3-~-GlcNAc -f

t

82

(367a) M. N. Fukuda, Biochemistry, 24 (1984) 2154-2163. (368) M. N. Fukuda and S. Hakomori, 1. Bid. Chem., 257 (1982) 446-455.

242

BARRY V. McCLEARY AND NORMAN K. MATHESON LY-L-FUC 1

3.

2 a-D-GalNAc-(1 + 3 ) - p - ~ - G a l1- + ( 4)-p-~-GlcNAc 1

3.

3 P-D-Gal-(I + 4)-P-~-GlcNAc-(l+3)-P-~-Gal-( 1+4 ) - ~ - G b C e r t 6

~-L-Fuc 1

t

1

2 1 a-D-GalNAc-(1 + 3)-P-D-Gal-(1 + ~)-P-D-GIcNAc 83

ceptible to hydrolysis by E. freundii endo-P-D-galactosidase at the arrows. The release of ceramide monohexoside and an oligosaccharide having a d.p. of 9-11 on enzymic hydrolysis favored structure 83. The structure of the Ad oligosaccharide chain was determined by using fragmentation with endo-P-D-galactosidase. This enzyme hydrolyzed the glycolipid to ceramide monohexoside and three oligosaccharides, the smallest of which had a d.p. of 5, and, after chromatographic separation, the structure of the pentasaccharide was established, by methylation analysis and sequential glycosidase degradation, to be 84. The second oligosaccharide had a d.p. of 8, and partial glycosidase sequencing, combined with WL-FUC 1

3.

2 P-D-Gal-(1 + 4)-p-~-GlcNAc-( 1 + 3)-D-Gal 3

t

1 LY-D-GIcNAc 84

methylation analysis, indicated that it had structure 85. The third oligosaccharide had a d.p. of 13, and appeared to be composed of a structure derived from the other two oligosaccharides. To determine the structure of the whole side-chain, the glycolipid was incorporated into a liposome, when, on hydrolysis with endo-P-D-galactosidase,it was then susceptible to hydrolysis in only one position, being converted into pentasaccharide 84, and another fraction that was still blood-group A-active. When released from liposome, the latter fraction could then be hydrolyzed by endo-P-D-galactosidase to ceramide monohexoside and a large oligosaccharide. Methylation analysis, and hydrolysis by N-acetyl-B-D-glucosaminidase, of the latter indicated a structure which led to a formula for Ad glycolipid of 86, which would be hydrolyzed by endo-P-D-galactosidase at the arrows.

P-D-GIcNAc 1

1 3 p-D-Gal-( 1 + 4)-p-~-GlcNAc-(1 + 3)-~-Gal

a-L-Fuc 1

6

1

t

2 1 p-D-Gal-( 1 + 4)-p-~-GlcNAc 3

t

1 a-D-GalNAc 85

a-L-FUC 1

1 p-D-Gal-( 2 1 + 4)-p-~-GlcNAc-( 1 + 3)-p-~-Gal-( 1+ J. 4)-p-~-GlcNAc

3

1

t i

1

a-D-GalNAc

C~-L-FUC

5.

3 1 + 3)-/3-~-Gal-(1 + 4)-~-Glc-Cer p-D-Gal-( 1 + 4)-p-~-GlcNAo(

1

6

1

t

2 1 p-D-Gal-( 1 + 4)-p-~-GlcNAc 3

t

1 a-D-GalNAc 86

244

BARRY V. McCLEARY AND NORMAN K. MATHESON

The specificity of the biosynthetic glycosyltransferases for the sugar being substituted, the hydroxyl position on that sugar, and the anomeric linkage formed223strictly control the structures that are synthesized. There are further effects, apparently associated with conformational factors, caused by glycosyl units both adjacent to, and farther removed from, the glycosyl residue being substituted. GDP-D-mannosyltransferases specific for the formation of four different types of a-D-mannosyl bonds, (1 + 2), ( 1 + 6), and (1 -* 3) to D-mannosyl groups, and another to D-xylosyl groups, have been distinguished as contributing to the biosynthesis of the cell wall of Cryptococcus l a ~ r e n t i i . ~ ~ ~ UDP-D-galactosyltransferases, specific for p-( 1 + 4) and a-(1+ 3) linkages to D-GlcNac-R and p-D-Gal( 1 + ~)-D-G~cNAc-R, respectively, have been purified from calf Collagen was the only protein found to be an acceptor3” for UDP-D-glucose-procollagen glucosyltransferase (EC 2.4.1.66). The specificities of the glycosyltransferases involved in the biosynthesis of asparagine-linked glycoprotein chains are consistent with the structures of the molecules produced. Branching, or extensions to existing branches, may depend on remote sugars. Extension of the oligosaccharide core of L-asparagine-linked glycoproteins by the addition of 2-acetamido-2-deoxyD-glucosyl groups after processing is effected by at least four separate tran~ferases.~”The initial reaction is substitution of one of the a-(1 -+ 3)-linked D-mannosyl groups in 72 to give 73, which is then hydrolyzed to 74. This can then be substituted at the a-(1+6)-linked D-mannosyl group by a second transferase, to give 87. Then, further substitution can occur P-D-GIcNAc-( 1 + 2)-cr-~-Man 1

1 3 I P-D-Man-(1 ~ 4 ) - P - ~ - G l c N A ~ A s n 6 * I

t

1

P-D-GICNAC-( I +2)-a-~-Man 87

with 2-acetamido-2-deoxy-~-glucose in one of two ways, to give a bisecting 2-acetamido-2-deoxy-~-glucosyl antenna (88), or a substituent on the Dmannosyl residue linked a-(1 + 3) (89). Both of the enzymes that catalyze (369) (369a) (370) (371)

J. S. Schutzbach and H. Ankel, J. Biol. Chem., 246 (1971) 2187-2194. N. M. Blanken and D. H. Van den Eijnden, J. Biol. Chem., 260 (1985) 12,927-12,934. H. Anttinen, R. Myllyla, and K. I. Kivirikko, Biochem. J., 175 (1978) 737-742. H. Schachter, S. Narasimhan, P. Gleeson. and G . Vella, Can. 1. Biochern. Cell. Biol., 61 (1983) 1049-1066.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

245

p - ~ - G l c N A c - (+ l 2)-a-D-Man 1

5. 3 I p - ~ - G l c N A c -1(+ 4 ) - p - ~ - M a n -1+4)-p-~-GIcNAc-.f;Asn ( 6 I

t

1 p - ~ - G l c N A c - (+ l 2)-a-D-Man

88

P-D-GIcNAc 1

5. 4 p - ~ - G l c N A c - ( l +2 ) - a - ~ - M a n 1

5. 3 I p-D-Man-(1+4)-p-~-GlcNAc+ sn 6 2 7

t

1

p - ~ - G l c N A c -1(+ 2 ) - a - ~ - M a n 89

these reactions are sensitive to substitution of the two existing antennae by 2-acetamido-2-deoxy-~-glucosyl groups. For optimal reaction, substitution of both of these is required. Furthermore, if the bisecting 2-acetamido-2deoxy-D-glucosyl group is present, there can then be no further substitution of the a-(1+3)-linked D-mannosyl residue. However, if a second 2acetamido-2-deoxy-~-glucosylgroup is in position on the a-(1+ 3)-linked group can D-mannosyl group, the bisecting 2-acetamido-2-deoxy-~-glucosyl still be attached. A conformational basis for this pattern has been proPOSeda371a,371b

N-Acetyl-P-D-glucosaminide-(1 + 4)-P-~-galactosyltransferase,from bovine colostrum, first substituted trisaccharide 90 (which is the partial structure of the branching point in blood-group I, antigenic structures) at the p - ~ - G l c N A c -1(+ 3 ) - ~ - G a l 6

t

1 p-o-GalNAc 90

(371a) J.-R. Brisson and J. P. Carver, Can. J. Biochem. Cell. Biol., 61 (1983) 1067-1078. (371b) J. P. Carver, Biochem. SOC.Trans., 12 (1984) 517-519.

246

BARRY V. McCLEARY AND NORMAN K. MATHESON

p-( 1 + 6)-linked 2-acetamido-2-deoxy-~-glucosyl group, and this substitution, in turn, enhanced the acceptor properties of the p-(1+3)-linked 2-acetamido-2-deoxy-~-glucosyl group in synthesis of the bis-substituted o l i g o ~ a c c h a r i d e The . ~ ~ ~D-galactosylation of the two chains of N-linked, complex, biantennary glycopeptide to give complex chains by reaction with UDP-D-galactose : N-acetyl-( 1 + 4)-p-~-galactosyltransferaseproceeds in a sequential manner, with the (1 + 3)-branch being substituted preferentially to the (1+ 6 ) - b r a n ~ h . ~ ~ ~ " The oligosaccharide structures responsible for the ABO blood-group system have been related by using the appropriate glycosyltransferases for Blood-group interconversion, and the results have been H substance (91) was converted374into an A-active substance (92) with a-~-Fuc-(l+ 2 ) - P - ~ - G a l +R 91

1

a-D-GalNAc 92

UDP-N-acetyl-D-galactosamine: (~-~-fucosy~-(1,2)-~-galactose-a-3-Nacetyl-D-galactosaminyltransferase(EC 2.4.1.40) and into B active substance (93) with UDP-D-galactose : a-L-fucose-( 1,2)-~-galactose-a-3-~-galactosyltransferase (EC 2.4.1.37). ~ - L - F u c -1(+ 2 ) - P - ~ - G a l +R 3

t

1 a-D-Gal

93

The high specificity of the glycosyltransferases can provide information about linkage type. A rabbit-liver glycoprotein reacted with CMP-N-acetylneuraminate D-galactosylglycoproteintransferase (EC 2.4.99.1) and CMPN-acetylneuraminate. As the enzyme was known to react with a p-D-Gal(1 + 4)-~-GlcNAc-sequence, but not where the D-galactosyl residue is p-(l+3)-linked, the nature of the D-galactosyl linkage-type in the glycoprotein could be deduced.375 (372) W. M. Blanken, G. J. M. Hooghwinkel, and D. H. van den Eijnden, Eur. J. Biochem., 127 (1982) 547-552. (372a) M. R. PBquet, S. Narasimhan, H. Schachter, and M. A. Moscarello, J. Biol. Chem., 259 (1984) 4716-4721. (373) W. M. Watkins, Froc. R. SOC.Londpn, Ser. B, 202 (1978) 31-53. (374) H. Schenkel-Brunner and H. Tuppy, Eur. 1. Biochem., 17 (1970) 218-222. (375) J. C. Paulson, R. L. Hill, T. Tanabe, and G. Ashwell, J. Biol. C h e m , 252 (1977) 8624-8628.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

247

XII, MISCELLANEOUS The presence of a-(1 + 4)-linked di- and tri-saccharides of D-galactosyluronic acid in enzymic hydrolyzates of partially acid-hydrolyzed gum , certain structural tragacanth, as well as p-D-Xyl-( 1 + 3 ) - ~ - G a l Aindicated features of the polymer.376The isolation of this disaccharide containing a D-xylosyl group was informative, as the glycosidic bond of a D-xylosyl unit is much more labile to acid than that of a D-galactosyluronic unit, and, hence, this oligosaccharide could not be isolated from an acid hydrolyzate. On incubation with coffee-bean arabinogalactan, which has a (1 + 3 ) - p - ~ galactan backbone with (1 + 3)-a-~-arabinofuranosyland p-( 1 + 6)-linked D-galactosyl units (type I1 a r a b i n ~ g a l a c t a n ) , ~(.1' ~+~3)-p-~-galactanase (EC 3.2.1.90) released heterosaccharides that contained L-arabinose and ~ - g a l a c t o s eGum . ~ ~ ~arabic, containing 27% of L-arabinosyl residues, was After partial only very slowly hydrolyzed by a-~-arabinofuranosidase.'~ hydrolysis by acid, which lowered the L-arabinose content to 4%, it was partially hydrolyzed by p-~-galactosidase,'~'and 58% of the D-galactose was released. A D-arabino-D-galactan (D-arabinose :D-galactose 5 :2) from the cell walls of Mycobacterium spp.378 was hydrolyzed by an enzymic extract from a soil bacterium (Aureobacterium sp.), to give a-(1+ 5)-linked arabinosaccharides, a mixed fraction of higher oligosaccharides, and a high-molecular-weight fraction that had a D-arabinose to D-galactose ratio of 2: 5.4, suggesting that the D-arabinosyl units occur in side chains. Polysaccharide fractions isolated from the soluble fraction of disintegrated Mycobacterium cells contained D-arabinosyl and D-mannosyl units (1-2 : l).378a Enzymic degradation with the Aureobacterium preparation gave a mixture of D-arabino-oligosaccharides (apparent hydrolysis, 20-25% as arabinose), and with Arthrobacter exo-a-D-mannanase released D-mannose in 20-30% yield. The high-molecular-weight fraction remaining after reaction with the former enzyme contained D-mannose and D-arabinose in the ratio of 35 : 1. In conjunction with methylation analysis and Smith degradation, a highly branched structure was proposed. Structural aspects of L-arabino-D-galactan glycoprotein from radish leaves have been studied by using P-D-galactonase, p-D-galactosidase, and a - ~ arabinofuranosidase, in conjunction with methylation analysis.378b A glycuronan called protuberic acid, from the fungus Kobayasia nip ponica, consists of L-idosyluronic and D-glucosyluronic residues in the ratio (376) (377) (378) (378a) (378b)

G . 0. Aspinall and J. Baillie, J. Chem. Soc., (1963) 1702-1714. Y. Hashimoto, Nippon Nogei Kagaku Kaishi, 45 (1971) 147-150. A. Misaki. N . Seto, and I. Azurna, .I. Biochem. (Tokyo),76 (1974) 15-27. A. Misaki, I. Azuma, and Y. Yamamura, J. Biochem. (Tokyo), 82 (1977) 1759-1770. Y. Tsumuraya, Y. Hashimoto, S. Yamamoto, and N. Shibuya, Carbohydr. Rex, 134 (1984) 215-228.

248

BARRY V. McCLEARY AND NORMAN K. MATHESON

of 1 :2. Hydrolysis by an extracted, endogenous enzyme-preparation released 4-O-~-idosyluronic-P -D-glucuronic acid, consistent with a heteroThe structure of the a-D-mannan of the cell wall of the yeast Succharomyces cerevisiue has been studied by using an exo-a-D-mannanase (EC 3.2.1.77) that cleaves a-(1 + 2) and a-(1 + 3) linkages, and an endo-( 1+ 6 ) - a - ~ -

mannanase. The nature of its structure, as a (1 + 6 ) - a - ~ chain to which are attached branches of short chains of a-(1 + 2)- and a-(1 + 3)-linked Dmannosyl units, was indicated by the production of an essentially unbranched (1 + 6)-a-~-mannanon incubation with exo-a-~-rnannanase.~~' The structure near the region of linkage to protein was established from a study of the mannan having the generalized structure 94, from a mutant yeast, having an unbranched, outer chain.380The side-chain linkages are a-(1 + 2) and a-(1 +3). The structure followed from hydrolysis of the unbranched section with endo-( 1+ 6)-a-~-mannanase, splitting of 1 + 4)-~-GlcNAc- linkage with endo-N-acetyl-p-Dthe -p-~-GlcNAc( glucosaminidase (see Section X),and sequential hydrolysis of the remainder with exo-a-D-mannanase, a-D-mannosidase, and, finally, p-Dmannosidase, giving, as products, compounds 95 to 99. In combination with acetolysis of 96, and identification of the oligosaccharide fragments, the generalized structure 94 was proposed. The structures of the cell-wall D-mannans of several other yeasts have been investigated3" by use of this exo-a-D-mannanase. Five were degraded to the (1 + 6)-a-~-mannanchain. Those which contained p-linked Dmannosyl units or a-D-galactosyl groups in the side chains were not significantly hydrolyzed. However, removal, by partial hydrolysis with acid, of the a-D-galactosyl units from five galactomannans, and of p-linked D-mannosyl units from three other D-mannans, rendered these polysaccharides partially susceptible to hydrolysis by exo-a-D-mannanase, consistent with an a-linked-D-mannan structure. Invertase from a Sacchuromyces cerevisiue mutant could be separated into two fractions on the basis of solubility in ammonium sulfate.382The soluble fraction reacted with endo-( 1+ 6)-a-mannanase, when it became insoluble. The results suggested that the insoluble fraction contained only the highly branched, core section, but the soluble fraction also had the (1+6)a-D-mannan chain attached. (378c) H. Tsuchihashi, T. Yadomae, and T. Miyazaki, 1. Biochem. (Tokyo), 96 (1984) 17991805. (379) G. H. Jones and C. E. Ballou, J. Biol. Chem., 244 (1969) 1043-1051; 1052-1059. (380) T. Nakajima and C. E. Ballou, 1. Bid. Chem., 249 (1974) 7685-7694. (381) P. A. J. Gorin, J. F. T. Spencer, and D. E. Eveleigh, Curbohydr. Res., 11 (1969) 387-398. (382) L. Lehle, R. E. Cohen, and C. E. Ballou, J. Bid. Chem., 254 (1979) 12,209-12,218.

a-D-Man 1

1 a-D-Man

a-D-Man 1

1

s.

1 a-D-Man 1

1

a-D-Man 1

1

CY-D-M~ 1

a-D-Man 1

1

n

1

6 ) - a - ~ - M a n -1(+ 6 ) - a - ~ - M a n - ( l +6 ) - a - ~ - M a n - ( l +6 ) - p - ~ - M a n - ( l +4)-/3-D-GlcNAc-(l+ 4)-p-~-GlcNAc--Asn

1

(AN, 94

I

endo-(1 + 6)-a-~-mannanase

a-D-Man 1

'1 a-D-Man

a-D-Man 1

1

1

1

a-o-Man 1

a-D-Man 1

a-D-Man 1

a-D-Man 1

1

1

1

1

a-D-Man-( 1 + 6 ) - a - ~ - M a n -1(+ 6)-a-~-Man-(1 + 6 ) - a - ~ - M a n -1(+ 6)-p-~-Man-( 1 + 4)-j?-~-GlcNAc-(l+ 4)-P-~-GlcNAc--Asn1 95

I

endo- N-acetyl-B-o-glucosaminidase

a-D-Man 1

1 a-D-Man a-D-Man

a-D-Man

1

1

1

1

a-D-Man

1

1

1

1

a-D-Man 1

1

a-D-Man 1

3.

a-DMan-( 1 + 6 ) - a - ~ - M a n -1(+ 6)-a-D-Man-(1 + 6)-a-D-Man-(1 + 6 ) - p - ~ - M a n -1( + 4)-D-GlcNAc %

I

exo-a-D- mannanase

a-D-Man-( 1 + 6 ) - a - ~ - M a n -1( + 6)-a-D-Man-( 1 + 6)-a-D-Man-(1 + 6 ) - p - ~ - M a n -1( + 4)-~-GlcNAc 97

I I

a-D-mannosidase

@+-Man-( 1 + 4)-D-GlcNAc 9%

6-D-mannosidase

D-Man+ D-GlcNAc 99

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

25 1

The carrageenans occur as a family of polymers in which D-galactosyl residues, linked alternately a-(1 + 3)- and p-( 1+ 4)-, are modified to various degrees by the formation of anhydro rings and sulfation. Their structures383 and conformational aspects384have been reviewed. Enzyme preparations hydrolyzing either the K or A fractions have been obtained.385Carrageenans from different sources are hydrolyzed to differing degrees by the same enzyme.386 K-Carrageenanase (EC 3.2.1.83) depolymerized the polysaccharide from Hypnea musciformis more rapidly, and to a greater extent, than that from Gigartina acicularis, and polymers from agarophytes were not attacked. Hydrolysis of carra rage en an,^^' which is endo, released products that included a resistant fraction and a series of oligosaccharides (such as 100 and 101) based on the 4-0-sulfo-neocarrabiose structure, up to the a - D - h G a l - (1 + 3)-~-Ga14SO, 100

a-D-AnGal-(l+ 3)-@-~-Ga14SO,-(1+4 ) - a - D - h G a l - ( l + 3)-~-Ga14SO, 101

octasaccharide (AnGal represents a 3,6-anhydro-~-galactosyl residue). Diand tetra-saccharides made up >95% of the oligosaccharide fraction isolated. The resistant fraction (20%) contained more D-galactosyl residues and sulfate groups than did the original polymer. Alkali treatment released 19% of the sulfate, with equivalent formation of 3,6-anhydro-~-galactosyl residues. After modification, the material was then degraded to the extent of 75% by K-carrageenanase. Because this enzyme cannot hydrolyze a p-( 1+ 4) linkage when it is adjacent to disaccharide units that contain 6-O-SUlfO-D-galaCtOSyl, disulfo-D-galactosyl, or 3,6-anhydro-2-0-sulfo-~galactosyl residues, it was proposed that the K-carrageenan examined consisted of 80% of /3-(1+ 4)-linked 4-0-sulfo-neocarrabiosyl units, and, in the remainder, the anhydro-D-galactosyl units were replaced by sulfated D-galactosyl residues. An enzyme that is involved in the biosynthesis of carrageenan has been detected in seaweed extracts.3s7 It converts 6-O-sulfo-~-galactosyl into 3,6-anhydro-~-galactosyl units at the polymer level, and this structural change significantly affects gelling properties that depend on conformati~n.~'~ (383) T. J. Painter, in Ref. 78, pp. 195-285. (384) D. A. Rees, E. R. Morris, D. Thom, and J. K. Madden, in G. 0. Aspinall (Ed.), The Polysaccharides, Vol. 1, Academic Press, New York, 1983, pp. 195-290. (385) J. Weigl and W. Yaphe, Can. J. Microbiol., 12 (1966) 939-947. (386) W. Yaphe and B. Baxter, Appl. Microbiol., 3 (1955) 380-383. (387) C. J. Lawson and D. A. Rees, Nature (London), 227 (1970) 392-393.

252

BARRY V. McCLEARY A N D NORMAN K. MATHESON

A fraction having a higher molecular weight, from a marine tunicate (Styela plicata), was sulfated, and contained a high level of D-galactose, with a lesser proportion of D-glucose and some amino sugar. Incubation with P-D-galactosidase released a small proportion (-2% ) of D-galactose and this was increased to -5% if the d.p. was slightly lower. a-D-Galactosidase had no eff e ~ t . ~ ~ ~ ~ XiII. WD-GLUCANS 1. Amylose and Branched (1 +4)(1+ 6)-cu-~-Glucans

Subsequent to publication of an earlier article,’ several enzymic procedures have been applied to studies on aspects of the structures of (1 + 4)(1 + 6)-a-~-glucans. Partial hydrolysis with alpha amylase (EC 3.2.1.1), followed by gel chromatography, has been used to study aspects of the physical structures of the amylose complexes formed with such organic compounds as 1butanol, and of retrograded amylose. Differences were detected.3s7b A method of determination of the amylose content of starches debranched the whole starch with isoamylase (EC 3.2.1.68), separated chains having a high d.p. (>135) from the remainder by gel chromatography, and estimated the amount of these.3s8 Values of 29, 0.9, and 38 were found for wheat, waxy maize, and amylomaize starches. In a variation of this procedure, after debranching, the longer (1 + 4)-achains were separated, by centrifuging, as the 1-butanol complex.389The average chain-length of the remaining (soluble) chains could then be determined. There have been additional illustrations of the use of debranching enzym e in the ~ characterization ~ ~ ~ of ~ the type (glycogen, phytoglycogen, or amylopectin) of (1 + 4)( 1 + 6)-a-~-glucan.This has been determined from the distribution of maltodextrin chain-lengths found by gel chromatography after debranching with isoamylase, and also the extent of debranching by pullulanase. The storage polysaccharides from the blue-green alga Anacystis nidulan~”~and the protozoan Gregarina blaberae39’ have been shown to

R. M. Albano and P. A. S . Mourio, J. Biol. Chem., 261 (1986) 758-765. J.-L. Jane and J. F. Robyt, Carbohydr. Rex, 132 (1984) 105-118. J. G. Sargeant, Staerke, 34 (1982) 89-92. S. Hizukuri, T. Kaneko, and Y . Takeda, Biochim. Biophys. Acta, 760 (1983) 188-191. D. J. Manners, in R. D. Hill and L. Munck (Eds.), New Approaches to Research on Cereal Carbohydrates, Elsevier, Amsterdam, 1985, pp. 45-53. (390) M. Weber and G. Wober, Carbohydr. Res., 39 (1975) 295-302. (391) C. Mercier, J. SchrCvel, and J. R. Stark, Comp. Biochem. PhysioL, B, 44 (1973) 1001-1010.

(387a) (387b) (388) (389) (389a)

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

253

have a phytoglycogen-like structure. Differences have also been detected3’la in the chain-length distributions of amylopectins from various starches. A re-examination of the determination of the A: B chain ratio of waxymaize amylopectin, by comparing the reducing sugar released from the @-limitdextrin3’* by isoamylase and by isoamylase plus pullulanase, showed that the value obtained is sensitive to the level of i~oamylase.~’~ A-Chains are defined as those that do not have other a-(1 + 4)-linked chains joined to them by way of a 6-hydroxyl group, and B-chains as those that do. The determination of this ratio depends on the ability of pullulanase to remove both maltosyl and maltotriosyl stubs from the @-limitdextrin, but of isoamylase to remove only the maltotriosyl units. However, isoamylase has been found to release maltosyl branches very slowly, and also to release maltotriosyl units much more slowly than maltosaccharide chains of higher d.p.3943395; hence, the amount of isoamylase added is critical. Also, the calculation involves a subtraction of two absorbance values, and quite small differences in either of these two readings can lead to large differences Re-estimation of the A: B chain-ratio in the calculated A: B ~hain-ratio.~’~”’~ with a higher level of isoamylase gave a value for waxy-maize amylopectin of slightly greater than one, similar to that obtained previously by debranching @-limit dextrins with pullulanase, and estimating maltose and maltotriose after paper-chromatographic ~eparation.~”When waxy-maize amylopectin was partly debranched with pullulanase, which preferentially removes outer chains, and the &limit dextrin was prepared, debranching of this by isoamylase and by pullulanase plus isoamylase, and comparison of the reducing sugar respectively released, gave an A: B chain ratio somewhat lower than for the original starch.393This is consistent with waxy-maize amylopectin’s having the more asymmetrical, cluster type of s t r u ~ t u r e , ~ ~ ~ * ~ ~ and is in agreement with physicochemical data and the bimodal distribution of chain lengths obtained on debranching. To account for the lowered A: B chain ratio of partly debranched m o 1 e ~ ~ lthe e ~ cluster , ~ model ~ ~ ~ was ~ ~ ~ ~ modified, so that B chains towards the outside of individual clusters carry more than one A-chain. S. Hizukuri, Carbohydr. Res., 141 (1985) 295-306. J. J. Marshall and W. J. Whelan, Arch Biochern. Biophys., 161 (1974) 234-238. D. J. Manners and N . K. Matheson, Carbohydr. Res., 90 (1981) 99-110. K. Kainuma, S. Kobayashi, and T. Harada, Carbohydr. Res., 61 (1978) 345-357. R. M. Evans, D. J. Manners, and J. R. Stark, Carbohydr. Res., 76 (1979) 203-213. W. A. Altwell, G. A. Milliken, and R. C. Hoseney, Sraerke, 32 (1980) 362-364. G . N . Bathgate and D. J. Manners, Biochem. J., 101 (1966) 3c-5c. M. Yamaguchi, K. Kainuma, and D. French, J. Ulirastruct. Rex, 69 (1979) 249-261. J. P. Robin, C. Mercier, R. Charbonniere, and A. Guilbot, Cereal Chem., 51 (1974) 389-406. (399a) D. J. Manners, Cereal Foods World, 30 (1985) 461-467.

(391a) (392) (393) (394) (395) (396) (397) (398) (399)

BARRY V. McCLEARY AND NORMAN K. MATHESON

254

When treated with a high level of pullulanase, rabbit-liver and oyster glycogens were partly debranched (-30% ). Gel chromatography indicated that outer chains had been preferentially r e m o ~ e d . ~The ~ ~released . ~ ~ ' chains had an average d.p. of 7.5 and 8.0, respectively. When the residual polysaccharides were completely debranched by isoamylase, the average d.p. values were 21 and 16, indicating that the exterior chains of these two glycogens are shorter than the interior chains. Isoamylase debranches glycogen by the preferential removal of exterior chain^,"^'*^^^ giving maltodextrin chains of increasing average d.p. as the degree of debranching increases. When the &limit dextrin of waxy-maize starch reacted with exomaltohexahydrolase (EC 3.2.1.98), which can by-pass some a-(1 + 6) linkages, the branched a-D-gluco-oligosaccharidesexpected, namely, 64-a-maltosylmaltopentaose (102), 63-a-maltotriosylmaltotetraose (103), 64-a-maltosylmaltohexaose (104), and 63-a-maltotriosylmaltopentaose G-G-G-G-G

t

G-G-G-G

t

G

G

G

G

I

102

I I

G 103

G-G-G-G-G-G

t

G-G-G-G-G

t

G

G

G

G

I

104

I

I

G 105

(105), were obtained with either one or two D-glucosyl units on the nonreducing side of the branch point403(- represents an a-(1 + 4 ) bond; +, an a-(1 + 6) bond; and G, a D-glucosyl unit, the reducing-end unit being italicized). These structures were determined by hydrolysis with pullulanase and alpha amylase. However, evidence was also obtained for the presence of 63-a-rnaltotriosylrnaltotriose(106) and 64-a-maltosylrnaltotetraose(107), H. Akai, K. Yokobayashi, A. Misaki, and T. Harada, Biochim. Biophys. Acta, 237 (1971) 422-429.

T. Harada, A. Misaki, H. Akai, K. Yokobayashi, and K. Sugimoto, Biochim. Biophys. Acta, 268 (1972) 497-505. T. N. Palmer, L. E. Macaskie, and K. K. Grewal, Carbohydr. Res., 115 (1983) 139-150. K. Kainuma, K. Wako, S. Kobayashi, A. Nogami, and S. Suzuki, Biochim. Biophys. A d a , 410 (1975) 333-346.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE G-G-G

G-G-G-G

G

G I

t

I

G

255

t

G

I

G

107 106

suggesting a possibility of unexpected structural features at the nonreducing end of some chains of the P-limit dextrin. Potato amylopectin contains phosphoric ester groups, and the disposition of these on the molecule has been established by enzymic degradation. After debranching with isoamylase, substituted chains were separated from the neutral chains on an ion-exchange The average d.p. of the phosphoric esterified chains was larger than that of the total chains, and the extent of @-amylolysisof the former suggested a statistical location of the phosphoric ester groups towards the middle of the chains. The &limit dextrin of the original amylopectin was then debranched with isoamylase, which has only a very low rate of action on maltosyl stubs, and the phosphoric esterified chains were collected. Further debranching of these with pullulanase, which removed maltosyl units, gave the molar ratio of branched to unbranched chains of 44 : 56. Half of the original, unbranched, phosphoric esterified chains would have been derived from B-chains substituted with maltotriose in the @-limit dextrin, which would have been. removed by isoamylase. Thus, at least 88% of the phosphoric ester groups are located on B-chains, with 12% or less on A-chains. From treatment of the phosphoric esterified fraction with beta amylase (EC 3.2.1.2), before and after pullulanase reaction, it was concluded that about one third of the phosphoric ester groups are on the inner section of the B-chains. There have been several examinations of the structure of Nageli dextrin,4°5-407which is prepared b y the prolonged action of acid on granular there was separated from waxy maize a branched starch. In one fraction that was resistant to pullulanase action. As this fraction contained some molecules having two branch points that were in close proximity, it was considered that this may have hindered hydrolysis, and that it could be of relevance to studies on the structure of the original amylopectin. In view of the close association of peptide with acid-insoluble mammalian-muscle glycogen that had been subjected to proteolysis, the possibil(404) Y. Takeda and S. Hizukuri, Carbohydr. Res., 102 (1982) 321-327. (405) R. S. Hall and D. J. Manners, Carbohydr. Res., 83 (1980) 93-101. (406) T. Watanabe, Y. Akiyama, A. Matsumoto, and K. Matsuda, Curbohydr. Res., 112 (1983) 171-177. (407) K. Umeki and K. Kainuma, Curbohydr. Rex, 96 (1981) 143-159.

256

BARRY V. McCLEARY AND NORMAN K. MATHESON

ity of a protein-carbohydrate linkage in this molecule was suggested:" Using proteolytic and amylolytic degradation, evidence has been found that the linkage is a-1- from D-glucosyl to the phenolic group of tyrosine.408a*408b A protein fraction (called glycogenin) has been prepared, and D-glucosylation of this has been demonstrated by using UDP-~-['~C]glucose and a rabbit-muscle The biosynthesis of amylopectin, which requires (1 -* 4)-a-~-glucan branching enzyme (EC 2.4.1.18), involves inter-chain transfer, although some intra-chain reaction could not be excluded.4w The minimum chainlength of a-(1 + 4)-linked substrate in this transglycosylation reaction was at least 40 D-glucosyl units, and it was proposed that this could be due to the enzyme's interacting with a maltosaccharide chain only when it was large enough to adopt a stable, helical conformation, or alternatively, a double-helical conformation.410The minimum length of chain needed for these conformations to exist is then relevant to the average chain-length in the amylopectin molecule. Although the branching enzyme that forms phytoglycogen has also been found in maize varieties that form normal and mutant starches, only the variety having the sugary gene forms phytoglycogen. An explanation of this behavior, has been provided by the finding that only granules from sugary maize are susceptible to attack by this enzyrne:'l Distributions of the multiple forms of branching enzymes present in high-amylose, differ from those in normal, starch 2. Pullulan

Pullulan is hydrolyzed by pullulanase at the a-(1 + 6) bonds, producing maltotriose plus some maltotetraose. Salivary alpha amylase cleaves at the maltotetraosyl units, when the a-(1 + 4) linkage next to the a'( 1 + 6) bond and towards the reducing end of the maltotetraose unit is split (dotted arrow marked A in 108). The size of units released by alpha amylase, as judged (408) N. A. Butler, E. Y. C. Lee, and W. J. Whelan, Carbohydr. Res., 55 (1977)73-82. I. R. Rodriguezand W. J. Whelan, Biochem. Biophys. Res. Commun., 132 (1985)829-836. (408b) M. A. Aon and J. A. Curtino, Biochem. J., 229 (1985)269-272. (408c) I. R. Rodriguez, J. S. Tandecan, B. R. Kirkman, and W. J. Whelan, Miami Winter Symp. (1986)96-99. (409) D. Borovsky, E. E. Smith, and W. J. Whelan, Eur. J. Biochem., 62 (1976)307-312. (410) D. Borovsky, E. E. Smith, W. J. Whelan, D. French, and S. Kikumoto, Arch. Biochem. Biophys., 198 (1979)627-631. (411) C. D. Boyer, E. K. G . Simpson, and P. A. Damewood, Sraerke, 34 (1982)81-85. (412) C. D. Boyer and J. Preiss, Plant Physiol, 67 (1981)1141-1145. (413) T. Baba, Y. Arai, T. Ono, A. Munakata, H. Yamaguchi, and T. Itoh, Carbohydr. Res., 107 (1982)215-230.

(408a)

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

257

by gel chromatography, has revealed that the distribution of maltotetraosyl units is not reg~lar.4'~

t

Two other enzymes active on pullulan have been isolated; these are isopullulanase (EC 3.2.1.57) from Aspergillus niger4" and Arthrobacter globiformis T6 (Ref. 416), and alpha amylase from Thermoactinornyces attacks a-(1 + 4) adjacent to a-(1 + 6) linkages ~ u l g a r i s . 4Isopullulanase ~~ and towards the nonreducing end of the repeating unit (at dotted arrows marked B in IOS), releasing a large proportion of isopanose (109) and small proportions of tetrasaccharide. T. vulgaris alpha amylase released mainly panose (110)(96.5%), with small proportions of maltose (1.5%), glucose

109

110

(0.7% ), isomaltose (0.3% ), and higher oligosaccharides (0.4% ). Cleavage at the a-(1+ 4) linkages next to the a-(1 + 6)bond and towards the reducing ends of both maltotriosyl and maltotetraosyl units (dotted arrows marked C in 108). Some a-(1 + 6) bonds in partially hydrolyzed pullulan may also be atta~ked.~" The products of hydrolysis by isopullulanase and T. vulgaris alpha amylase are in agreement with the structure previously established for pullulan. G . Carolan, B. J. Catley, and F. J. McDougal, Carbohydr. Rex, 114 (1983) 237-243. Y. Sakano, M. Higuchi, andT. Kobayashi, Arch. Eiochem. Eiophys., 153 (1972) 180-187. M. Tago, M. Aoji, Y. Sakano, T. Kobayashi, and T. Sawai, Agric. Eiol Chem, 41 (1977) 909-910. M. Shimizu, M. Kanno, M. Tamura, and M. Suekane, Agric. Eiol. Chem., 42 (1978) 1681-1688. Y. Sakano, S. Hiraiwa, J. Fukushima, and T. Kobayashi, Agric. EioL Chem., 46 (1982) 1121-1 129.

BARRY V. McCLEARY AND NORMAN K. MATHESON

258

3. Dextrans Enzymic hydrolysis of dextrans has provided information both on the linkage types and the disposition of different linkages as established by methylation. Since the publication of the earlier article,' enzymic studies on Leuconostoc dextrans have continued, and investigations of Streptococcus a-D-glucans have shown that there are significant, structural differences between some of these and those of Leuconostoc spp. Reviews on dextrans have The enzymes mainly employed in studies on dextran structure have endo-( 1 + been endo-( 1 + 6)-a-~-glucanase(dextranase, EC 3.2.1.11),"21*422 3)-cu-~-glucanase (EC 3.2.1.59),"23 exo-( 1 + 6)-a-~-glucohydrolase ( g l u c o d e ~ t r a n a s e ~dextrangluc~sidase~~~; ~~*~~~; EC 3.2.1.70), and exo-isoEC 3.2.1.94). The two maltohydrolase (isornaltode~tranase,"~~-~~~ endo-a-D-glucanases detect chains of either sequential a-(1+ 6) or a-(1 + 3) linkages, and any resulting, branched oligosaccharides provide information about branching in the polysaccharide: high levels of branching restrict hydrolysis. The exo-enzymes provide information about sequences from the nonreducing ends of chains. Other enzymes that have been used include exo-hydrolases for a-(1 + 2) (Ref. 430) and a-(1 + 3) linkages!31 The composition of some dextrans as having almost entirely a-(1 + 6) bonds, for example, dextran (T-2000) and B-512 (Refs. 421 and 422), is shown by their essentially total-possible hydrolysis to a limit oligosaccharide mixture by endo-( 1 + 6)-a-~-glucanase.Dextran B-1355 (L), having 88% of a-(1 + 6) linkages, gave 84% of the hydrolysis p~ssible.~"The degree of hydrolysis (57-16% of isomaltose equivalents) of seven dextrans having mainly a-(1 + 6) bonds was directly correlated with the fraction of a-(1 + 6) linkages.422 Fractionation, and identification, of the oligosaccharides released have given data about branching. Hydrolysis of Leuconostoc B(419) (420) (421) (422)

R. L. Sidebotham, Adu. Carbohydr. Chem. Biochem., 32 (1974) 371-444. G. J. Walker, in Ref. 173, pp. 75-126. A. Pulkownik and G. J. Walker, Carbohydr. Res., 54 (1977) 237-251. A. L. Minakova and M. E. heobrazhenskaya, Biochemistry ( U S S R ) , 42 (1977) 1264-

(423) (424) (425) (426) (427) (428) (429)

G. J. Walker and M. D. Hare, Carbohydr. Res., 58 (1977) 415-432. T. Ohya, T. Sawai, S. Uemura, and K. Abe, Agric. Bid. Chem., 42 (1978) 571-577. T. Sawai, T. Yamaki, and T. Ohya, Agric. Biol. Chem., 40 (1976) 1293-1299. G. J. Walker and A. Pulkownik, Carbohydr. Res., 36 (1974) 53-66. T. Sawai, T. Tohyama, and T. Natsume, Carbohydr. Res., 66 (1978) 195-205. A. Misaki, M. Toni, T. Sawai, and 1. J. Goldstein, Carbohydr. Res., 84 (1980) 273-285. T. Sawai, S . Ohara, Y. Ichimi, S. Okaji, K. Hisada, and N. Fukaya, Carbohydr. Res.,

1273.

89 (1981) 289-299. (430) Y. Mitsuishi, M. Kobayashi, and K. Matsuda, Carbohydr. Res., 83 (1980) 303-313. (431) G. J. Walker and M. D. Hare, Carbohydr. Res., 77 (1979) 289-292.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

259

5 12( F) and Bacillus dextrans with Penicillium and Streptococcus sp. ~ ~ ~ ~ ~ ~33-a-~-glucosylisomaItosac~~ endo-( 1 + 6 ) - a - ~ - g l u c a n a s e sproduced charides and 33-a-isomaltosylisomaltosaccharides(110a and 1lob, respectively, where + is a (1 + 3)-a linkage and - is (1 + 6)-a linkages ( n = 0 to 4). G

G-G

1

1

( G ) -G-G-G

( G ) -G-G-G

110n

110b

The proportion of products having one-unit side-chains indicated that at least half of the (1 + 3) linkages were to single D-glucosyl groups. The tetrasaccharide products422of dextran LU-122, which has 68% of a-(1 + 6) and 32% of a-(1 + 2) bonds, was examined b y using an a-D-glucosidase lacking an ability to hydrolyze 2-linked a-D-glucosyl groups. This showed that 25% of the tetrasaccharide fraction was isomaltotetraose, and 75% was branched: the structure of the latter was established by methylation (111). analysis as 22-a-~-g~ucosyIisoma~totriose n-D-GlC 1

1 L

a-D-GlC-( 1 + 6)-a-D-GIC-(1 + 6)-D-Gk

111

The amount of hydrolysis by endo-( 1 + 6)-a-~-glucanase,in conjunction with the percentage of (1 + 6) linkages as determined by methylation analysis, indicates the degree of consecutiveness of these linkages. Endo-( 1+ 3)-a-~-glucanasecan be used in the same way to determine the disposition of a-(1 + 3) bonds. The resistance to hydrolysis of B-l355(S) dextran by latter enzyme,423*428*432*433 despite the presence of 40% of (1 + 3) linkages, combined with the low extent of hydrolysis by endo-( 1 + ~ ) - c Y - D g l ~ c a n a s e , 4is~ ~consistent with a structure containing alternating (1 + 3) and (1 + 6) bonds, as assigned from chemical evidence. On the other hand, dextran B-l355(L) was extensively hydrolyzed by endo-( 1 + 6 ) - a - ~ g 1 u c a n a s e , 4 ~ and ~ * ~ an ~ ~ endo-( 1 + 3)-a-~-glucanasegave no hydrolysis. These results characterized this fraction as a dextran having a-(1 + 6) main chains and a-(1 + 3) branches. Dextranglucosidase hydrolyzes only nonreducing, terminal a-(1 + 6) linked D-glucosyl units in an exo manner, including those adjacent to a (431a) C. Taylor, N. W. H. Cheetham, and G . J. Walker, Carbohydr. Res., 137 (1985) 1-12. (432) M. D. Hare, S. Svensson, a9d G. J. Walker, Carbohydr. Res., 66 (1978) 245-264. (433) G . L. C8tt and J. F. Robyt, Carbohydr. Res., 101 (1982) 57-74.

260

BARRY V. McCLEARY A N D NORMAN K. MATHESON

non-U-( 1 + 6) linkage, provided that a branch point is not involved. a-(1 + 6) Linkages at branch points are not hydrolyzed, and non-a-( 1 + 6) bonds cannot be bypassed. In a dextran, it does not release D-glucose from two-unit ~ i d e - c h a i n s The . ~ ~ ~extent of reaction with five dextrans was inversely proportional to the percentage of non-a-( 1 + 6) linkages.434Synthetic dextran having 2 % of non-( 1 + 6) bonds gave 35% conversion into D-glucose equivalents, but a B-1335 dextran having 35% of non-(1 + 6) bonds released insignificant amounts, and dextran B-1415, having 14% of a-(1 + 4 ) bonds gave -17% hydrolysis. The degree of hydrolysis (25%) of B-512(F) dextran, which has 5% of branch linkages, was explained by proposing that side chains that are longer than two D-glucosyl units have an average chain-length of 33. Data on side-chain length, obtained chemically, showed that 40% of the chains contained one D-glucosyl unit, and 45% had two, and that 15% were longer than two. Another possibility considered was the existence of a range of polymeric molecules differing in the extent of branching. Hydrolysis by isomaltodextranase of Leuconostoc dextrans having a-( 1 + 6) linkage contents of 57 to 96% an approximate, direct correlation of these percentages with the degree of hydrolysis. For the same polysaccharide, it was generally higher than with glucodextranase, as isomaltodextranase can bypass some non-a-( 1 + 6) linkages. Soluble B-1355 was hydrolyzed extensively by i s o r n a l t o d e x t r a n a ~ e , releasing ~ ~ ~ - ~ ~ ~isomalt(112) in the ratio428of 5.6: 1, consistent ose and 32-a-~-glucosylisomaltose a-D-GlC 1

.1 3 a-D-GlC-(1+ 6)-D-Glc 112

with the alternating (1 + 6)( 1 + 3) structure. The limit dextran remaining was shown by methylation analysis to be highly branched, and a model was proposed of ramified chains of alternating a-(1 + 6) and a-(1 + 3) bonds, with linkage between chains to 0 - 3 or 0 - 6 . Incubation of this dextran with exo-( 1 + 3)-a-~-glucanasereleased 1% of ~-glucose:~' The product was then hydrolyzed by isomaltodextranase, to give mainly isomaltose, with much less of 112 than from the untreated glucan, indicating that the trisaccharide released from the untreated dextran was mainly derived from the nonreducing ends of chains. The extent of hydrolysis by the two enzymes together was no greater than with isomaltodextranase alone (61 Yo). An enzyme that specifically removed a-(1 + 2)-linked D-glucosyl branches ~ ' a-(1 + 2) linkage has been isolated from a Flavobacteriurn ~ p p . ~The

-

(434) G . J. Walker and A. Pulkownik, Carbohydr. Res., 29 (1973) 1-14.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

261

contents, as determined by methylation analysis, for three Leuconostoc dextrans [B-l298(S),B-l299(S), and B-l397(S)]were generally proportional to the extent of enzymic hydrolysis. Kojibiose and other gluco-disaccharides were not hydrolyzed, suggesting that the enzyme may need to recognize the a-(1 + 6) chain. Partial hydrolysis of L. mesenteroides B-l299(S) d e ~ t r a n ~ ~ ~ ~ with this (1 + 2)-hydrolase released 3% of D-glucose. Treatment of the nondialyzable material with (1 + 6)-endo-dextranase gave a degree of hydrolysis of lo%, and fractionation of the oligosaccharide mixture gave three branched products (A, B, and C). Amyloglucosidase converted B into D-glucose and A. The (1 + 2)-hydrolase converted A into D-glucose and isomaltotriose, B into D-glucose and isomaltotetraose, and C initially into D-glucose and B. The structures were assigned as A, 2 3 - ~ - ~ - g I ~ ~ ~ s y l i s o m a l totriose (112a); B, 23-a-~-glucosylisomaltotetraose(112b); and C, 23,24-dia-D-glucosylisomaltotetraose (112c). Isolation of the last compound provided evidence for the occurrence of adjacent m ~ -1+= ( 2) branch-points. a-D-GIC 1

.1

2 a - ~ - G l c -1(+ 6)-a-D-Glc-(l+ 6)-D-GlC 112a

LY-D-GIC 1

.1

2 a-D-GlC-(1 + 6)-a-D-Glc-(1 + 6)-a-D-GlC-(1 -D 6)-D-GIC

112b ff-D-GlC 1

a-D-GlC 1

.1

.1

2 2 a-D-GlC-(1 -+ 6)-a-D-GlC-(1 -i 6)-a-D-GlC-(1 + 6)-D-Gk

ll2c

On exhaustive hydrolysis of this d e ~ t r a [B-l299(S)] n ~ ~ ~ ~ with the (1+= 2)-hydrolase, the degree of hydrolysis was 31.5%, indicating that about one third of the D-glucosyl units are single a - ~ 1-+=( 2)-linked branches. Treatment of the original dextran with Arthrobacter glucodextranase gave 3.0% hydrolysis, and a combination of both enzymes, 74%. After prior (1 + 2)-hydrolase reaction, the degree of hydrolysis by glucodextranase was (434a) Y. Mitsuishi, M. Kobayashi, and K. Matsuda, Curbohydr. Res., 127 (1984) 331-337. (434b) M. Kobayashi, Y. Mitsuishi, S. Takagi, and K. Matsuda, Curbohydr. Res., 127 (1984) 305-317.

262

BARRY V. McCLEARY A N D NORMAN K. MATHESON

greatly increased over the value obtained without prior (1 + 2)-hydrolase action, consistent with (1 + 6 ) - a - ~chains being substituted with single a-~1+ ( 2)-linked D-glucosyl units. Further treatment with (1 + 2)-hydrolase released more D-glucose, and then glucodextranase released another quantity, until the fourth cycle, when no more appeared. A resistant core remained. This was considered to result from occasional a - ~ - ( l + 2) linkages ~ and (1 + 2)-branches further substituted by a a-(1+ 3) in (1 + 6 ) - a - chains, linkage, both of which would stop action by either enzyme. In conjunction with methylation analysis and 13C-n.m.r. spectroscopy, a generalized model ( 6)-linked Dstructure was proposed. This consists of chains of a - ~ -1+ glucosyl units containing occasional (1 + 2) links, joined together by a-D(1 + 2) linkages. Many of the (1 + 6)-linked units have single a - ~ -1(+ 2)linked D-glucosyl groups attached, with a few of these having an additional a - ~ -1(+ 3)-linked D-glucosyl group attached (112d). Similar studies have been made434cwith dextran B-l298(S). exo-( 1+ 4)-a-D-GlUCanaSe (amyloglucosidase) hydrolyzes a-(1+ 6) linkages in the vicinity of a-(1 + 4) bonds. The ability to hydrolyze these linkages in relation to the location of a-(1+ 4) bonds varies with the source of the enzyme.435Extents of hydrolysis of dextran of up to 33% have been reported.436 A number of dextrans have been examined with a pig-spleen a-Dg l u c ~ s i d a s eThis . ~ ~ ~enzyme could differentiate exterior a-(1 + 2) linkages, because it readily hydrolyzed a-(1+ 3) and a-(1+ 6), but more slowly split a-(1+ 2), linkages in the glucans. Soluble dextran, synthesized by one of two D-glucosyltransferases isolated from S. mutans, contained 32% of a-(1 + 3) branch linkages. It was very ~~ after slightly hydrolyzed (<1%) by exo-( 1 + 3 ) - a - ~ - g l u c a n a s e ?However, this treatment, it underwent 16% hydrolysis by isomaltodextranase, whereas prior to treatment it was resistant. It was concluded that exo-(1 + 3 ) - a - ~ glucanase removed one or more a-(1 + 3)-linked D-glucosyl units from nonreducing termini, allowing the isomaltodextranase to recognize the a- ( 1+ 6)-linked main-chain. Hydrolysis by endo-(1+ 3)-a-~-glucanasehas demonstrated the presence of consecutive a-(1+ 3) linkages in a number of streptococcal a-D-glucans (mutans). Insoluble a-D-glucans produced by S. mutans OMZ 176 and OMZ 65 were hydrolyzed by endo-( 1+ 3)-a-~-glucanase, whereas the soluble polysaccharides from the same sources, and dextran T-2000, were (434c) (435) (436) (437)

M. Kobayashi, S. Takagi, and K. Matsuda, Agric. Biol. Chem., 49 (1985) 773-777. B. V. McCleary and M. A. Anderson, Carbohydr. Res., 86 (1980) 77-96. M. M. McCabe and E. E. Smith, Carbohydr. Res., 63 (1978) 223-239. M. E. Preobrazhenskaya, A. L. Minakova, and E. L. Rosenfeld, Carbohydr. Rer, 38 (1974) 267-277.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE GcG I G I GcG I G

G

GcG

G

4

G I

4

I I

I I G I

G I G

I GcG I G I G G-G

I

G

I

G I G-G I G-tG I G G-A

I

G

I

G-tG

1

-

(1+6)-~r

+

(1+2)-(r

_--

G

I G

(1+3)-~

I I G I

GcG

I

I

G

GcG

1 1 G+G I G I

I I G G G G G 1 .1 1J. I GcGcG-G-G-G-G-G-G-G-G-G I

b1

G

GcG

G

GtG---G

I I

G+G

G G+G-G-G-G

I

GcG

I

GcG

F;?

1

G

I

GcG I G+G 1

I

263

&

F; +I

c,

I GcG I G

ll2d

not.438Enzymic degradation of the insoluble fractions indicated that they had a structure made up of a main chain of a-(1 + 3) linkages and a-(1+ 6) branch points, in agreement with chemical evidence. Conversely, the soluble polysaccharides were extensively hydrolyzed by endo-( 1+ 6)-a-~-glucanase, and not attacked by endo-( 1 + 3)-a-~-glucanase,consistent with these having a-(la+6) chains with a-(1 + 3) branches. An examination of insoluble cu-D-glucans produced by strains of Strep tococcus mutans, S. salivarius, and S. sanguis, and D-glucosyltransferases (438) S. Ebisu, K. Kato, S. Kotani, and A. Misaki, J. Bacreriol., 124 (1975) 1489-1501.

264

BARRY V. McCLEARY A N D NORMAN K. MATHESON

from some of showed that high proportions of the (1 + 3) linkages established by methylation analysis were susceptible to hydrolysis with endo-( 1 + 3)-a-~-glucanase,in agreement with the a-(1 + 3) linkages being arranged in sequence. The extent of hydrolysis by endo-( 1 -* 3)-a-~-glucanase of the insoluble glucan synthesized by one glucosyltransferase from S. mutans OMZ 176 was 84%, and the methylation analysis mixture contained 88% of 2,4,6-tri-O-methyl-~-glucose.The insoluble glucan from s. sanguis 804, which contained fewer ( 1 + 3) bonds (21%), was 17% hydrolyzed. a-D-Glucan fractions from S. mutans PK-1 were also partly hydrolyzed by endo-( 1 -* 3 ) - a - ~ - g l u c a n a s e . ~ ~ ~ On incubation with endo-( 1 + 6)-a-~-glucanaseof the insoluble Strep tococcal polysaccharides, a high proportion of the (1 + 6) linkages detected by methylation analysis was hydrolyzed, indicating that the a-(1 + 6) linkages were consecutive as well. Chromatographic examination of the enzymic-hydrolysis products gave substituted isomaltosaccharides, showing that branching occurred on the a-(1 + 6) chains. None of the insoluble streptococcal a-D-glucans were susceptible to any significant extent to hydrolysis by dextranglucosidase. Isomaltohydrolase, which can bypass, or hydrolyze, certain non-a-( 1 + 6) linkages, gave432only a slight reaction (1-9%). Generalized structures for the polymers synthesized by Streptococcus spp. and by isolated D-glucosyltransferases have been proposed from this enzymic evidence, in conjunction with chemical information. They were depicted (113) as having a-(1-*6)- and a-(1+3)-linked

G

113

chains interlinked by bonds to 0 - 3 or 0-6, respectively. Single, 3-linked a-D-glucosyl residues are attached to the a-(1 + 6) chains [- represents an a-(1 + 6) and + an a-(1 + 3) linkage].

(439) K. Irnai, M. Kobayashi, and K. Matsuda, Agric. Biol. Chem., 41 (1977) 1889-1895.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

265

4. (1 + 3),(1+ 4)-cu-~-Glucans

On exhaustive hydrolysis with mycodextranase (EC 3.2.1.61), nigeran [agave 3-5% of trisaccharide, in addition to nigerose and d i ( n i g e r ~ s e ) ~ ~' ~ - G l c -1(+ 3) - a - ~-Glc-( 1 + 4 )-a-~ -Glc-( 1+ 3)-~-Glc]. The trisaccharide fraction was a mixture of three parts of ~-D-GIc-( 1 + 3)-a-D-GlC-(1 + 4)-D-GlC and one part of a - ~ - G l c -1(+ 4)-a-D-Gk-(I + 3 ) - ~ - G l cindicating , that 25% of the chains had a-(1+ 4) linkages at the nonreducing terminus, and that the enzyme preferentially hydrolyzes a-(1 + 4) D-glucosidic linkages adjacent to D-glucosyl residues linked &-( 1+ 3). A comparison of the oligosaccharide fragments from mycodextranase hydrolysis of sodium borotritide end-reduced, as well as uniformly labelled, nigeran, revealed that about half of the chains had a-(1 + 4 ) linkages at the reducing end. On hydrolysis by mycodextranase, isolichenan, which has an 11 :9 ratio of a-(1+ 4) to a-(1 + 3) linkages, released nigerose and a - ~ - G l c -1(+ 3)- a - ~- G l c -1(+ 3 ) - ~ - G l cbut , no di(nigerose), and it was concluded that 1+ 4 ) - a - ~ - G l c the polymer lacks segments of - a - ~ - G l c -1(+ 3)-&-~-Glc-( (1 + 3)-a-D-GlC-(1 + 4 ) - a - ~ - G l c 1- (+ 3 ) - a - ~ - G l c -1(+ and nonreducing, terminal segments of ~ -D-GIC-( 1 + 3 )-a-~ -Glc -( 1 + 4 ) - a - ~ - G k -1(+ 3)-a-D1+ ,and therefore contains a majority of one, or two, Glc-( 1+ 4) - a - ~-Glc-( consecutive a-(1+ 3) bonds surrounded by a-(1+ 4) linkages, in agreement with chemical evidence. When pseudonigeran and Lentinus a-D-glucan, having 100 and 85% of a-(1+ 3) linkages, were incubated with an endo-( 1 + 3 ) - a - ~ - g l u c a n a s e , ~ ~ ~ the degrees of hydrolysis were 28.5 and 14.3% of the total glycosidic bonds. Elsinan, isolated from Elsinoe leucospila, consists of maltotriosyl and maltotetraosyl units, joined by a-(1 + 3) linkages, and its structure was determined by enzymic hydrolysis with two alpha amylases having differing hydrolytic capacities."' Incubation with salivary alpha amylase released 32-a-~-glucosylmaltose (114) in high yield, with some 32-a-maltosylmaltose (115). An alpha amylase from Aspergillus oryzae could split the a - ( l + 4 ) U-D-GlC 1

1 3 a-D-GlC-(1 + 4)-D-GlC 114

3 U-D-GlC-(1 + 4)-D-GlC 11s

linkage in the maltotetraosyl, but not the maltotriosyl, segments, producing 33-a-~-glucosylmaltotriose(116), a heptasaccharide (117), and higher (440) K.K. Tung, A. Rosenthal, and J. H. Nordin, J. Biol. Chem., 246 (1971) 6722-6736. (441) A. Misaki, H. Nishio, and Y. Tsumuraya, Carbohydr. Rer, 109 (1982) 207-219.

266

BARRY V. McCLEARY AND NORMAN K. MATHESON

CC-D-GIC 1

.1 3 CY-D-GIC-( 1 + 4)-CC-D-GIC-(I + 4)-CC-D-GIC 1

5. 3

CC-D-GIC-(~+~)-CC-D-GIC-(I + 4)-D-GIC 117

oligosaccharides having d.p. values of 10, 13, and 16, based on the same structure. Gel chromatography gave ratios of these of 28: 10:6: 3: 1. A fraction of higher molecular weight, having an average d.p. of 30-35, was also isolated (in 33% yield), and it was degraded by salivary alpha amylase almost exclusively to 116, showing that it consisted of maltotriosyl units joined by a-(1 + 3) linkages. These results revealed a nonregular disposition of maltotetraosyl sections along the polymer chain. XIV. P-D-GLVCANS* 1. D-Glucans Based on a (1 +3)-/3-Backbone and on (1 +3)-&Chains

More structures of ( 1-* 3)-/3-~-glucansthat are substituted by single, O-6-linkedYD-glucosyl residues, similar to those of Sclerotium species’99uz and of laminaran,” described in the earlier article,’ have been examined. Trivial names have been given to members of this series of polymers (from different sources) that are substituted to various extents by (1 + 6)/3-D-glucosyl groups. This structure was derivedu2 from methylation analysis and Smith degradation, and also by hydrolysis of both the original polysaccharide and of the Smith-degraded product with exo-( 1+ 3)p-D-glucanase. This en~yme’~+’~@removes D-glucosyl groups sequen(442) J. Johnson, S. Kirkwood, A. Misaki, T. E. Nelson, J. V. Scaletti, and F. Smith, Chem. Ind. (London), (1963) 820-822. (443) S. Nagasaki, K. Saito, and S. Yamamoto, Agric. B i d . Chem., 41 (1977) 493-502. (444)T. G. Villa, V. Notario, T. Benitez, and J. R. Villanueva, Can. J. Biochem., 54 (1976) 927-934.

* Cellulose, succinoglycan, and xyloglucans are discussed in Sections 11,l; X,2; and II,2.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

267

tially from the nonreducing end, and also hydrolyzes a p-( 1+ 3)-linked D-glucosyl residue substituted at 0-6 by a single D-glucosyl group, releasing gentiobiose. The Smith-degraded polymer releases D-glucose. Further evidence for the structure of these glucans has resulted from hydrolysis (EC 3.2.1.39) of both the native and the with endo-(1 + 3)-fi-~-glucanase"~ Smith-degraded polymers. The ratio of main chain to side chain units varies (1.5 : 1 to 9 : 1) between species, and changes with the developmental stage.445 p-D-Glucans from Schizophyllum communew and Pythium acanthicum"' have been studied. Reaction with exo-(1 + 3)-p-~-glucanaseof an extracelMar fraction from Sclerotinia libertiana,"' which had a structure similar to that in an extract from sclerotia, gave D-glucose and gentiobiose in the ratio of 2: 1; only D-glucose was released after Smith degradation. During hydrolysis of the polymer up to a level of 73%, the ratio of D-glucose to gentiobiose was constant, indicating that branching by the p-( 1+ 6)-linked D-glucosyl groups occurs in a relatively regular manner on 0 - 6 of each third D-glucosyl residue in the main chain. D-Glucose and gentiobiose were also produced on hydrolysis by exo-( 1+ 3)-p-~-glucanaseof p-D-glucans extracted by alkali from sclerotia of Grifora umbellata"' and fruit bodies of Dictyophora hydrolysis of G. umbellata p-D-glucan, and of that i n d u s i ~ t aon ~ ~enzymic ~: ~ ~ ' ~sodium ~' hydroxide, the ratio of extracted from D. i n d u ~ i a t a ~ with D-glucose to gentiobiose remained relatively constant, indicating that the distribution of (1 + 6)-linked residues was reasonably uniform. In contrast, a water-soluble, (1 + 6)-branched (1 + 3)-p-~-glucan,which was part of a mixture isolated from a 2% sodium carbonate extract of the fruiting bodies In the earlier of D. indusiata, appeared to show heterogeneity in ~tructure.4~~ stages of hydrolysis of this polysaccharide by exo-( 1+ 3)-p-~-glucanase, there was a very high proportion of gentiobiose released; the ratio of gentiobiose to glucose was -5.5: 1. As the reaction proceeded, the ratio gradually decreased and reached a constant value of 0.8: 1 at later stages of hydrolysis. However, a hot-water-soluble extract had a ratio of 2 :5 of (1 + 6)- to (1 -+ 3)-p linkages, and enzymic hydrolysis indicated a uniform distribution of side chain^."^'" The location of sulfoacetyl groups in an acidic p-D-glucan from an Aureobasidium sp. was assisted by the use of (445) P. A. J. Gorin and E. Barreto-Bergter, in Ref. 78, pp. 365-409. (446)J. H. Sietsma and J. G . H. Wessels, Biochim. Biophys. Acra, 496 (1977) 225-239. (447) J. H. Sietsma, J. J. Child, L. R. Nesbitt, and R. H. Haskins, J. Gen. Microbiol., 86 (1975) 29-38. (448) Y. Ueno, Y. Hachisuka, H. Esaki, R. Yamauchi, and K. Kat6, Agric. Biol. Chem., 44 (1980) 353-359. (449) Y. Ueno, M. Abe, R. Yamauchi, and K. Kat6, Carbohydr. Res., 87 (1980) 257-264. (450) S. Ukai, C. Hara, and T. Kiho, Chem. Phann. BulL, 30 (1982) 2147-2154. (451) C. Hara, T. Kiho, and S. Ukai, Carbohydr. Res., 117 (1983) 201-213. (451a) C. Hara, T. Kiho, and S. Ukai, Carbohydr. Res., 145 (1986) 237-246.

268

BARRY V. McCLEARY AND NORMAN K. MATHESON

exo-( 1+ 3 ) - P - ~ - g l u c a n a s e Treatment .~~~ of the polymer with this enzyme gave D-glucose and gentiobiose (- 1 :3). Paper chromatography of the hydrolysis products indicated that the sulfoacetic acid group was associated with a component of d.p. 2, suggesting that it is attached to the p - ( l + 6)-linked D-glucosyl branch-units. Water-soluble glucan from the mycelia and zoospores of the fungus Phytophthora palrniuora gave gentiobiose and D-glucose on treatment with and it was concluded that there were one, or exo-( 1 + 3)-p-~-glucanase,4’~ two, branching residue(s) per molecule, which had a d.p. of -30. Enzymic hydrolysis of a p-D-glucan fraction from the cell wall of h’ricularia oryzae454*455 released 3-O-gentiobiosyl-~-glucose(118) and gentiotriose, indicating possible differences in structure from that of the polymer separated from Sclerotium sp. On reaction of the alkali-insoluble glucan P-D-GIC-(1 + 6)-P-D-Glc 1

.1

3

D-Glc 118

from Schizophyllum communea6 with exo-( 1 -* 3)-p-~-glucanase,84% of the anthrone-positive material became soluble, and the major fractions were D-glucose and gentiobiose (-30%), but gentiosaccharides of d.p. of up to at least six were also separated. In conjunction with chemical evidence, a structure was proposed. The skeletal glucan of Lentinus ed~des;’~ remaining after trichloroacetic acid and alkali extraction, was digested with exo-( 1+ 3)-p-~-glucanase,whereupon -86% of the material that gave a positive reaction with phenol-sulfuric acid was solubilized. The degree of hydrolysis was 22%, and the products included a homologous series of gentio-oligosaccharides up to gentioheptaose. These gentiosaccharides were presumed to be derived from a (1 46)-p-~-glucan present in the preparation and released by an endo-( 1-* 6)-P-~-glucanasecontaminant in the exo-(1+ ~ ) - P - D glucanase employed. The insoluble residue contained 23% of D-glucose, 65% of N-acetylhexosamine, and 1.2% of amino acid, and gave an X-ray diffraction pattern almost identical to that of chitin. The ratio of p-( 1-P 3) (452) N. Hamada and Y. Tsujisaka, Agric. Bid. Chem., 47 (1983) 1167-1172. (453) M. C. Wang and S. Bartnicki-Garcia, Carbohydr. Res., 37 (1974) 331-338. (454) T. Nakajima, K. Tamari, K. Matsuda, H. Tanaka. and N. Ogasawara, Agric. B i d . Chem., 34 (1970) 553-560. (455) T. Nakajima, K. Tamari, K. Matsuda, H. Tanaka. and N. Ogasawara, Agric. Bid. Chem., 36 (1972) 11-17. (456) M. Shida, Y . Ushioda, T. Nakajima, and K. Matsuda, J. Eiochem. (Tokyo),90 (1981) 1093-1 100.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

269

linkages in the fractions formed by successive stripping by exo-( 1 + 3 ) - p - ~ glucanase of the alkali-insoluble, skeletal material was determined, and the products were examined by chromatography. Partial structures of glucan fractions, and a model of the structure of the core material, were proposed. endo-( 1 + 3)-P-~-Glucanaseswhich lyse yeast cell-walls have been the subject of considerable s t ~ d y . These ~ ~ ~ can - ~ be ~ ~grouped into endoenzymes that produce oligosaccharides of either low d.p. or of high d.p., and exo-( 1 + 3)-p-~-glucanases.Some endo-( 1 + 3)-P-~-glucanasesdo not lyse cell walls, and these characteristically release oligosaccharides of low d.p. on hydrolysis of laminaran or p a ~ h y m a n . ~ ~ ~ - ~ ~ ~ Extraction of Saccharomyces cerevisiae cell-walls produced three p-Dglucan fractions: an alkali-insoluble, predominantly ( 1 + 3)-P-~-glucan; an alkali-insoluble, acetic acid-solubilized, branched (1 + 6)-p-~-glucan having (1 + 3) linkages; and an alkali-soluble (1 + 3)-P-~-glucanhaving &D-( 1 + 6) Chemical analysis of the alkali-insoluble (1 + 3)P-D-glucan showed that it is branched, containing 3% of ( 1 + 6 ) - p - ~ glycosidic, interchain linkages.469 Treatment with endo-( 1+ ~ ) - P - D glucanase gave D-glucose, laminarabiose, and laminaratriose, but not gentiobiose, as reaction products of low d.p. A partial structure (119) was G-(G),-G

3.

G-(G),-G-(G),-G

.1

...G-G-G.. . 119

(457) J. S. D. Bacon, A. H. Gordon, D. Jones, I. F. Taylor, and D. M. Webley, Biochem. J., 120 (1970) 67-78. (458) K. Doi, A. Doi, and T. Fukui, Agric. Biol. Chem., 37 (1973) 1619-1627. (459) S. Nagasaki, J. Fukuyama, S. Yamamoto, and R. Kobayashi, Agric. Biol. Chem., 38 (1974) 349-357. (460) Y. Kobayashi, H. Tanaka, and N. Ogasawara, Agric. Biol. Chem., 38 (1974) 973-978. (461) F. M. Rombouts and H.J. Phaff, Eur. J. Biochem., 63 (1976) 109-120. (462) S. Nagasaki, H. Mori, and S. Yamamoto, Agric. Biol. Chem., 45 (1981) 2689-2694. (463) T. W. Jeffries and J. D. Macmillan, Curbohydr. Res., 95 (1981) 87-100. (464) D. J. Manners and G. Wilson, Biochem. J., 135 (1973) 11-18. (465) M. Mandels, F. W. Parrish, and E. T. Reese, Phytochemistry, 6 (1967) 1097-1100. (466) D. J. Manners and J. J. Marshall, J. Insr. Brew., 75 (1969) 550-561. (467) J. S. D. Bacon and V. C. Farmer, Biochem. J., 110 (1968) 3 4 ~ - 3 5 ~ . (468) J. S. D. Bacon, V. C. Farmer, D. Jones, and I. F. Taylor, Biochem. J., 114 (1969) 557-567. (469) D. J. Manners, A. J. Masson, and J. C. Patterson, Biochem. J., 135 (1973) 19-30. (470) D. J. Manners, A. J. Masson, J. C. Patterson, H. Bjorndal, and B. Lindberg, Biochem. J., 135 (1973) 31-36. (471) G. H. Fleet and D. J. Manners, J. Gen. Microbiol., 94 (1976) 180-192. (472) G. H. Fleet and D. J. Manners, J. Gen. Microbiol., 98 (1977) 315-327.

270

BARRY V. McCLEARY A N D NORMAN K. MATHESON

proposed that was consistent with the results of chemical and enzymic studies[-isaP-(l+3) and +,aP-(1+6),linkage,anda+b+ccomprise -60 D-glucosyl residues]. The alkali-soluble P-D-glucan contained 80435% of P-D-(1 + 3) linkages, 8-l2% of &D-( 1 + 6) linkages, and 3-4% of branched residues linked471T472 through 0-1, 0-3, and 0-6. This glucan was extensively hydrolyzed by endo-( 1 + 3)-P-~-glucanase,yielding D-glucose, laminarasaccharides, gentiotriose (but not gentiobiose), a fraction excluded on Bio-Gel P-2 chromatography, and 25% of insoluble, resistant material which was not hydrolyzed by endo-( 1+ 6)-P-~-glucanase.Hydrolysis of the original polywas much less extensive, giving mainly mer with endo-( 1 + 6)-P-~-glucanase D-glucose, gentio-biose, -triose, and -tetraose, 32-P-~-glucosylgentiobiose (120), a mixed-linkage tetrasaccharide, and small proportions of laminarasaccharides. Oligosaccharide 120 was partly characterized with exo-( 1 + 3)-P-~-glucanase,which gave D-glucose and gentiobiose. The proportion of gentio-triose and -tetraose, compared to the two mixed-linkage P-D-GIC 1

.1

3 P-D-GIC-(1 + 6)-D-GIC

120

oligosaccharides, decreased with time-period of hydrolysis. The insoluble material remaining after ( 1 + 6)-P-~-glucanasehydrolysis was extensively degraded by endo-( 1 + 3)-P-~-glucanase,yielding an insoluble residue plus a series of oligosaccharides similar to those released on hydrolysis of the original polymer by this enzyme. The insoluble material produced on treatment of the original, alkali-soluble glucan with endo-( 1 + 6)-PD-glucanase was shown by methylation analysis to be mainly a (1 + 3)~ 6) linkages. P-D-glucan having a small percentage (-1”/0) of P - D - ( + The resistance of the P-D-(1 + 6) linkages to hydrolysis by endo-( 1 + 6)-P-~-ghcanasewas presumed to indicate that these linkages occur as isolated di- or tri-saccharide units in larger blocks of P-D-(1 + 3)-linked residues. The isolation of both (1 + 3)- and (1 + 6)-linked oligosaccharides on endo-enzymic hydrolysis means that both linkage types are found as sequences. It was concluded that the alkali-soluble glucan has a (1+ 3)-P-~-glucancore, with occasional (1 + 6)-linked residues, and a low degree ~ sideof branching (-2%). To this are attached mainly P - D - ( +3)-linked chains, which may contain secondary branching, also in the form of (1 + 3)-linked chains. Side chains containing either mainly (1+ 6)-linked residues or a mixture of (1 + 6)-linked and (1 + 3)-linked residues are also present.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

27 1

The cell-wall p-D-glucan fractions, insoluble in alkali and acid, from wild and from a mutant S. cereuisiae have been compared. Methylation analysis indicated more 1,3- and 1,3,6-linked D-glucosyl residues, and fewer 1,6linked in the latter. The wild type was hydrolyzed less by exo-(1+ 3)-p-~-glucanaseand more by endo-( 1 + 6 )-p -~ - g h c a n a s ethan was the glucan from the mutant, consistent with the methylation data.473a Two cell-wall fractions were obtained from mycelia of Neurospora crassa by differential extraction with alkali (soluble in 4%, and insoluble in 24%); on incubation with endo-( 1+ 3)-p-~-glucanase,both gave473D-glucose, a 18). b i o ~exoe laminara-biose and -triose, and 6 2 - p - ~ - g l ~ ~ ~ ~ y l l a m i n a r(1 (1 + 3)-p-~-Glucanasereleased D-glucose and gentiobiose. Structures were proposed for both fractions when this evidence and the results of methylation analysis were considered. Laminarans contain a small and varying percentage of (1+ 6) linkages.474 exo-( 1+ 3)-P-~-Glucanasehydrolysis of three water-soluble fractions from Peluetia canaliculata gave similar amounts of D-glucose, small proportions of gentiobiose, and traces of other g l u c o s a ~ c h a r i d e s Hydrolysis .~~~ by exo(1 -+ 3)-P-~-glucanaseof laminaran (from Laminaria hyperborea) after Smith degradation gave less gentiobiose than prior to degradation, indicative of the presence of “side chains” made up of single, D-glucosyl One of the products was 1-0-p-laminarabiosyl-D-mannitol. Gentiobiose was obtained in only a trace from the insoluble fraction, consistent with the presence of fewer (1 + 6) linkages. The P-D-glucan from Eisenia bicyclis apparently has a structure consisting of p-( 1+ 3)-linked and some p-(1+ 6)-linked D-glucosyl residues, with some of the latter located in the chain as blocks and others as ’~ Treatment of the polysaccharide with endo-( 1 + 6 )-p -~ - g lu c a n a s e ~gave D-glucose, laminarabiose, and gentiobiose, as well as a series of 6-plaminara-D-glucosaccharides(121, where n was 1 to 3). Characterization P-D-GIc-(lfr3)-P-D-GlC-(Ik3)-P-D-GlC 1

3.

6 D-Glc 121

(473) N. Hiura, T. Nakajima, and K. Matsuda, Agric. Bid. Chem., 47 (1983) 1317-1322. (473a) M. Shiota, T. Nakajima, A. Satoh, M. Shida, and K. Matsuda, J. Biochem. (Tokyo), 98 (1985) 1301-1307. (474) D. J. Manners and R. J. Sturgeon, in Ref. 77, pp. 472-514. (475) J. R. Stark, Carbohydr. Res., 57 (1977) c l l - c 1 2 . (476) T. E. Nelson and B. A. Lewis, Carbohydr. Res., 33 (1974) 63-74. (477) Y. Shibata, J. Biochem. (Tokyo),75 (1974) 85-92.

212

BARRY V. McCLEARY A N D NORMAN K. MATHESON

of these oligosaccharides employed p- D-glucosidase followed by chromatographic separation of partial-hydrolysis products. Evidence that the p-Dglucanase could not only split p-( 1 + 6)-linked D-glucosyl residues in (1 + 6)-P-~-glucans,but could also cleave p-( 1+ 3) linkages of 6-0-substituted D-glucosyl units, was provided by the hydrolysis of 32-p-gentiobiosylgentiobiose (122) to gentiobiose. Hydrolysis of the original laminaran with endo-( 1 + 3)-P-~-glucanasegavellc oligosaccharides of d.p. 3, 5, 7, and 9. P-D-GIc-(1 -+ 6)-P-D-GlC 1

3.

3 P-D-GIc-(1 -+ 6)-D-Glc 122

An endo-( 1 + 6)-P-~-glucanasefrom Bacillus circulans WL-12 hydrolyzed laminaran, and the activity increased as the &D-( 1 + 6)-linked Dglucosyl content increa~ed.4’~ The main products were gentiobiose and D-glucose, with no laminarasaccharides. There were traces of mixed-linkage oligosaccharides, including 121, where n = 0, and, probably, where n = 1. This enzyme could not hydrolyze the (1 + 3)-P-~-glucanfrom Sclerotinia spp., which has one-third of the main-chain units substituted at 0 - 6 with single D-glucosyl groups. Reaction of Eisenia bicyclis laminaran with exo(1 + 3)-/3-~-glucanase””gave glucose, gentiobiose, 6-0-/3-laminarabiosylD-glucose (121with n = 0), and 122,as well as11b33-~-~-glucosylgentiotriose (122a), 33-p-gentiobiosylgentiotriose (122b), and 3 4 - / 3 - ~ - g l ~ ~ ~ ~ y I - 3 2 - P gentiobiosylgentiobiose (122c). Combined with those from methylation P-D-GIc 1

3.

3 P - ~ - G l c - ( l -6)-P-D-GIc-( + 1 -+6)-D-GIC 122a

P - ~ - G l c - ( l -6)-P-D-Glc + 1

-

3. 3

P-D-GIc-(1 + 6 ) - p - ~ - G k -1 ( 6)-D-Glc 122b

(478) F. M. Rombouts, G . H. Fleet, D. J. Manners, and H. J. Phaff, Carbohydr. Res., 64 (1978) 237-249.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

273

P-D-GlC 1

1 3 B-D-GlC-(1 -+ 6)-P-D-GIC 1

1

3 P-D-GIC-(l -P 6)-D-GIC 122c

analysis and 13C-n.m.r. spectroscopy, the results were consistent with the proposed structure containing (1+6) linkages in both the main ( l + 3)-p-chain and as branches of single, (1 + 6)-linked D-glucosyl groups. Land plants contain a p-( 1 + 3)-~-glucan(called callose). On incubation with exo-p-( 1+ 3)-~-glucanase,a D-glucan isolated from a maize-shoot preparation was hydrolysed 92%, and the only sugar liberated at all stages of hydrolysis was D-glucose, indicating little, if any, 2. (1 + 3x1+4)-P-~-Glucans

Lichenan and the (1 + 3)( 1 + 4)-p-~-glucans from cereals are susceptible to hydrolysis by three types of endo-glucanase, cellulase; (1 + 3 ) ( 4 ) - p - ~ glucanase (EC 3.2.1.6); and lichenase [( 1 + 3)( 1 + 4)-4-glucanohydrolase (EC 3.2.1.73)].'*479Treatment of lichenan and cereal P-D-glucans with celIulase gives 32-p-~-glucosylcellobiose(123), 33-p-~-glucosylcellotriose (124), and 32-~-cellobiosylcellobiose (125) as major r e a c t i o n - p r o d ~ c t s . ~ ~ ~ The enzyme has no action on reduced Pneumococcus SIII poly~accharide,"~~ P- D-GlC 1

.1 3 P-D-GlC-(1 -+ 4)-D-GlC 123

P-D-GIc 1

3

3 P-D-GlC-(1 -P 4)-P-D-GIC-(1 + 4)-D-GlC 124

(478a) Y. Kato and D. J. Nevins, Plant Physiol., 78 (1985) 20-24. (479) M. A. Anderson and B. A. Stone, FEES Lett., 52 (1975) 202-207. (480) A. S . Perlin and S. Suzuki, Can. J. Chem., 40 (1962) 50-56.

274

BARRY V. McCLEARY AND NORMAN K. MATHESON P-D-GlC-(1+4)-P-D-Gk 1

3.

3 P-D-GIC-(1+ 4)-D-Glc

125

which consists of alternating P-D-(1 + 3)- and P-D-(1 + 4)-glucosyl residues. These results indicated that it only hydrolyzes the substrate when at least two (1 + 4) links are adjacent. The ratio of trimer to tetramer released was 4.0: 1 for lichenan and 2.2: 1 for oat g l ~ c a n . ~ " (1 + 3)(4)-p-~-Glucanase (nonspecific endo-( 1 + 3)-B-~-glucanase) can hydrolyze both the p-( 1+ 3) bonds in laminaran and a p-( 1+ 4) bond . ~ ~laminarabiose ~ was adjacent to a p-( 1 + 3) linkage in cereal g l ~ c a n sOnly produced on extensive hydrolysis of Pneumococcul RSIII polysaccharide by this enzyme,479consistent with hydrolysis of p-( 1 + 4) bonds adjacent to p-( 1 + 3)-linked D-glucosyl residues. The third enzyme that hydrolyzes lichenan and cereal p-D-glucans is Pneumococcul RSIII polysaccharide, but has l i ~ h e n a s e . ~It~also ~ * ~cleaves ~' no action on ( 1 + 4)-~-(CM-cellulose)or (1 + 3)-p-~-glucan(laminaran). Hydrolysis of barley-grain p-D-glucan by different lichenase preparations482-484 gave 3-O-~-cellobiosy~-~-glucose (126) and 3- O-p-cellotriosylD-glucose (127) in the molar ratio of -2 : 1,together with small proportions of oligosaccharides of higher d.p.482*484a*484b These two oligosaccharides P-D-GIC-( 1 + 4)-P-D-GlC 1

3. 3 D-GIc 126

(481) D. J. Huber and D. J. Nevins, Plant Physiol., 60 (1977) 300-304. (482) W. W. Luchsinger, S.-C. Chen, and A. W. Richards, Arch. Biochem. Biophys., 112 (1965) 524-530; 531-536. (483) J. R. Woodward and G. B. Fincher, Carbohydr. Res., 106 (1982) 111-122. (484) H. Suzuki and T. Kaneko, Agric. Bid. Chem., 40 (1976) 577-586. (484a) J. R. Woodward, G. B. Fincher, and B. A. Stone, Carbohydr. Polym., 3 (1983) 207-225. (484b) Y. Kato and D. J. Nevins, Carbohydr. Res., 147 (1986) 69-85.

ENZYMIC ANALYSIS OF POLYSACCHARIDE STRUCTURE

275

together made up 90% of the sugars produced. Sequence dependence of cello-triosyl and -tetraosyl units was estimated from the release of oligosaccharides during enzymic hydrolysis.484cHydrolysis of oat-bran p-D-glucan also released484d126 and 127. Hydrolysis of p-glucan from rice seedlings gave a ratio of 2.6: 1, indicating 30.5% of (1 + 3) linkage^.^'" Treatment of p-D-glucans from the coleoptiles of five cereals with lichenase of (1 + 3)(4)P-D-glucanase gave the tri- and tetra-saccharides in a ratio of 2.5-3.3: 1, indicating that all of the polysaccharides contained 30-31% of p-( 1+ 3) linkages, and that only a small percentage of consecutive p-( 1 + 3) linkages could be present.485 On hydrolysis with lichenase, a preparation from immature barley coleoptiles and primary leaves released486tri- and tetrasaccharides in the ratio of 2.5: 1. p-D-Glucan from Zea mays shoots, extracted by hot water, reacted with lichenase, and the products were fractionated by gel chromatography. The major fractions were 126 (65.5%) and 127 (26.8%) (ratio 2.4: 1) with much lower proportions of disaccharide (2.9% ), pentasaccharide (1.6% ), and smaller proportions (<1YO each) of hexa- and oligo-saccharides of higher d.p. These were then treated with cellulase, which indicated that they were mixtures. In conjunction with methylation analysis, the presence of small amounts of two to four contiguous (1+ 3) linkages, blocks of more than four (1+ 4) linkages and a region of alternating (1 + 3) and (1 +4) linkages was pr0posed.4'~~Significant proportions of oligosaccharides of higher d.p. (-8-9%) have been found in barley p-D-glucan h y d r o l y ~ a t e s . 4 ~ ~ ~ ~ ~ ~ "

3. p-D-Glucans Based on (1 +6)-p-Chains The (1 + 6)-p-~-glucansinclude pustulan and lutean, which contain only units, and a minor fraction of Saccharomyces cerevisiae (1 + 6)-p-~-glucosyl cell-wall glucan which has a high proportion of p-( 1+ 6) linkages with -19% of the D-glucosyl residues /3-(1+ 3). Some endo-(1 + 6 ) - p - ~ - g l u c a n a s e s ~ ~ are ~ . ~highly ~ ~ . ~ specific ~ ~ - ~ ~ for ~ (1 + 6)-p-~-glucans, whereas others can hydrolyze either (1+ 6)-linked /3-D-glucosyl residues or (1 + 3) linkages adjacent to a p-( 1+ 6)-linked (484c) R. G. Staudte, J. R. Woodward, G. B. Fincher, and B. A. Stone, Carbohydr. Polym., 3 (1983) 299-312. (484d) G. 0. Aspinall and R. C. Carpenter, Carbohydr. Polym., 4 (1984) 271-282. (485) D. J. Nevins, R. Yamamoto, and D. J. Huber, Phytochemirtry, 17 (1978) 1503-1505. (486) Y. Kato, K. Iki, and K. Matsuda, Agric. Biol. Chem., 45 (1981) 2737-2744. (487) J. Abe, A. Amemura, and T. Harada, Agric. Biol. Chem., 44 (1980) 1877-1884. (488) S. Katohda, F. Suzuki, S. Katsuki, and T. Sato, Agric. BioL Chem, 43 (1979) 2029-2034. (489) S. Yamamoto, R. Kobayashi, and S. Nagasaki, Agric. Biol. Chem, 38 (1974) 1493-1500. (490) E. T. Reese, F. W. Parrish, and M. Mandels, Can. J. Microbiol., 8 (1962) 327-334.

276

BARRY V. McCLEARY AND NORMAN K. MATHESON

The minor component isolated from alkali-insoluble, yeast glucan by selective enzymolysis with endo-( 1 -* 3)-p-~-glucanase,or by chemical fractionation, was hydrolyzed by an endo-( 1 + 6)-p-~-glucanase,to give a mixture of D-glucose and gentiosaccharides having degrees of polymerization of 2-5, as well as 120 and a mixed-linkage tetrasa~charide.4~~'~~' The polymer was considered to have a highly branched structure with a high proportion of &D-( 1 + 6)-glucosidic linkages, as there was no hydrolysis by endo-( 1 -* 3)-P-~-glucanase. The small proportion of &D-( 1 + 3)glucosidic linkages were considered to occur mainly as inter-chain and, perhaps to a lesser degree, as inter-residue linkages. The glucan from a mutant, which appeared to lack this component, was much less hydrolyzed by endo-( 1 -* 6 ) - P - ~ - g l u c a n a s e than ~ ~ ~ "was the wild type. 4. Cyclic (1 +2)-/3-~-Glucans Agrobacterium and Rhizobium species synthesize cyclic (1 -* 2)-P-~-glucanshaving a d.p. of -20 units. On incubation with these polymers, adaptively produced (1 -* 2)-p-~-glucanase from fungi released sophorose and higher oligo~accharides.~~~ Hydrolytic capacity has also been a~~~ The lack of hydrolysis found in ~ n a i l - g u and t ~ ~C~ y t ~ p h a g preparations. of the native polysaccharide by almond and Aspergillus niger p-Dglucosidase, but hydrolysis of the partially hydrolyzed polymer, is consistent with a cyclic structure.

(491) E.T. Reese, F. W.Parrish, and M. Mandels, Can. J. Microbid., 7 (1961) 309-317. (492) A. Zorreguieta. M. E. Tolmasky, and R. J. Staneloni, Arch. Biochem. Biophys., 238 (1985) 368-372. (493) A. Amemura, Agric. Biol. Chem., 48 (1984) 1809-1817.

ADVANCES IN CARBOHYDRATE CHEMISTRY A N D BIOCHEMISTRY, VOL. 44

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS COMPOSED OF REPEATING UNITS

BY VLADIMIRN. SHIBAEV N. D. Zelinsky Institute for Organic Chemistry, Academy of Sciences of the U.S.S.R., Moscow GSP-I, 117913, U.S.S.R

I. INTRODUCTION A number of complicated polymers having chains composed of monosaccharide residues are present at the surface of bacterial These polymers often serve as characteristic antigens of bacteria, and are very diverse in their structure. They may be divided into three main groups: ( 1 ) Cell-wall polymers characteristic mainly of Gram-positive bacteria, where different glycan chains are interconnected by covalent linkages, producing an enormous, cross-linked macromolecule. Teichuronic acids and neutral glycans are typical polymers in this group. Monosaccharide residues are also present in teichoic acids, in which monomeric units are linked through phosphoric diester linkages, and in bacterial peptidoglycans. (2) Polymers having carbohydrate chains linked to a lipid anchor embedded in a bacterial membrane; the term “bacterial amphiphiles” was suggested’ for these polymers. Lipopolysaccharides of Gram-negative bacteria constitute the best-known example of these polymers. (3) Exocellular polysaccharides, which are produced by strains of both Gram-positive and Gram-negative bacteria. They include capsular and extracellular (slime) polysaccharides. ( 1 ) I. W. Sutherland (Ed.), Surface Carbohydrates of the Prokaryotic Cell, Academic Press, London, 1977. (2) D. J. Tipper and A. Wright, in J. C. Gonsalus (Ed.), The Bacteria, Vol. 7 (Mechanism of Adaptation, Vol. Eds., J. R.Sokatch and L. N. Ornston), Academic Press, New York, 1979, pp. 291-426. (3) A. Wright and D. J. Tipper, in Ref. 2, pp. 427-486. (4) H. J. Rogers, H. R. Perkins, and J. B. Ward, Microbial Cell Walls and Membranes, Chapman and Hall, London, 1980. ( 5 ) A. J. Wicken and K. W. Knox, Biochim. Biophys. Acta, 604 (1980) 1-26.

277

Copyright @ 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.

278

VLADIMIR N. SHIBAEV

In the polymers of groups ( 1 ) and ( 2 ) ,polysaccharide chains composed of oligosaccharide repeating-units (sometimes, partially modified) are usually linked to a unique oligosaccharide unit present near the point of attachment of the chain to another polymeric chain, or to a lipid anchor. This unit is called the “linkage region” in the polymers of bacterial cell-wall, and the “core region” in lipopolysaccharides. The present chapter concentrates on the biosynthesis of polysaccharide chains composed of oligosaccharide repeating-units that are linked through glycosidic bonds. The main enzymic reaction during assembly of these chains is the glycosylation reaction, that is, the transfer of a glycosyl group from an activated monosaccharide derivative, the glycosyl donor, onto a hydroxyl group present in a molecule of a glycosyl acceptor. Contrary to the situation with other biopolymers, there are many possibilities for the formation of inter-monomeric linkages in polysaccharide chains, and strict regio- and stereo-specificity of the glycosylation reaction is necessary for correct assembly of these polymers. The answers to three questions seem the most essential to an understanding of the mechanism of polysaccharide biosynthesis from the chemical point of view: (a) What is the nature of the glycosyl donors in the glycosylation reactions, and how are these donors formed in bacterial cells? (b) Which mechanisms determine the formation of definite, intermonomeric linkages in a polymeric chain? (c) What is the sequence of enzymic reactions during assembly of the polymer; that is, how is the chain initiated, elongated, terminated, and further processed? The present state of our knowledge concerning these questions is discussed in the present chapter, with special attention given to classification of the available data, and to making generalizations whenever possible. The literature was systematically examined up to April, 1983, and some subsequent papers have also been incorporated. Biosynthesis of bacterial polysaccharides was described in the aforementioned books’-4 and in several reviews6-’ published after 1977. These texts may be used as a source of information on topics not considered in the present article. They include: ( i ) the biosynthesis of unique, oligosaccharide sequences of bacterial polymers, such as the linkage region, the core region of lipopolysaccharides, and oligosaccharide units present in glycoproteins (6) (7) (8) (9)

I. W. Sutherland, Trends Biochem. Sci., 4 (1979) 55-59. I. W. Sutherland, Ado. Microb. Physiol., 23 (1982) 79-150. S. J. Tonn and J. E. Gander, Annu. Rev. Microbiol., 33 (1979) 169-199. F. A. Troy, Annu. Reu. Microbiol., 33 (1979) 519-560.

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279

of archaebacteria, ( i i ) the biosynthesis of polymeric chains that contain phosphoric diester linkages, particularly of teichoic acids, (iii) the biosynthesis of dextrans and levans, ( i u ) regulation of the amount of polysaccharides produced in the cell, and ( u ) subcellular localization of biosynthetic systems, and transportation of the polymers from the site of the synthesis to the cell surface. 11. GLYCOSYL ESTERSOF NUCLEOTIDES AND POLYPRENYL PHOSPHATES I N POLYSACCHARIDE BIOSYNTHESIS GLYCOSYL

Monosaccharides are activated for participation in glycosylation reactions by conversion into diphosphoric or phosphoric ester derivatives. Three types of activated sugar intermediate are known to play an important role in the biosynthesis of carbohydrate chains; their structures and enzymic interconversions are shown in Scheme 1 for D-glucopyranose 0

ic.. $-

0

0

I1

I1

-P-0-P-

HO

OH

I

I

0-

0-

HO

OH

1

Bpr-OPO,Hz UDP CH,OH

0

CH2OH

OH

OH

0

0

P-0-

P-0-Bpr

0-

0-

I1 I

3

2

SCHEME 1

II I

280

VLADIMIR N. SHIBAEV

derivatives. These include glycosyl esters of nucleotides (mainly those of nucleoside diphosphates), such as “uridine diphosphate glucose” (1: UDPGlc),” polyprenyl glycosyl monophosphates, exemplified by the p-Dglucopyranosyl derivative 2 (p-Glc-p-Bpr)” and polyprenyl glycosyl diphosphates similar to the a-D-glucopyranosyl derivative 3 (a-Glc-ppBPd. A brief description of enzymic reactions leading to de novo synthesis of these intermediates, and a summary of their biological functions, are presented in this Section. 1. Primary Glycosyl Nucleotides The “sugar nucleotides” (an uninformative name that has been used for glycosyl nucleotides, or more strictly, glycosyl esters of nucleoside di- or mono-phosphates) were discussed in this Series” in 1973. Since then, accumulation of new data about these derivatives has continued, and now, about 35 representatives of this class are known to participate in the biosynthesis of polysaccharide chains of bacterial polymers (for a survey, see Ref. 13). These include glycosyl esters of uridine 5’-diphosphate (UDP), thymidine 5’-diphosphate (dTDP), guanosine 5‘-diphosphate (GDP), cytidine 5’-diphosphate (CDP), cytidine 5’-monophosphate (CMP), and adenosine 5’-diphosphate (ADP). It has been suggested” that a distinction be made between the “primary” and the “secondary” glycosyl nucleotides. The former derivatives are produced as a result of the interaction of nucleoside 5‘-triphosphates with glycosyl phosphates for nucleoside glycosyl diphosphates) or monosaccharides (for cytidine glycosyl monophosphates). The enzymes that catalyze these reactions belong to the group of nucleotidyltransferases (10) The condensed IUPAC-IUB system of terminology for sugar residues, oligosaccharide chains, and polysaccharides [Eur. J. Eiochem., 126 (1982) 433-437, 439-4411 is used in somewhat modified form in the present article. The abbreviations Glc, Gal, and Man stand for pyranoses of the D series, and Rha and Fuc for pyranoses of the L series, unless stated otherwise. The abbreviation NDP for nucleotides means nucleoside 5’-diphosphate, with nucleoside residues abbreviated as recommended by the IUPAC-IUB. (11) In abbreviations for polyprenyl glycosyl phosphates, “p” stands for phosphate, and “pp” for diphosphate groups. The abbreviation Bpr means a bacterial undecaprenyl group. For the moraprenyl group (see Ref. 57), the abbreviation Mpr is used, and the symbol Pre stands for any unidentified, lipid acceptor having the properties of polyprenol. (12) N.K. Kochetkov and V. N. Shibaev, Adu. Carbohydr. Chem. Eiochem., 28 (1973) 307-399. (13) 0. Gabriel, Methods Enzymol, 83 (1982) 332-353.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

281

(EC 2.7.7 group). Secondary glycosyl nucleotides are formed from the primary derivatives through enzymic reactions leading to modification of their monosaccharide fragments; these conversions are discussed in Section 111. In bacteria, primary glycosyl nucleotides i n c l ~ d e ' ~UDP-Glc, -~~ as well as other nucleotide derivatives of the same monosaccharide. Important representatives of this class are 16-21*23-25 dTDp-Glc and 17-19.21.26-29 C D P-Glc, which serve as precursors for a number of secondary glycosyl nucleotides, whereas similar enzymic reactions have not been demonstrated for GDP~ l18.19.2 ~ 1.30, and ADp_G1C~7-19,21.31-38 seems to participate only in the biosynthesis of bacterial glycogen, a subject that is beyond the scope of this article.

(14) E. E. B. Smith, G. T. Mills, H. P. Bernheimer, and R. Austrian, J. Gen. Microbiol., 20 (1959) 654-669. (15) A. Kamogawa and K. Kurahashi, 1. Biochem. (Tokyo), 57 (1965) 758-765. (16) R. L. Bernstein and P. W. Robbins, J. Biol. Chem., 240 (1965) 391-397; P. W. Robbins and R. L. Bernstein, Methods Enzymol., 8 (1966) 253-256. (17) K. Kimata and S. Suzuki, J. Biol. Chem., 241 (1966) 1099-1113. (18) T. Chojnacki, T. Sawicka, and T. Korzybski, Acta Biochim. Pol., 15 (1968) 293-297. (19) T. Chojnacki, W. Jankowski, and E. Janczura, Acra Biochim. Pol., 18 (1971) 347-351. (20) T. Nakae and H. Nikaido, J. Bid. Chem., 246 (1971) 4386-4396. (21) D. Lapp and A. D. Elbein, J. Bacteriol., 112 (1972) 327-336. (22) L. Lee, A. Kimura, and T. Tochikura, Biochim. Biophys. Acta, 527 (1978) 301-304; J. Biochem. (Tokyo),86 (1979) 923-928. (23) J. H. Pazur and E. W. Shuey, J. Bid. Chem., 236 (1961) 1780-1785. (24) S. Kornfeld and L. Glaser, J. Biol. Chem., 236 (1961) 1791-1795. (25) A. Melo and L. Glaser, J. Biol. Chem., 240 (1965) 398-405. (26) V. Ginsburg, P. J. O'Brien, and C. W. Hall, Biochem Biophys. Res. Commun., 7 (1962) 1-4. (27) R. M. Mayer and V. Ginsberg, J. Biol. Chem., 240 (1965) 1900-1904; Methods Enzymol., 8 (1966) 256-258. (28) H. Nikaido and K. Nikaido, J. Biol. Chem., 241 (1966) 1376-1385. (29) P. A. Rubenstein and J. L. Strominger, J. Bid. Chem., 249 (1974) 3789-3796. (30) K. Kawaguchi, S. Tanida, K. Matsuda, Y. Tani, and K. Ogata, Agric. Biol. Chern., 37 (1973) 75-81. (31) L. Shen and J. Preiss, J. Biol. Chem., 240 (1965) 2334-2340; Methods Enzymol., 8 (1966) 262-266. (32) C. E. Furlong and J. Preiss, J. Biol. Chem., 244 (1969) 2539-2548. (33) H. Ozaki and J. Preiss, Methods Enzymol., 28 (1972) 406-413. (34) T. Haugen, A. Ishaque, A. K. Chatterjee, and J. Preiss, FEES Lett., 42 (1974) 205208. (35) M. Lehmann and J. Preiss, J. Bacteriol., 143 (1980) 120-127. (36) S.-G. Yung and J. Preiss, J. Bacteriol., 147 (1981) 101-109. (37) W. K. Kappel and J. Preiss, Arch. Biochem Biophys., 209 (1981) 15-28. (38) S.-G. Yung and J. Preiss, J. Bacteriol., 151 (1982) 742-749.

282

VLADIMIR N. SHIBAEV

Guanosine (D-mannosyl diphosphate) (GDP-Man, 4)39-42and uridine (2-acetamido-2-deoxy-~-glucosyldiphosphate) (UDP-GlcNAc, 5)43,44are other important examples of primary glycosyl nucleotides. Although formation of ADP and dTDP derivatives of the latter monosaccharide was observed,I7 their functions remain

HO

OH

4

0

-0CH2 0

A ~ N H

6-

O-

HO

OH

5

(39) J. Preiss and E. Wood, J. Bid. Chem., 239 (1964) 3119-3126; J. Preiss, Methods EnzymoL, 8 (1966) 271-275. (40) R. H. Kornfeld and V. Ginsburg, Biochim. Biophys. Acfa, 117 (1966) 79-87. (41) S. M. Rosen and L. D. Zeleznick, Merhods Enzymol., 8 (1996) 145-147. (42) J. Preiss and E. Greenberg, Anal. Biochem., 18 (1967) 464-471; Methods EnzymoL, 28 (1972) 281-284. (43) J. L. Strominger and M. Smith, 1. B i d . Chem., 234 (1959) 1822-1827. (44) D. Filer, S. H. Kindler, and E. Rosenberg, J. Bacteriol., 131 (1977) 745-750. (45) Several cases of synthesis of a-D-galactosyl nucleotides from a-D-galaCtOpyranOSyl phosphate with bacterial enzymes have been reported. These included formation of UDP-Gal through reaction with uridine 5’-triphosphate2’ or U D P - G I C , ’ ~ *and ~ ~ -of~ ~ dTDP-Gal through interaction with thymidine S’-triphosphate.’’ (46) L.-J. Lee, A. Kimura, and T. Tochikura, Agric. B i d . Chem., 42 (1978) 723-730. (47) K. Kurahashi and A. Sugimura, J. Bid. Chem., 235 (1960) 940-945. (48) S. Saito, M. Ozutsumi, and K. Kurahashi, J. Biol. Chem., 242 (1967) 2362-2368. (49) L.-J. Wong, K.-F. Sheu, S.-L. Lee, and P. A. Frey, Biochemistry, 16 (1977) 1010-1016. (50) J. H. Pazur and J. S . Anderson, J. Biol. Chem., 238 (1963) 3155-3160.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

283

The monosaccharide groups in all of the primary glycosyl nucleotides mentioned have the same configuration at C-1, that is, they all belong to the a- Dseries. Conversion of the primary glycosyl nucleotides into the secondary ones occurs without changes at the anomeric center, and, as a consequence, the resulting derivatives contain a-D-glycosyl groups if the conversion does not include epimerization at C-5 (or p-L-glycosyl groups if such epimerization occurs). Among nucleoside glycosyl monophosphates, the primary derivatives include cytidine (N-acetylneuraminic monophosphate) (CMP-NeuAc),” in which the D-glycero-P-D-galacto configuration of the monosaccharide group has been confirmed,52 and the analogous derivative of 3-deoxy-~manno-2-octulosonic Glycosyl nucleotides constitute the main, activated form of monosaccharides for their incorporation into polysaccharide chains; this occurs through glycosylation reactions, with release of nucleoside 5’-diphosphates or nucleoside 5‘-phosphates. These reactions are catalyzed by membranebound glycosyltransferases (the EC 2.4 group). Glycosyl acceptors in the biosynthesis of bacterial, polysaccharide chains are also membrane-bound, and the glycosylation reactions with water-soluble glycosyl nucleotides have to occur at the surface of biological membranes. Formation of glycosidic linkages as a result of glycosyl transfer from glycosyl nucleotides may lead to inversion, as well as to retention, of the configuration at C-1 of the glycosyl group transferred; the two types of glycosidic linkage seem to be almost equally frequent in bacterial polysaccharides (see Section IV). The net retention of configuration may actually be the result of double glycosyl transfer, each with inversion. In some cases, intermediate formation of polyprenyl glycosyl monophosphates from glycosy1 nucleotides (see Section II,2) seems to be in accord with this proposal, but such a mechanism is definitely not general. The ability of nucleoside glycosyl diphosphates to serve as glycosyl phosphate donors in the biosynthesis of polyprenyl glycosyl diphosphates (see Section II,3) is well documented. It is not yet known whether they may function as immediate precursors of glycosyl phosphate residues not infrequently present as components of bacterial polymers, or whether some other intermediates are involved. (51) L. Warren and S. Blacklow, J. Bid. Chem., 237 (1962) 3527-3534. (52) J. Haverkamp, T. Spormaker, L. Dorland, J. F. G. Vliegenthart, and R. Schauer, J. Am. Chem. SOC.,101 (1979) 4851-4853. (53) M. A. Ghalarnbor and E. C. Heath, J. Biol. Chem., 241 (1966) 3216-3221; Methods Enzymd., 8 (1966) 221-224. (54) P. H. Ray, C. D. Benedict, and H. Grasmuk, J. BacterioL, 145 (1981) 1273-1280; P. H. Ray and C. D. Benedict, Methods EnzyrnoL, 83 (1982) 535-540.

VLADlMlR N. SHIBAEV

284

2. Polyprenyl Glycosyl Monophosphates Polyprenyl glycosyl monophosphates are formed from glycosyl nucleotides by nucleophilic attack at C-1 of the glycosyl group by the phosphate group of bacterial polyprenyl phosphate (compare the conversion of 1 into 2 in Scheme 1). A glycosyl acceptor in the reaction is a derivative of C,,-polyprenol having eight internal 2- and two internal E-isoprenic units (see Refs. 55 and 56 for review^)'^; predominance of Cso-polyprenol is characteristic for mycobacteria. The reaction was observed for several primary glycosyl nucleotides, and generally occurs with inversion of the configuration at C-1. Formation of P-Glc-p-Bpr was demonstrated with membrane preparations containing the corresponding glucosyltransferases from several strains of Salmonella,6°-62Escherichia c0li,6~Shigella j l e ~ n e r i , 6 ~Bacillus ,~~ lichen if or mi^,^'*^^ Mycobacterium smegrnati~;~and Streptococcus sanguix6* Analogous derivatives of D-mannopyranose, presumably of the p configuration, were identified in several b a ~ t e r i a . ~ ’ .In ~ ~the - ~case ~ of reaction with UDP-GlcNAc, both anomers of GlcNAc-p-Bpr were formed in membranes (55) F. W. Hemming, in T. W. Goodwin (Ed.), Biochemistry oflipids, Buttenvorths, London, 1974, pp. 39-97. (56) V. N. Shibaev, Usp. Biol. Khim., 17 (1976) 187-216. (57) Readily available plant polyprenols from leaves of ficus (ficaprenol) or mulberry

(moraprenol) are frequently used in biosynthetic studies as substitutes for bacterial undecaprenol. These polyprenols are composed of 10-12 isoprenic units (undecaprenol is the main component) with three internal E-isoprenic units localized at the w en d of the carbon hai in.^'.^^ See This Volume, pp. 341-385. (58) G. I. Vergunova, I. S. Glukhoded, L. L. Danilov, G. I. Eliseeva, N. K. Kochetkov, M. F. Troitsky, A. I. Usov, A. S. Shashkov, and V. N. Shibaev, Bioorg. Khim., 3 (1977) 1484-1492. (59) (60) (61) (62) (63) (64)

J. Tanaka and M. Takagi, Biochem. J., 183 (1979) 163-165. A. Wright, J. Bacteriol., 105 (1971) 927-936. H. Nikaido and K. Nikaido, J. Biol. Chem., 246 (1971) 3912-3919. T. Sasaki, T. Uchida, and K. Kurahashi, J. Bid. Chem., 249 (1974) 761-772. W. Jankowski and T. Chojnacki, Acra Biochim. Pol., 19 (1972) 51-69. W. Jankowski, T. Mankowski, and T. Chojnacki, Biochim. Biophys. Acra, 337 (1974)

153-162. (65) I. C. Hancock and J. Baddiley, Biochem. J., 127 (1972) 27-37. (66) W. Sasak and T. Chojnacki, Arch. Biochem. Biophys., 181 (1977) 402-410. (67) J. Schultz and A. D. Elbein, Arch. Biochem. Biophys., 160 (1974) 311-322. (68) D. J. Mancuso and T.-H. Chiu, J Bacteriol, 152 (1982) 616-625. (69) M. Scher, W. J. Lennan, and C . C. Sweeley, Roc. Narl. Acad Sci. USA, 59 (1968) 1313-1320. (70) M. Lahav, T.-H. Chiu, and W. J. Lennan, 1. Bid. Chem., 244 (1969) 5890-5898. (71) K. Takayama and D. S. Goldman, J. Bid. Chem., 245 (1970) 6251-6257. (72) K. Takayama and E. L. Armstrong, FEBS Len, 18 (1971) 67-69. (73) M. F. Mescher, U. Hansen, and J. L. Strominger, J. Biol. Chem., 251 (1976) 7289-7296.

BIOSYNTHESIS O F BACTERIAL POLYSACCHARIDE CHAINS

285

of B. ~ e r e u s . ~ Polyprenyl ~.~’ monophosphate ester of N-acetylneuraminic acid having an unidentified configuration at C-2 was detected in E. c01i.’~ In addition to polyprenyl glycosyl monophosphates formed from primary glycosyl nucleotides, synthesis of the P-D-galactopyranosyl derivative from in a system from Acetobacter xylinum. UDP-Gal was Polyprenyl glycosyl monophosphates have been shown to serve as glycosyl donors in the biosynthesis of side chains of some polysaccharides. Contrary to glycosyl nucleotides, they represent membrane-bound glycosyl donors which may be preferable for some glycosyltransferases.

3. Polyprenyl Glycosyl Diphosphates Formation of polyprenyl glycosyl diphosphates (compare the conversion of 1 into 3 in Scheme 1) is a result of attack by polyprenyl phosphate on a phosphorus atom linked to the glycosyl group; that is, the reaction is a glycosyl phosphate transfer, and the corresponding enzymes belong to the EC2.7.8 group. The configuration at C-1 of the glycosyl group in these derivatives may be expected to be the same as in the glycosyl nucleotides. The reaction seems characteristic for uridine glycosyl diphosphates. In addition to G l ~ - p p - B p r , ~identified ~ * ~ ~ * polyprenyl ~~ glycosyl diphosphates include derivatives of a-~-galactopyranose,~~-~’ 2-acetamido-2~ ~ ’ ~2-acetamido-2-deoxy-a-~-galac~-~~ deoxy-a-~ - g l u c o p y r a n o s e , ~ ~ ’and topyran~se.’~Also, several analogous N-acetylmuramylpentapeptide (74) S. Yamamori, N. Murazumi, Y. Araki, and E. Ito, J. Biol. Chem., 253 (1978) 6516-6522. (75) N. Murazami, S. Yamamori, Y. Araki, and E. Ito, J. Biol. Chem., 254 (1979) 11,79111,793. (76) F. A. Troy, I. K. Vijay, and N. Tesche, J. Biol. Chem., 250 (1975) 156-163. (77) R. C. Garcia, E. Recondo, and M. Dankert, Eur. J. Biochem., 43 (1974) 93-105. (78) P. Romero, R. C. Garcia, and M. Dankert, Mol. Cell. Biochem., 16 (1977) 205-212. (79) I. W. Sutherland and M. Norval, Biochem. J., 120 (1970) 567-576. (80) C. Weisgerber and K. J a m , Eur. J. Biochem., 127 (1982) 165-168. (81) M. Dankert, A. Wright, W. S. Kelley, and P. W. Robbins, Arch. Biochern Biophys., 116 (1966) 425-435. (82) M. J. Osborn and R. Yuan Tse-Yuen, J. Biol. Chem., 243 (1968) 5145-5152. (83) M. J. Osborn, M. A. Cynkin, J. M. Gilbert, L. Muller, and M. Singh, Methods Enzymol., 28 (1972) 583-601. (84) F. A. Troy, F. Frerman, and E. C. Heath, J. Biol. Chem., 246 (1971) 118-133. (85) M. E. Tolmasky, R. J. Staneloni, R. A. Ugalde, and L. F. Leloir, Arch. Biochem. Biophys., 203 (1980) 358-364. (86) L. J. Douglas and J. Baddiley, FEES Lerr., 1 (1968) 114-116. (87) D. Brooks and J. Baddiley, Biochem. J., 115 (1969) 307-314. (88) H. Hussey and J. Baddiley, Biochem. J., 127 (1972) 39-50. (89) H. A. I. McArthur, F. M. Roberts, I. C. Hancock, and J. Baddiley, FEES Lerr., 86 (1978) 193-200. (90) J. 9 . Ward and C. A. M. Curtis, Eur. J. Biochem., 122 (1982) 125-132.

286

VLADIMIR N. SHIBAEV

derivatives participate in the biosynthesis of the peptidoglycan of bacterial cell-wal~.~'-~~ Polyprenyl glycosyl diphosphates operate mainly as membrane-linked glycosyl acceptors in the biosynthesis of carbohydrate chains of bacterial polymers. In reactions of polymerization of repeating units during polysaccharide synthesis, the polyprenyl diphosphate derivatives serve as donors of growing polysaccharide chain, but monosaccharide transfer from a polyprenyl glycosyl diphosphate has never been detected.

111. BIOSYNTHESIS OF MONOSACCHARIDE COMPONENTS, AND THEIRACTIVATION FOR POLYM ERIC-CH AI N FORMATION A great diversity of monosaccharide structures is a characteristic feature of bacterial polysaccharide chains, especially of 0-specific chains of lipopolysaccharides from Gram-negative bacteria. Several surveys of monosaccharide components of bacterial polysaccharides have been p~blished.~~-~' The aim of this Section is to summarize present data on the structure of the monosaccharide constituents of bacterial polymers, their pathways of biosynthesis, and their modes of activation for subsequent incorporation into polymeric chains. Most of the uncommon monosaccharides arise as the result of transformations of the glycosyl group in glycosyl nucleotides. These transformations were discussed at length in a previous article on glycosyl nucleotides in this Series'*; it, as well as reviews by G l a ~ e or r~~ Gabriel and van Lenten,'" may be consulted for details omitted in the following discussion. (91) J. S. Anderson, M. Matsuhashi, M. A. Haskin, and J. L. Strominger, J. Biol. Chem., 242 (1967) 3180-3190. (92) C. P. Dietrich, A. V. Colucci, and J. L. Strominger, J. Eiol. Chem., 242 (1967) 3218-3225. (93) Y. Higashi, J. L. Strominger, and C. C. Sweeley, J. Biol. Chem., 245 (1970) 3697-3702. (94) J. N. Umbreit and J. L. Strominger, J. Bacteriol., 112 (1972) 1306-1309. (95) G. Ashwell and J. Hickman, in G. Weinbaum, S. Kadis, and S. J. Ajl (Eds.), Microbial Toxins, Vol. 4, Academic Press, London, 1971, pp. 235-266. (96) K. Jann and 0. Westphal, in M. Sela (Ed.),The Antigens, Vol. 3, Academic Press, New York, 1975, pp. 1-125. (97) S. G. Wilkinson, in Ref. 1, pp. 97-176. (98) L. Kenne and B. Lindberg, in G. 0.Aspinall (Ed.), The Polysaccharides, Vol. 2, Academic Press, New York, 1983, pp. 287-363. (99) L. Glaser, in G. 0. Aspinall (Ed.), Carbohydrates, Butterworths, London, 1973, pp. 191-212. (100) 0. Gabriel and L. van Lenten, in D. J. Manners (Ed.), Biochemistry of Carbohydrates 11, Univ. Park Press, Baltimore, 1978, pp. 1-36.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

287

In this Section, deoxy sugars, aminodeoxy sugars, and glycuronic acids are treated as modification products of simple aldoses that are classified here according to the structure of their formal, carbohydrate precursors. 1. D-Fructose-derived Aldoses

The most divergent group of monosaccharide components of bacterial polysaccharides is a group of aldohexopyranoses. The structures of sugars belonging to the group may be formally derived from D-glucopyranose through the following modification reactions: ( a ) epimerization at a chiral center of the monosaccharide residue, (b) exchange of a hydroxyl group for a hydrogen atom, leading to a deoxy sugar, ( c ) exchange of a hydroxyl group for an amino group, together with the N-acetylation that may follow (aminodeoxy and acetamidodeoxy sugars are formed as a result of this modification), and (d) oxidation at C-6, producing a hexuronic acid.'" In most cases, this formal operation actually corresponds to the real biosynthetic pathway of monosaccharide formation. Three monosaccharides of the group are constituents of primary glycosyl nucleotides (see formulas 1, 4, and 5). D-Glucose, D-mannose, and 2acetamido-2-deoxy-~-glucose 6-phosphates are formed through enzymic modifications of D-fructose 6-phosphate, and, after their isomerization to the corresponding a-D-hexopyranosyl phosphates, serve as substrates for synthesis of the primary glycosyl nucleotides which, in turn, are converted into secondary glycosyl nucleotides having modified'hexosyl groups.

a. D-gluco-, D-galacto-, and D-manno-Hexoses.-The derivatives of this group that are most widely distributed in Nature possess the D-gluco, D-galacto, and D-manno configurations of the hexosyl groups; their structures are summarized in Table I. Transition from the D-gluco to the D-galacto configuration occurs through enzymic epimerization at C-4 of the hexosyl group in glycosyl nucleotides. ~ *D- P ' ~-~G ~ C N A C , ~ * ~ ~ ' - ~ ~ ~ Such reactions were observed for U D P - G ~ C , ' ~ * ' U (101) The following abbreviations are used in the Tables for these modifications: En for epimerization at C-n, H" for exchange of OH + H at C-n, N" for exchange of OH + NH2 (NHAc) at C-n, and A for the conversion CH20H+ C02H. (102) D. B. Wilson and D. S. Hogness, J. B i d . Chem., 239 (1964) 2469-2481; Methods Enzyrnol., 8 (1966) 229-240. (103) Y. Imae, N. Morikawa, and K. Kurahashi, 1. Biocbern. (Tokyo), 56 (1964) 138-144. (104) L.-J. Lee, A. Kirnura, and T. Tochikura, Agric. Bid. Chem., 42 (1978) 731-737. (105) L. Glaser, Biochim. Biopbys. Acta, 31 (1959) 575-576; J. Biol. Chem., 234 (1959) 28012805. (106) H. Kawai, K. Yamamoto, A. Kirnura, and T. Tochikura, Agric. B i d . Chern., 37 (1973) 1741-1743. (107) K. Yamamoto, H. Kawai, and T. Tochikura, Appl. Enoiron. Microbiol., 41 (1981) 392-395.

288

VLADIMIR N. SHIBAEV

and UDP-G~CA."~ All of the monosaccharides formed are quite common in bacterial polysaccharides. Formation of the a-~-xylo-hexosyl-4-ulose derivatives (6a-c) was suggested as an intermediate step in the epimerization at C-4. Convincing evidence for its presence as a component of a tightly bound enzyme-substrate complex was pre~ented'~'-"~ in the case of 6a.

R' 6

R' =OH, R2 = CH,OH b R' = NHAc, R2= CHzOH c R ' = O H , R2=C0,H a

Epimerization at C-2 of UDP-GlcNAc, leading to the UDP derivative of 2-acetamido-2-deoxy-~-mannose, was also ob~erved"~-"~; the monosaccharide was identified as a constituent of several capsular polysaccharides and Neisseria meningitidis group A from Streptococcus (Ref. 123), the teichuronic acid of Bacillus cereus,L24and the 0-specific (108) E. E. B. Smith, G. T. Mills, H. P. Bernheimer, and R. Austrian, Biochim. Biophys. Acra, 29 (1958) 640-641. (109) G. L. Nelsestuen and S. Kirkwood, J. Biol. Chem., 246 (1971) 7533-7543. (110) Y. Seyama and H. M. Kalckar, Biochemisrry, 1 1 (1972) 40-44. ( 1 1 1 ) U. S. Maitra and H. Ankel, Roc. Narl. Acad. Sci. USA, 68 (1971) 2660-2663. (112) U. S. Maitra and H. Ankel, J. Biol. Chem., 248 (1973) 1477-1479. (113) W. L. Adair, 0. Gabriel, D. Ullrey, and H. M. Kalckar, J. Biol. Chem., 248 (1973) 4635-4639. ( 1 14) U. S. Maitra, M. A. Gaunt, and H. Ankel, J. Bid. Chem., 249 (1974) 3075-3078. ( 1 15) T. Kawamura, N. Iochihara, N. Ishimoto, and E. Ito, Biochem. Biophys. Res. Commun., 66 (1975) 1506-1512. ( 1 16) T. Kawamura, N. Kimura, S. Yamamori, and E. Ito, J. B i d . Chem., 253 (1978) 3595-3601; T. Kawamura, N. Ishimoto, and E. Ito, Methods Enzymol., 83 (1982) 515-519. (117) H. C. Lew, H. Nikaido, and P. H. Makela, J. BacrerioL, 136 (1978) 227-233. (118) T. Yoneyama, Y. Koike, H. Arakawa, K. Yokoyama, Y. Sasaki, T. Kawamura, Y. Araki, E. Ito, and S. Takao, J. Bacteriol., 149 (1982) 15-21. (119) L. G. Bennett and C. T. Bishop, Can. J. Chem., 58 (1980) 2724-2727. (120) N. Ohno, T. Yamodae, and T. Miyazaki, Carbohydr. Res., 80 (1980) 297-304. (121) P.-E. Jansson, B. Lindberg, and U. Lindqvist, Carbohydr. Res., 95 (1981) 73-80. (122) M. B. Perry, V. Daoust, and D. J. Carlo, Can. J. Biochem., 59 (1981) 524-533. (123) D. R. Bundle, I. C. P. Smith, and H. J. Jennings, J. Biol. Chem., 249 (1974) 2275-2281. (124) K. Amano, S. Hazama, Y. Araki, and E. Ito, Eur. J. Biochem., 75 (1977) 513-522.

BIOSYNTHESIS O F BACTERIAL POLYSACCHARIDE CHAINS

289

TABLEI Structures and Activated Forms of D-gluco-. D-galacto-, nod D-manno-Hexoses Present in Bacterial Polysaccharidesa Configuration of hexosyl residue Modification Unmodified NZ N2N' A N ~ A N'N'A H6 H6H3 H~N* H6N3 H6N4 H6N2N4

D-&CO

UDP' UDP' X UDP UDP X dTDPd CDP UDP

X dTDP X

D-galacto

D-manno

UDP UDP UDP X dTDPd CDP UDP dTDPd dTDP UDPd

GDP' UDP GDP UDP X GDP CDP

The symbol X in the Table means that an activated form of the monosaccharide is not known; a dash shows that the monosaccharide has not been identified as a component of bacterial polysaccharides. For references, see the text. * See Ref. 101 for abbreviations. Primary glycosyl nucleotide. See comments in the text.

polysaccharide of Shigellu dy~enten'ae.'~~ It was suggested that the mechanism of the epimerization includes intermediate oxidation at C-3 of the hexosyl group.126 A general pathway of biosynthesis of nucleotide-linked hexuronic acids is oxidation of nucleoside hexosyl diphosphates catalyzed by NAD-linked, four-electron-transferring dehydrogenases (for a review, see Ref. 127). The best known reaction of this type is oxidation of the UDP-Glc leading to UDP-GlcA. ''*128*129 An activated derivative of D-mannuronic acid, which is a component of the extracellular polysaccharide of Arthrobucter ~ i s c o s u s 'and ~ ~ of an alginic acid-like polysaccharide"' produced by (125) B. A. Dmitriev, Yu. A. Knirel, 0.K. Sheremet, N. K. Kochetkov, and I. L. Hofman, Bioorg. Khim., 3 (1977) 1219-1225. (126) W. L. Salo, Biochim. Biophys. Acra, 452 (1976) 625-628. (127) D. S. Feingold and J. S. Franzen, Trends Biochem. Sci., 6 (1981) 103-106. (128) E. E. B. Smith, G. T. Mills, R. Austrian, and H. P. Bernheimer, J. Gen. Microbiol., 22 (1960) 265-271. (129) A. Bdolah and D. S. Feingold, J. Bacteriol., 96 (1968) 1144-1149. (130) I. R. Siddiqui, Carbohydr. Rer, 4 (1967) 277-283. (131) P. A. Sandford, Adu. Carbohydr. Chem. Eiochem., 36 (1979) 265-314.

290

VLADIMIR N. SHIBAEV

Azotobacter vinelandii and Pseudomonas aeruginosa, is formed through oxidation of GDP-Man. 1 3 2 ~ 1 3 32-Acetamido-2-deoxyhexuronicacids having ~ ~ ~~ ' ~ - r' n a n n o ~ ~ ~configurations -'~' are synthesized the D - ~ ~ U C O ~ and as UDP derivatives by oxidation of U D P - G ~ C N A C 'and ~ ~ UDPM ~ ~ N 115.1 A 17.140-142 ~ . For an analogous compound having the D-galacto c~nfiguration,'~~ an activated form has not been identified, but it seems reasonable to suggest that the most plausible way to formation of the UDP derivative of the monosaccharide is by oxidation of UDP-GalNAc, or epimerization at C-4 of UDP-GlcNAcA. Three monosaccharides of the group were identified which contain an amino group at both C-2 and C-3. These include acylated 2,3-diamino-2,3dideoxy-D-glucose, which is a component of the lipid A region of lipopolyand 2,3-di(acetamido)-2,3saccharides from Rhodopseudomona~,'~~ dideoxy-~-glucuronic~~~ and - ~ - m a n n u r o n i c Iacids, ~ ~ identified as constituents of P. aeruginosa 0-specific polysaccharides. The pathway of their biosynthesis, and their activated forms, remain unknown. The key intermediates in the biosynthesis of 6-deoxy sugars are the nucleoside 6-deoxyhexosyl-4-ulose diphosphates (7), formed through enzymic reactions catalyzed by NDP-sugar 4,6-dehydratases (EC 4.2.1.4547) from primary glycosyl nucleotides. These reactions were observed

(132) J. Preiss, J. Bid. Chem., 239 (1964) 3127-3132; Merhods Enzymol., 8 (1966) 285-287. (133) B. A. Pugashetti, L. Vadas, H. S. Prihar, and D. S. Feingold, J. BacrerioL, 153 (1983) 1107-1110. (134) S. Hanessian and T. H. Haskell, J. Bid. Chem., 239 (1964) 2758-2764. (135) E. J. Smith, J. Bid. Chem., 243 (1968) 5139-5144. (136) H. Mayer, Eur. J. Biochem., 8 (1969) 139-145. (137) H. R. Perkins, Biochem. J., 86 (1963) 475-483. (138) T. C. M. Wu and J. T. Park, J. Bacteriol, 108 (1971) 874-884. (139) D.-F. Fan, C. E. John, J. Zalitis, and D. S. Feingold, Arch. Biochem. Biophys., 135 (1969) 45-49; D.-F. Fan and D. S. Feingold, Merhods Enzymol., 28 (1972) 435-437. (140) N. Ichihara, N. Ishimoto, and E. Ito, FEES Leu., 39 (1974) 46-48. (141) T. Kawamura, N. Ishimoto, and E. Ito, J. Bid. Chem., 254 (1979) 8457-8465; Merhods Enzymol., 83 (1982) 519-522. (142) J. S. Anderson, R. L. Page, and W. L. Salo, J. Biol. Chem., 247 (1972) 2480-2485. (143) K. Heyns and G. Kiessling, Carbohydr. Res., 3 (1967) 340-353. (144) J. Weckesser, G. Drews, J. Roppel, H. Mayer, and I. Fromme, Arch. Microbiol, 101 (1974) 233-245. (145) B. A. Dmitriev, N. A. Kocharova, Yu. A. Knirel, A. S. Shashkov, N. K. Kochetkov, E. S. Stanislavsky, and G. M. Mashilova, Eur. J. Biochem., 125 (1982) 229-237. (146) Yu. A. Knirel, E. V. Vinogradov, A. S. Shashkov, B. A. Dmitriev, N. K. Kochetkov, E. S. Stanislavsky, and G. M. Mashilova, Eur. J. Biochem., 128 (1982) 81-90,

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

29 1

CH,

l a

R'=OH, R2=H, Z=dTDP

b R'=OH, R2=H, Z = C D P c R ' = H , R ~ = O HZ , =GDP d

R'=NHAc, R 2 = H , Z = U D P

~, 158 GDP-Man, '59-16 and UDPfor ~ T D P - G ~ c , ' ~ 'c- D ' ~P~- G ~148,150,156G~CNAC?~ (7a) at C-4 of the Reduction of dTDP-6-deoxy-~-xylo-hexos-4-ulose hexosyl group should lead to dTDP derivatives of 6-deoxy-~-glucose(Dquinovose) and 6-deoxy-~-galactose(D-fucose). Although D-quinovose and D-fucopyranose have not been found as components of bacterial polysaccharides, the corresponding glycosyl nucleotides were identifiedI4' in extracts of Escherichia coli Y-10. Non-stereospecific reduction of GDP-6-deoxy-~-lyxo-hexos-4-ulose(7c) to derivatives of 6-deoxy-~-mannose(D-rhamnose) and 6-deoxy-~-talose (see Section III,l,c) was found to occur with an enzyme from an unidentified, (147) R. Okazaki, T. Okazaki, J. L. Strominger, and A. M. Michelson, J. Biol. Chem., 237 (1962) 3014-3026. (148) M. Matsuhashi, J. M. Gilbert, S. Matsuhashi, J. G. Brown, and J. L. Strominger, Biochem. Biophys. Res. Commun., 15 (1964) 55-59. (149) J. M. Gilbert, M. Matsuhashi, and J. L. Strominger, J. Biol. Chem., 240 (1965) 1305-1308. (150) A. Melo, H. Elliot, and L. Glaser, J. Biol. Chem., 243 (1968) 1467-1474. (151) 0. Gabriel and L. C. Lindquist, J. Biol. Chem., 243 (1968) 1479-1484. (152) S.-F. Wang and 0. Gabriel, J. Biol. Chem., 244 (1969) 3430-3437. (153) H. Zarkowsky and L. Glaser, J. Biol. Chem., 244 (1969) 4750-4756; L. Glaser, H. Zarkowsky, and L. Ward, Methods EnzymoL, 28 (1972) 446-454. (154) S.-F. Wang and 0. Gabriel, J. Biol. Chem., 245 (1970) 8-14. (155) C. E. Snipes, G. U. Brillinger, L. Sellers, L. Mascaro, and H. G. Floss, 1 Biol. Chem., 252 (1977) 8113-8117. (156) S. Matsuhashi, M. Matsuhashi, J. G. Brown, and J. L. Strominger, J. Biol. Chem., 241 (1966) 4283-4287. (157) A. E. Hay and A. D. Elbein, J. Biol. Chem., 241 (1966) 5473-5478. (158) R. D. Bevill, Biochem. Biophys. Res. Commun., 30 (1968) 595-599. (159) V. Ginsburg, J. Biol. Chem., 236 (1961) 2389-2393; Methods Enzymol., 8 (1966) 293-295. (160) A. Markovitz, J. Biol. Chem., 239 (1964) 2091-2098; Methods EnzymoL, 8 (1966) 296-300. (161) A. D. Elbein and E. C. Heath, J. Biol. Chem., 240 (1965) 1926-1931. (162) J. Distler, B. Kaufman, and S. Roseman, Arch. Biochem. Biophys., 116 (1966) 466-478.

292

VLADIMIR N. SHIBAEV

Gram-negative b a ~ t e r i u m . ' ~D-Rhamnose ~*'~~ is a constituent of 0-specific polysaccharides from Xanthomonas ~arnpestris'~~ and Pseudomonas cepacia.I6' An analogous reaction of UDP-2-acetamido-2,6-dideoxy-~-xyZohexos-4ulose (7d) results in formation of the derivatives of 2-acetamid0-2~6-dideoxyD-glucose and -D-galactose.166These amino sugars were identified as components of several 0-specific polysaccharides (for reviews, see Refs. 95 and 167). CDP-6-deoxy-~-xylo-hexos-4-ulose (7b) serves as a precursor in the biosynthesis of CDP-3,6-dideo~yhexoses~'*'~' having the D-rib0 (paratose, 3,6dideoxy-"D-glucose"), D-xylo (abequose, 3,6-dideoxy-"~-galactose"),and D-arabino (tyvelose, 3,6-dideoxy-"~-mannose") configurations. These monosaccharides are characteristic components of 0-specific polysaccharides from Salmonella and Yersinia pseudotuberculosis. The conversion includes at least three enzymic reacti~ns.'~~-'''In the first stage, which requires pyridoxamine 5'-phosphate as a ofa actor,'^'^^^^ dehydration of 7b occurs through intermediate formation of the Schiff base.'73 Reduction of the resulting, unsaturated derivative with NADPH, the mechanism of which is not completely clear,'74 leads to CDP-3,6dideoxy-D-erythro-hexos-4-u10se,'~~ and, in the third stage, further reduction of the latter at C-4 of the hexosyl group produces the derivatives of paratose or abequose; the stereochemistry of the reaction is determined by the source of the enzyme.I6' The tyvelose derivative is formed as a result of enzymic epimerization at C-2 of the hexosyl group in CDP-paratose."' 3-Amino-3,6-dideoxyhexoses having the D-gluco and D-galacto configurations were identified as constituents of several lipopolysaccharides. In extracts of Xanthomonas campestris, conversion of 7a into a dTDP-3(163) N. M. Winkler and A. Markovitz, J. Biol. Chem., 246 (1971) 5868-5876. (164) J. Hickman and G . Ashwell, J. Biol. Chem., 241 (1966) 1424-1428. (165) Yu. A. Knirel, A. S. Shashkov, B. A. Dmitriev, N. K. Kochetkov, N. V. Kasyanchuk, and I. Ya. Zakharova, Bioorg. Khim., 6 (1981) 1851-1859. (166) A. Daniel, R. A. Raff, and R. M. Wheat, J. Bacteriol., 110 (1972) 110-116. (167) B. Jann, K. Jann, and G . 0. Beyaert, Eur. J. Biochem., 37 (1973) 531-534. (168) S. Matsuhashi, M. Matsuhashi, and J. L. Strominger, J. Biol. Chem., 241 (1966) 42674274; S. Matsuhashi and J. L. Strominger, Merhods Enzymol., 8 (1966) 310-316. (169) S. Matsuhashi and J. L. Strominger, J. Bid. Chem., 242 (1967) 3494-3500. (170) H. Pape and J. L. Strominger, J. BioL Chem., 244 (1969) 3598-3604. (171) P. Gonzalez-Porque and J. L. Strominger, J. Bid. Chem., 247 (1972) 6748-6756. (172) P. Gonzalez-Porque and J. L. Strominger, Proc. Narl Acad. Sci. USA, 69 (1972) 16251628. (173) P. A. Rubenstein and J. L. Strominger, J. Biol. Chem., 249 (1974) 3776-3781. (174) P. A. Rubenstein and J. L. Strominger, J. Bid. Chem., 249 (1974) 3782-3788. (175) S. Matsuhashi, J. Biol. Chem., 241 (1966) 4275-4282.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

293

acetamido-3,6-dideoxyhexosewas L-glutamate served as the amino-group donor and pyridoxal phosphate as the coenzyme. The configuration of the hexosyl group in the glycosyl nucleotide was not determined directly, but the polysaccharide from this strain contains 3-acetamido-3,6dideoxy-D-galactosyl units. L64*177 Three different 4-amino-4,6-dideoxy sugars have been found to be present in bacterial polysaccharides. Viosamine (4-amino-4,6-dideoxy-~-glucose) and thomosamine (4-amino-4,6-dideoxy-~-galactose) are constituents of 0-specific polysaccharides in different strains of E. ~ o l i , " whereas ~ the D-manno derivative (perosamine) is a characteristic component of the Biosynthesis of dTDP derivatives polysaccharide from Vibrio ch01erae.I~~ of N-acetylviosamine and N-acetylthomosamine occurs through transamination of 7a with L-glutamate and pyridoxal phosphate, followed by Nacetylation, with acetyl-CoA as the acetyl-group donor.180-182The stereochemistry at C-4 in the product is determined by the source of the transaminase. For perosamine, the mechanism of biosynthesis remains unclear, and the activated form has not been identified. It may be suggested that this monosaccharide is formed through transamination of GDP-6deoxy-~-lyxo-hexos-4-ulose, or through epimerization at C-2 of dTDP-4acetamido-4,6-dideoxy-~-glucose. Similar transamination of the hexosylulose group in 7d seems a possible way for the biosynthesis of activated derivatives of 2-acetamido-4-amino2,4,6-trideoxyhexoses. The reaction was demonstrated'62 in extracts of Strep tococcuspneumoniae type 14. The configuration of the hexosyl group formed was not determined, but the presence of a monosaccharide of the D-galucto configuration was demonstrated in polysaccharides of several strains of S. p n e ~ m o n i a e ' ~and ~ , 'Shigella ~~ sonnei,IS5and an analogous D-ghco-hexose was identified in polymers of Bacillus licheniformis'86 and P. a e r ~ g i n o s a . ' ~ ~ (176) W. A. Volk and G . Ashwell, Biochem. Biophys. Res. Commun., 12 (1963) 116-120. (177) G. Ashwell and W. A. Volk, J. Biol. Chem., 240 (1965) 4549-4555. (178) D. N. Dietzler and J. L. Strominger, J. Biol. Chem., 248 (1973) 104-109. (179) J. W. Redmond, FEES Letr., 50 (1975) 147-149. (180) M. Matsuhashi and J. L. Strominger, J. Biol. Chem., 239 (1964) 2454-2463; Methods Enzymol., 8 (1966) 317-323. (181) M. Matsuhashi and J. L. Strominger, J. Biol. Chem., 241 (1966) 4738-4744. (182) H. Ohashi, M. Matsuhashi, and S. Matsuhashi, J. Bid. Chem., 246 (1971) 2325-2330. (183) H. J. Jennings, C. Lugowsky, and M. N. Young, Biochemistry, 19 (1980) 4712-4719. (184) B. Lindberg, B. Lindqvist, J. Lonngren, and D. A. Powell, Carbohydr. Rex, 78 (1980) 111-1 17. (185) L. Kenne, B. Lindberg, K. Peterson, E. Katzenellenbogen, and E. Romanowska, Carbohydr. Res., 78 (1980) 119-126. (186) U. Zehavi and N. Sharon, J. Biol. Chem., 248 (1973) 433-438. (187) S. G. Wilkinson, Biochem. J., 161 (1977) 103-109.

VLADIMIR N. SHIBAEV

294

TABLEI1 Structures and Activated Forms of L-glum-, L-galaeto-, and L-mannoHexoses Present in Bacterial Polysaccharides" ~~

~

Configuration of hexosyl residue Modification N ~ A H6 H6H3 H~N* H6N3

L-gluco

L-galado

L-manno

X X

X GDP GDP

dTDP CDP X -

X -

'See footnote a to Table I, and Ref. 101.

b. L-gluco-, L-galacto-, and L-manno-Hexoses.-Monosaccharides of this group, listed in Table 11, are much less widely distributed in bacterial polymers than their D enantiomers. Among them only 6-deoxy-~-mannose (L-rhamnose) and 6-deoxy-~-galactose(L-fucose) may be considered to be common constituents of bacterial polysaccharides. Nucleoside 6-deoxyhexosyl-4-ulose diphosphates (7) formed from dTDP-Glc and GDP-Man serve as precursors of these monosaccharides. An enzyme was purified that catalyzes epimerization at C-3 and C-5 of the hexosylulose group in dTDP-6-deoxy-~-xylo-hexos-4-ulose (7a), resultd e r i v a t i ~ e ' ~(8a). ~-'~~ ing in formation of the 6-deoxy-~-lyxo-hexosyl-4-ulose Further reduction of the latter with NADPH leads to dTDP-L-rhamnose (dTDP-wa).23,147,190,192

8 a R' =OH, R2 = H, Z = dTDP b R'=H, R2=OH, Z = G D P

(188) (189) (190) (191) (192)

A. Melo and L. Glaser, J. Bid. Chem., 243 (1968) 1475-1478. 0. Gabriel, Methods Enzymol., 28 (1972) 454-461. R. W. Gaugler and 0. Gabriel, J. Bid. Chem., 248 (1973) 6041-6049. H. P. Wahl and H. Grisebach, Biochim. Biophys. Acta, 568 (1979) 243-252. L. Glaser and S. Kornfeld, J. Biol. Chem., 236 (1961) 1795-1799; Methods Enzymol., 8 (1966) 302-306.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

295

A similar mechanism seems probable for conversion of GDP-6-deoxy-~*’~’ in lyxo-hexos-4-ulose (7c) into GDP-L-fucose ( G D P - F U C ) , ~ ~although, this case, the enzymes that participated in the process were not separated, and intermediate formation of the 6-deoxy-~-xylohexosyl-4-dose derivative (8b) was not demonstrated. The hexosylulose derivative 7c may also be converted into the G D P 3,6-dideoxy-“~-galactderivative of colitose (3,6-dideoxy-~-xylo-hexose, a component of lipopolysaccharides from several enterobacterial strains. Another monosaccharide of this group, ascarylose (3,6-dideoxy-~arabino-hexose, 3,6-dideoxy-“~-mannose”),which is present in Yersinia pseudotuberculosis lipopolysaccharides, was found to be synthesized as the CDP derivative through epimerization and reduction of CDP-B-deoxy-~xylo-hexos-4-ulose. 168-171 The origin of other monosaccharides of the group remains unclear. 2-Acetamido-2,6-dideoxy-hexoses having the manno no'^^ and ~-galacto” configuration can probably be formed from UDP-GlcNAc and UDPManNAc through reactions similar to those involved in the biosynthesis of dTDP-Ma and GDP-Fuc. The analogous L-gluco d e r i ~ a t i v e ” may ~ be related to the L-galacto sugar. It seems much more difficult to suggest a reasonable pathway for the biosynthesis of the 3-acetamido-3,6-dideoxy-~glucose present in the core region of Aeromonas hydrophila lipopolysachar ride,''^ and of the 2-acetamido-2-deoxy-~-galacturonic acid identified as a component of lipopolysaccharides from P. aeruginosa. 187,197 c. Hexopyranoses of Other Configurations.-A number of hexopyranosyl components (of bacterial polysaccharides) having configurations different from gluco, galacto, and manno have been identified; their structures are summarized in Table 111. In most cases, it is quite easy to see how these monosaccharides may be derived from monosaccharides present in primary glycosyl nucleotides, or formed from them through the enzymic reactions already discussed. Experimental evidence for activated forms of the sugars of this group is presented only in the case of 6-deoxytalose, which is an epimer, at C-4, of 6-deoxymannose. In the D series, the GDP derivative of the monosaccharide is formed, together with GDP-D-rhamnose, through nonspecific, enzymic (193) E. C. Heath and A. D. Elbein, Proc. Nafl. Acad. Sci. USA, 48 (1962) 1209-1216; A. D. Elbein and E. C. Heath, Merhods EnzymoL, 8 (1966) 300-302. (194) B. Jann and K. Jann, Eur. J. Biochem., 5 (1968) 173-177. (195) B. A. Dmitriev, L. V. Backinowsky, N. K. Kochetkov, and N. A. Khomenko, Eur. J. Biochem., 31 (1973) 513-518. (196) J. H. Banoub and D. H. Shaw, Carbohydr. Res., 98 (1981) 93-103. (197) S. G. Wilkinson and A. P. Welbourn, Biochem. J., 149 (1975) 783-784.

VLADIMIR N. SHIBAEV

296

TABLE111

Hexose Components of Bacterial Polysaccharides Having a Configuration Different from gluco, galacto, or manno

Monosaccharide D- NlOSe 6-Deoxy-~-altrose 6-Deoxy-~-talose 6-Deoxy-~-talose 4-Deoxy-~-arabino-hexose 2-Amino-2,6-dideoxy-~-talose L-Guluronic acid L-Iduronic acid 2-Amino-2-deoxy-~-altruronic acid 2-Amino-2-deoxy-~-taluronic acid 2,3-Diamino-2,3-dideoxy-~-guluronic acid 4-Deoxy-~threo-hex-4-enuronic acid

a See footnote a to Table I. meric level.

See Ref. 101.

Activated form"

Possible relation to common hexoses

X X GDP dTDP

D-gluco-E' ~-galacto-H~E' o-manno-H6E4 L-manno-H6E4 o-manno-H4E3 L- manno-H6N2E4 D-manno-AES ~-gIuco-AE' D-galacto-N2AE' L-manno- N'AE" ~-manno-N'N~AE' dehydration of D-galacturonic acid

X X

-e X

X X X X

' Formed through modification at the poly-

reduction of the carbonyl group in GDP-6-deoxy-~-lyxo-hexos-4u ~ o s ~ . ' In ~ ~the. ' L~series, ~ the dTDP derivative of the monosaccharide is a product of reduction of dTDPd-deoxy-~-lyxo-hexos-4-ulose;in this case, the stereochemistry of the reduction is determined by the source of the enzyme. Most bacteria produce L-rhamnose,but, in some strains, B-deoxy-~talose is formed. 189*190*198 The analogous pathway starting from UDP2-acetamido-2,6-dideoxy-~-xylo-hexos-4-ulose seems quite probable for ( N-acetylpneumosamthe biosynthesis of 2-acetamido-2,6-dideoxy-~-talose ine), a component of Streptococcus pneumoniae capsular polysaccharide~.'~~ Epimerization at C-3 and C-5 of the hexosyl group in the hexosyl-4-ulose derivatives seems to be a quite common transformation of glycosyl nucleotides. The assumptions that these two reactions may occur independently, and that the process may be completed after inversion at only one chiral center, allow suggestion of an explanation for the origin of the D-allosezoO

(198) C. A. Tylenda, D. Charon, F. P. Lombardi, and 0. Gabriel, Infect. Irnmun., 23 (1979) 3 12-3 19. (199) 0. Larm and B. Lindberg, Adv. Carbohydr. Chem. Biochem., 33 (1976) 295-322. (200) A. Misaki, Y. Tsumuraya, M. Kakuto, H. Takemoto, and T. Igarashi, Carbohydr. Res., 75 (1979) c8-cl0.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

297

found in the extracellular polysaccharide of Pseudomonas species, and for 6-deoxy-~-altrose:~' a component of Yersinia lipopolysaccharides. It is necessary to postulate the existence of a previously unknown, modification reaction of glycosyl nucleotides, that is, deoxygenation at C-4, in order to explain the formation of 4-deoxy-~-arabino-hexose, a monosaccharide constituent of Cirrobacter lipopolysaccharide.202rl-Deoxy-~-fhreohex-4-enuronic acid, found in Klebsiella K22 polysac~haride:~~ may be formed through dehydration of the D-galacturonic acid group or residue, either in the glycosyl nucleotide, or in the polysaccharide. Formation of L-guluronic acid, a component of the alginic acid-like polysaccharide produced by P. aeruginosa and Azorobacfer vinelandii, requires special comment. In this case, a polymer built from /3-(1+ 4)linked D-mannosyluronic acid residues serves as an intermediate in the bio~ynthesis.*@'*~~~Part of the D-mannosyluronic acid residues in the polymer is subjected to an epimerization at C-5 catalyzed by an exocellular enzyme of the micro-organism,205-207 producing a polysaccharide composed of structural blocks that contain only D-mannosyluronic acid or only Lgulosyluronic acid residues, as well as some having both. The mechanism of the epimerization remains unclear. It seems probable that other L-hexuronic acids listed in Table 111, such as L-iduronic,208 2-amino-2-deoxy-~-altruronic,~~ and 2,3-diamino-2,3dideoxy-L-guluronic2" acids may arise in bacterial polymers as a result of epimerization at C-5 at the polymer level. Such a pathway was demonstrated for the biosynthesis of L-idosyluronic acid residues in glycosaminoglycans of higher animals.211*212 The origin of 2-amino-2-deoxy-~-taluronic acid:" a constituent of pseudomurein of Mefhanobacreriurn, is difficult to explain at present. (201) J. Hoffman, B. Lindberg, and R. R. Brubaker, Carbohydr. Res., 78 (1980) 212-214. (202) E. Romanowska, A. Romanowska, C. Lugowski, and E. Katzenellenbogen, Eur. J. Biochern., 121 (1981) 119-123. (203) H. Niemann, H. Friebolin, and S. Stirm, unpublished data cited in Ref. 98. (204) F. Couperwhite and M. F. McCallum, Arch. Microbiol.. 97 (1974) 73-80. (205) D. F. F'indar and C. Bucke. Biochem J., 152 (1975) 617-622. (206) 9. Larsen and A. Haug, Carbohydr. Res., 17 (1971) 287-296. (207) A. Haug and B. Larsen, Carbohydr. Res., 17 (1971) 297-308. (208) L. Lee and R. Cherniak, Carbohydr. Res., 33 (1974) 387-390. (209) T. Kontrohr, Carbohydr. Res., 58 (1977) 498-500. (210) Yu. A. Knirel. E. V. Vinogradov, A. S. Shashkov, B. A. Dmitriev, N. K. Kochetkov, E. S. Stanislavsky, and G. M. Mashilova, Eur. 1. Biochem. 134 (1983) 289-297. (211) U. Lindahl, in G. 0. Aspinall (Ed.), Carbohydrates, Ser. 2, Buttenvorths, London, 1976, pp. 283-312. (212) L. R o d h and M. I. Horowitz, in M. 1. Horowitz and W. F'igman (Eds.), 7 l e Glycoconjugales. Vol. 2. Academic Press, New York, 1978, pp. 3-71. (213) H. Konig and 0. Kandler, Z. Physiol. Chern., 361 (1980) 981-983.

298

VLADIMIR N. SHIBAEV

d. Other Monosaccharides, Structurally Related to D-Fructose.-Pentoses are rather infrequent components of bacterial polysaccharides. Part of these monosaccharides may be considered to be biogenetically related to D-ribose ~ ’ ~ ~-arabinose”~ should be (see Section 1142). Nevertheless ~ - x y l o s e and treated as D-fructose-derived monosaccharides. In higher plants, the pathway of their biosynthesis consists in decarboxylation of the D-glucosyluronic acid group of UDP-GlcA, followed by epimerization at C-4 of the resulting pentosyl group. These reactions were not demonstrated with bacterial enzymes, although, in a prokaryotic, blue-green alga, formation of the UDP derivatives of the pentoses through decarboxylation of UDP-GlcA and UDP-GalA was shown.216UDP-Glc 4-epimerase of E. coli can use UDP-Dxylose as a substrate.’” 4-Amino-4-deoxy-~-arabinose,a component of the lipid A fragment of bacterial lipopolysaccharides, may probably be included in this group, although its biosynthesis has not been investigated. Several monosaccharides mentioned in this Section are present in polysaccharide chains not only as pyranoses but also as furanoses. From the biogenetic point of view, a furanosidic form of a monosaccharide must be considered to be an additional component, as no ready interconversions of cyclic forms may be expected for monosaccharide residues incorporated into oligosaccharide chains, or in the activated form used for their formation. Such a situation is rather common for D-galactose and the structurally related monosaccharides L-arabinose2’* and D-fucose; thus far, the latter monosaccharide has been identified only as the furanose form in the 0specific polysaccharide of Eubacterium ~ a b u r r e u m . 6-Deoxy-~-altrose ~’~ is present as the pyranose in the lipopolysaccharide of Yersinia enterocolitica,201but as the furanose in a similar polymer2*’ of Y. pseudotuberculosis type VB. Paratose, which is usually present in polysaccharides as the a-pyranose, was identified as the P-furanose in the 0-specific polysaccharide221*2’2 of Y. pseudotuberculosis serotype IB. (214) M. B. Perry, V. Daoust, B. B. Diena, F. E. Ashton, and R. Wallace, Can. J. Biochem., 53 (1975) 623-629. (215) R. E. Hurlbert, J. Weckesser, H. Mayer, and I. Fromme, Eur. J. Biochem., 68 (1976) 365-371. (216) D.-F. Fan and D. S. Feingold, Arch. Biochem. Biophys., 148 (1972) 576-580; Methods Enzymol, 28 (1972) 438-439. (217) H. Ankel and U. S. Maitra, Biochem. Biophys. Res. Commun., 32 (1968) 526-532. (218) S. G. Wilkinson, L. Galbraith, and W. J. Anderson, Carbohydr. Res., 112 (1983) 241-252. (219) J. Hoffman, B. Lindberg, T. Hofstad, and H. Lygre, Carbohydr. Res., 58 (1977) 439-442. (220) N. I. Korchagina, R. P. Gorshkova, and Yu. S. Ovodov, Bioorg. Khim., 8 (1982) 1666- 1669. (221) S. V. Tomshich, R. P. Gorshkova, Yu. N. Elkin, and Yu. S. Ovodov, Eur. J. Biochem., 65 (1976) 193-199. (222) V. V. Isakov, R. P. Gorshkova, S. V. Tomshich, Yu. S. Ovodov, and A. S. Shashkov, Bioorg. Khim., 7 (1981) 559-562.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

299

The nature of the activated forms of furanose monosaccharides and the route of their formation still remain a mystery. During an investigation of the biosynthesis of Salmonella T1 antigen, a polysaccharide composed of D-galactofuranosyl and D-ribofuranosyl residues, the D-galactopyranosyl group of UDP-Gal was shown to serve as the precursor of the D-galactofuranosyl residues in the p o l y ~ a c c h a r i d e .Isomerization ~ ~ ~ * ~ ~ ~ of the Dgalactopyranosyl group of UDP-Gal into a D-galactofuranosyl residue without splitting of the glycosyl-phosphate linkage was found to be catalyzed by an enzyme from the mold Penicillium charlesii.225Intermediate formation of the hexosyl-2-ulose derivative was s ~ g g e s t e d , ~but ~ ~further *~~’ details of the mechanism remain unclear. A similar process was not observed with enzymes of bacterial origin. Branched-chain monosaccharides have now been detected as components of bacterial polysaccharides. The known examples include yersiniose [3,6~ ~ ~ Y. ] pseudotuberculosis, dideoxy-4- C-(hydroxyethy1)-D-xylo-h e x o ~ e from ~ ~ ~6a 3 -C-(hydroxymethy1)pentofuranose from Coxiella b ~ r n e t t i ,and deoxy-3-C-methylhexoses from the same organism and from Nitrobacter h a m b u r g i e n s i ~ Several . ~ ~ ~ branched-chain monosaccharides were identified as components of antibiotics, and the pathways of their biosynthesis in bacteria were studied. These investigations were discussed in detail by Grisebach in this Series.230The usual precursors for the formation of the monosaccharides of this group are the nucleoside 6-deoxyhexosyl-4-ulose diphosphates 7a and 7b. 2. D-Ribose-derived Aldoses

Bacterial polysaccharides frequently contain D-ribofuranosyl residues as components of their polymeric chains. D-Ribdose 5-phosphate was shown to serve as a precursor for the D-ribofuranosyl units in Salmonella T1 antigen,224but details of the enzymic reactions and the nature of the activated (223) H. Nikaido and M . Sarvas, J. BacterioL, 105 (1971) 1073-1082. (224) M. Sarvas and H. Nikaido, J. Bacterial., 105 (1971) 1063-1072. (225) A. G. Trejo, J. W. Haddock, G . J. T. Chittenden, and J. Baddiley, Biochem. J., 122 (1971) 49-57. (226) W. S. Fobes and J. E. Gander, Biochem. Biophys. Res. Commun., 49 (1972) 76-83. (227) M. T. Johnson and J. E. Gander, Biochim. Biophys. Acta, 523 (1978) 9-18. (228) R. P. Gorshkova, N. A. Komandrova, V. A. Zubkov, and N. I. Korchagina, Abstr. Eur. Symp. Carbohydr. Glycoconjug., Znd, Budapest, (1983) C-26; R. P. Gorshkova, V. A. Zubkov, V. V. Isakov, and Y. S. Ovodov, Carbohydr. Res., 126 (1984) 308-312. (229) S. Schramek, J. Radziejewska-Lebrecht, and H. Mayer, Abstr. Eur. Symp. Carbohydr. Glycoconjug., 2114 Budapest, (1983) C-4. (230) H. Grisebach, Adu. Carbohydr. Chem. Biochem., 35 (1978) 81-126.

VLADIMIR N. SHIBAEV

300

form of the monosaccharide remain unclear. In a note23’published in 1961, the detection of dTDP-ribose, in addition to other dTDP-sugars, in extracts of Streptomyces was described, but confirmatory evidence for the presence of the glycosyl nucleotide and for its enzymic reactions has not yet been presented. Other monosaccharide components (of bacterial polysaccharides) that are structurally related to D-ribose include D-riburonic identified in the exocellular polysaccharide produced by a strain of Rhizobium meliloti, and D-arabinose, frequently present as the furanose, in polysaccharides of mycobacterial ~ e l l - w a l l . ~ * ~- ~~~ y~ ~l ~o sshould e ~ ~probably ~ * ~ ~be~included in the group, as it may be derived from D-arabinose through epimerization at C-4. Biosynthesis of these monosaccharides was not investigated.

3. D-Sedoheptulose-derived Aldoses

In this group of monosaccharide components of bacterial polysaccharides, a primary glycosyl nucleotide is ADP-D-glycero-D-manno-heptose ( 9 ) , identified in extracts of a mutant strain of Shigella s~nnei.’~’ CHZOH

I

HOCH

w HO

OH

9

Formation of a possible precursor of 9, namely, D-glycero-D-mannoheptose 7-phosphate, from D-sedoheptulose 7-phosphate was demonstrated in Salmonella typhim~rium.~~‘ (231) J. Baddiley, N . Blumsom,A. DiGiloramo, and M. DiGiloramo, Biochim. Biophys. Acra, 50 (1961) 391-392. (232) A. Amemura, M. Hisamatsu, S. K. Ghai, andT. Harada, Carbohydr. Res.,91(1981) 59-65. (233) A. Misaki, N. Seto, and I. Azuma, J. Biochem. (Tokyo),76 (1974) 15-27. (234) A. Misaki, I. Azuma, and Y. Yamamura, J. Biochem. (Tokyo),82 (1977) 1759-1770. (235) J. Weckesser, G . Rosenfelder, H. Mayer, and 0. Liideritz, Eur. J. Biochem., 24 (1971) 112-1 15. (236) D. J. Neal and S. G . Wilkinson, Carbohydr. Res., 69 (1979) 191-201. (237) T. Kontrohr and B. Kocsis, J. Biol. Chem., 256 (1981) 7715-7718. (238) L. Eidels and M. J. Osborn, J. Biol. Chem., 249 (1974) 5642-5648.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

301

Biosynthesis of the most common constituent of the bacterial-lipopolysaccharide core, L-glycero-D-manno-heptose,was to occur through enzymic epimerization at C-6 of the heptosyl group in 9. Genetic supports this pathway of biosynthesis. Three other heptoses present in bacterial lipopolysaccharides are probably biogenetically related to the heptoses mentioned, although experimental evidence for enzymic reactions leading to their formation has not been described. These monosaccharides include D-glycero-D-galacto-heptose, found in Eubacterium saburreum lipopolysaccharides in both the pyranofrom s i d i ~ and ~ ~ 'furanosidic2" forms, 6-deoxy-~-manno-heptopyranose~~~ the same source, and 6-deoxy-~-altro-heptofuranose,identified as a component of Yersinia lipop~lysaccharides.~~~ 4. Ketoses and 3-Deoxyaldulosonic Acids

Two of the most frequent monosaccharide components of bacterial polymers belonging to this group have been the subjects of articles in this Series. They are 3-deoxy-~-manno-2-octulosonic a normal constituent of the core region of bacterial lipopolysaccharides that is also present in some other polymers, and N-acetylneuraminic found in several capsular polysaccharides. Enolpyruvate phosphate serves as the precursor of the C-1-C-3 fragment of the monosaccharides, with D-arabinose 5-phosphate or 2-acetamido-2-deoxy-~-mannose 6-phosphate being an acceptor for transfer of the three-carbon unit. Characteristic, activated forms of these monosaccharides are the CMP derivatives. 3-Deoxy-~-glycero-pentulosonicacid was found to be present in Klebsiella K38 p o l y ~ a c c h a r i d eand ~~~ 3-deoxy-~threo-hexulosonic acid was identified .~~~ polyas a component of Azotobacter capsular p o l y ~ a c c h a r i d e0-Specific saccharides of Pseudomonas aeruginosa and Shigella boydii were shown2" (239) T. Kontrohr and B. Kocsis, Absrr. Eur. Symp. Carbohydr. Glycoconjug., 2nd, Budapest, (1983) C-9; B. Kocsis and T. Kontrohr, 1.Bid. Chem., 259 (1984) 11,858-1 1,860. (240) W. G . Coleman, J. Bid. Chem., 258 (1983) 1985-1990. (241) L. Eidels and M. J. Osborn, Proc. Natl. Acad. Sci. USA, 68 (1971) 1673-1677. (242) V. Lehmann, G . Hammerling, N. Nurminen, F. Minner, E. Ruschmann, 0. Liideritz, T. T. Kuo, and B. A. D. Stocker, Eur. J. Biochem., 32 (1973) 268-275. (243) J. Hoffman, B. Lindberg, S. Svensson, and T. Hofstad, Carbohydr. Res., 35 (1974) 49-53. (244) W. Kondo, F. Nakazawa, M. Sato, and T. Ito, Carbohydr. Rex, 117 (1983) 125-131. (245) K. Samuelson, B. Lindberg, and R. Brubaker, J. Bacreriol., 117 (1974) 1010-1016. (246) J. Hoffman, B. Lindberg, and J. Liinngren, Carbohydr. Res., 47 (1976) 261-267. (247) F. M. Unger, Ado. Carbohydr. Chem. Biochem., 38 (1981) 323-388. (248) R. Schauer, Adu. Carbohydr. Chem. Biochem., 40 (1982) 132-234. (249) B. Lindberg, K. Samuelsson, and W. Nimmich, Carbohydr. Res., 30 (1973) 63-70. (250) D. Claus, Biochem. Biophys. Res. Commun., 20 (1965) 745-751. (251) Yu. A. Knirel, E. V. Vinogradov, A. S. Shashkov, N. K. Kochetkov, V. L. L'vov, and B. A. Dmitriev, Carbohydr. Res., 141 (1985) c l - c 3 .

302

VLADIMIR N. SHIBAEV

to contain N-acylated 5,7-diarnino-3,5,7,9-tetradeoxy-~-glycero-~-mannononulosonic acid. The biosynthesis of these monosaccharides and the nature of their activated forms have not yet been clarified. The ketohexoses identified as components of bacterial polysaccharides include D-fructose, found in Vibrio lipopoly~accharide'~~ and Hemophilus influenzae capsular poly~accharide,*~* and D-xylulose ( D- threo-2-pentulose), identified in Pseudomonas lipopoly~accharide.~~~ Activated forms of the monosaccharides were not determined. 5. Monosaccharide Structures and the Nature of Their Activated Forms Table IV, which briefly summarizes the material described in previous sub-sections, shows the total of the different monosaccharide components identified in bacterial polysaccharides, and our present knowledge about their activated forms. It may be seen that identification of the activated forms has been achieved for only approximately half of the monosaccharides known to be involved. The most striking gap in the information available is the lack of data about the activated forms of D-ribose-derived monosaccharides and of most of the D-fructose-derived aldoses having configurations other than gluco, galacto, and manno. Nevertheless, some generalizations concerning the favored structures of the activated forms for different monosaccharides seem possible. Thus, the known D-sedoheptulose-derived monosaccharides are activated as ADP derivatives, and 3-deoxyaldulosonic acids as CMP derivatives. In a group of D-fructose-derived aldoses, activation as the UDP derivatives is favored for monosaccharides of the D-gluco and D-gulacto configuration. The exceptions are 6-deoxyhexoses, which are activated as the dTDP derivatives, and 3,6-dideoxyhexoses, where activation as the CDP derivatives is characteristic. The monosaccharides having the D-munno and L-galucto configuration are activated mainly as GDP derivatives. In the first group, nevertheless, the 3,6-dideoxyhexoseis present as the CDP derivative, and 2-acetamido-2deoxyhexoses are activated as the UDP derivatives. 6. Modifications of Functional Groups in Monosaccharide Units

Many bacterial polysaccharides contain, in addition to the monosaccharide constituents summarized in Table IV, a number of noncarbohydrate (252) P. Branefors-Helander, L. Kenne, B. Lindberg, K. Peterson, and P. Unger, Carbohydr. Res., 88 (1981) 77-84. (253) S. G . Wilkinson, Carbohydr. Res., 98 (1981) 247-252.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

303

TABLEIV Activation of Monosaccharides for Biosynthesis of Polymeric Chains Number of different monosaccharides known Activated forms" Biogenetic POUP

dT

U

1. D-Fructosederived aldoses 2 D-glUC0 Series 5 3 D-galacro Series 5 D-manno Series 2 L-gluco Series L-galacro Series L-manno Series - 1 Others 2 1

Total

1

4

2. D-Ribose-derived nldoses Total

G

C

-

1 1 1

3

_

-

2

1

-

_

1 7

6

4

A

-

_ _

Total known

Unknown

Total

8

4

9 6

1 2 2 2

12 10

-

8 2 4

2 2 4

1 10

14

-

31

22

53

-

_

3

-

-

-

-

-

4

4

3. D-Sedoheptulose-derived aldoses _ _ Total

_

_

2

2

3

5

2

5

7

35

34

69

4. Ketoses and 3-deoxyaldulosonicacids - _ _ Total

2

Total

6

1

4

7

6

2

Abbreviations denote nucleoside residues of activated forms.

components that are usually linked to oxygen atoms of hydroxyl groups in monosaccharides; the amino group in aminodeoxy sugars, and the carboxyl group in glycuronic acids may also be modified. A modification of the monosaccharide units of polysaccharides may obviously be effected at different stages of the biosynthesis of a polymer: ( a ) prior to formation of the activated form of a monosaccharide, (b) at the level of glycosyl nucleotides, ( c ) at the stage of formation of oligosaccharide intermediates, and ( d ) after the synthesis of a polymeric chain. The most complete list of modified monosaccharide units of bacterial polysaccharides may be found in a review by Kenne and Lindberg." The present discussion is limited only to examples where some data on biosynthesis are available.

304

VLADIMIR N. SHIBAEV

Two known types of modification of monosaccharide units with formation of ether bonds include 0-methylation and 0-(1-carboxyethy1)ation;both ( R ) - and (S)-lactyl ethers have been identified. 0-Methylated sugars are frequent constituents of 0-specific chains of bacterial polysaccharides. Weckesser and coworkers254compared the content of 0-methylated sugars in different polymers, and concluded that, in some cases, they constitute integral components of repeating units of the polymer; that is, all of the monosaccharide residues of a certain type in the polymer are modified. It seems reasonable to suggest that, in these examples, methylation takes place prior to formation of the polymeric chains. The opposite case, when only part of the monosaccharide residues of the polymer is modified, also exists. In a mannan, the 0-specific polysaccharide of Klebsiellu 05, 3- 0-methyl-D-mannose is present only at the nonreducing end of the polymeric chain,”’ and the same is true for the 3-O-methyl-~rhamnosyl group in a rhamnan,2s6 the polymer of Klebsiellu 010. For homopolysaccharides, growth of the polymeric chain by transfer of monosaccharide groups to its nonreducing end is very typical (see Section V), and methylation of a terminal monosaccharide group may be a signal for cessation of chain elongation. The biosynthesis of two polysaccharides, from Mycobucteriu, that contain 0-methylated sugar residues was investigated. The methyl group was found to originate from a methionine residue. The structures of several precursors polysaccharide allow the suggesof the 3- 0-methyl-D-mannose-containing tion that methylation occurs at the polymer level at the same time as the process of chain elongati~n.~’~ In the case of the acylated polysaccharide, which contains residues of 6-O-methyl-~-glucose,both modifications take place after formation of the polymeric chain, but the acylation reaction precedes incorporation of the methyl 0-(1-Carboxyethyl) ethers of monosaccharides are integral components of the polysaccharide repeating-units in all such cases investigated. This fact allows the suggestion that the modification should occur prior to polymeric chain-formation. Experimental evidence for modification at the glycosyl nucleotide level was obtained only for the most widely distributed monosaccharide of this group, namely, N-acetylmuramic acid, which is a component of bacterial peptidoglycan. In this case, enolpyruvate phosphate serves as a modifying agent, and UDP-GlcNAc as a substrate, for (254) (255) (256) (257) (258)

J. Weckesser, H. Mayer, and J. Fromrne. Bioehem. J., 135 (1973) 293-297. B. Lindberg, J. Liinngren, and W. Nimmich, Acfa Chem. Scand., 26 (1972) 2231-2236. H. Bjorndal, B. Lindberg, and W. Nimmich, Acta Chem. Scand., 24 (1970) 3414-3415. H. Yamada, R. E. Cohen, and C. E. Ballou, J. Biol. Chem., 254 (1979) 1972-1979. E. Grellert and C. E. Ballou, J. Bid. Chem., 247 (1972) 3236-3241.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

305

m o d i f i ~ a t i o n . *The ~ ~ *enol ~ ~ ~ether initially formed is further reduced in a NADPH-dependent r e a ~ t i o n . ' ~ ' - ~ ~ ~ Similar enol ethers probably serve as intermediates in another common modification-reaction of monosaccharide units especially characteristic of exocellular polysaccharides, namely, the formation of cyclic acetals of pyruvic acid. In the biosynthesis of the capsular polysaccharide from Xunthomonus cumpestris, the modification was shown265to occur at the level of a polyprenyl pentasaccharide diphosphate intermediate prior to polymerization of the repeating units, and enolpyruvate phosphate was a precursor of the pyruvic acid residues. A similar observation was made during a study of the biosynthesis of Rhizobium meliloti exopolysaccharide.266 0-Acylation also provides a frequent modification of monosaccharide residues in bacterial polysaccharides, and, in most cases, substitution with acyl groups is only partial. Acetyl-coenzyme A was shown to serve as a precursor of 0-acetyl groups in polysaccharides. The reaction takes place at the polymer 1eve1,267-271 or in oligosaccharide intermediates of the biosynthesis of the poly~accharide.'~~.~'~

IV. INTER-MONOMERIC LINKAGESI N BACTERIALPOLYSACCHARIDES As already mentioned, formation of glycosidic linkages between monomeric units of the carbohydrate-containing polymers of the bacterial cell-surface is catalyzed by membrane-bound glycosyltransferases, and glycosyl nucleotides are the usual glycosyl donors in the reaction. (259) (260) (261) (262) (263)

K. G. Gunetileke and R. A. Anwar, J. Bid. Chem., 241 (1966) 5740-5743. R. I. Zernell and R. A. Anwar, 1. Bid. Chem., 250 (1975) 3185-3192; 4959-4964. A. Taku, K. G . Gunetileke, and R. A. Anwar, J. Bid. Chem., 245 (1970) 5012-5016. A. Taku and R. A. Anwar, J. Bid. Chem., 248 (1973) 4971-4976. G. G. Wickus, P. A. Rubenstein, A. D. Warth, and J. L. Strorninger, J. Bacteriol., 213

(1973) 291-295. (264) R. A. Anwar and M. Vlaovic, Can. J. Biochem., 57 (1979) 188-196. (265) L. Ielpi, R. 0. Couso, and M. Dankert, Biochem. Biophys. Rex Commun., 102 (1981) 1400- 1408. (266) M. E. Tolrnasky, R. J. Staneloni, and L. F. Leloir, J. Bid. Chem., 257 (1982) 6751-6757. (267) P. W. Robbins, J. M. Keller, A. Wright, and R. L. Bernstein, J. Bid. Chem., 240 (1965) 384-390. (268) C. G. Hellerqvist, U. Rudbn, and P. H. Makela, Eur. J. Biochem., 25 (1972) 96-101. (269) K.-K. Tung and C. E. Ballou, J. Biol. Chem., 248 (1973) 7126-7133. (270) U. Rudbn and P. H. Makela, Eur. J. Biochem., 48 (1974) 11-20, (271) W. F. Vann, T. Y. Liu, and J. 9. Robbins, J. Bacteriol., 133 (1978) 1300-1306. (272) 1. W. Sutherland and J. F. Wilkinson, Biochem. J., 110 (1968) 749-754.

306

VLADIMIR N. SHIBAEV

Inter-monomeric linkages in polysaccharide chains may differ. Consequently, the enzymes that operate in the biosynthesis of these chains must be specific for the type of inter-monomeric linkage formed; that is, they must be able to catalyze formation of a glycosidic linkage of defined configuration with a definite hydroxyl group of a glycosyl acceptor. The proper sequence of inter-monomeric linkages in a polymer structure may be achieved only if the enzymes can, in addition, recognize the structures of both the glycosyl donor and the glycosyl acceptor. A minimal requirement here is an ability of the enzyme to identify, without error, monosaccharide residues of both partners of the reaction. As a result, at least one specific enzyme should exist for the synthesis of each disaccharide fragment that is present in a polymeric chain. Actually, several different enzymes may be responsible for the synthesis of the same disaccharide fragment in different polymers, if other details of the acceptor or the donor structure are essential for their proper interaction with the enzyme. This concept, known as273-275 the “one enzyme-one linkage concept,” has been widely used in discussions of the biosynthesis of carbohydrate chains in animal glycoproteins and proteoglycans. It implies that the structure of a carbohydrate chain is dictated by the specificity of the enzymes that participate in glycosidic-bond formation. Application of the same concept for discussion of the biosynthesis of bacterial polysaccharides seems worth while. Approximately 400 different glycosyltransferases are necessary in order to ensure the synthesis of those bacterial polysaccharides whose structures have thus far been elucidated. This estimate is based on the results of an analysis of the structures, made in order to ascertain how many different disaccharide fragments are present. An example of such an analysis is shown in Table V for the disaccharide sequences L-rhamnopyranosyl-D-galactopyranose, D-mannopyranosyl-L-rhamnopyranose,and D-galactopyranosylD-mannopyranose that are characteristic for the 0-specific polysaccharides of Salmonella serogroups A, B, D, and E, the objects of many biosynthetic studies. Full details of similar analyses for other disaccharide sequences will be published elsewhere, as the resulting Tables are too voluminous for inclusion in this Chapter, but the most interesting results are summarized in Tables VI and VII. (273) H. Schachter and L. Rod& in W. H. Fischman (Ed.), Metabolic Conjugation and Metabolic Hydrolysis, Vol. 3, Academic Press, New York, 1973, pp. 1-149. (274) L. Rodin and N . B. Schwartz, in W. J. Whelan (Ed.), Biochemistry of Carbohydrates, Butterworths, London, 1975, pp. 95-152. (275) H. Schachter and S. Roseman, in W. J. Lennan (Ed.), The Biochemistry of Glycoproteins and Ploteoglycans, Plenum Press, New York, 1980, pp. 85-110.

BIOSYNTHESIS O F BACTERIAL POLYSACCHARIDE CHAINS

307

TABLEV Some Disaccharide Fragments of Bacterial Polysaccharides" Structures of the fragments 1. Sequence L-Rha-D-Gal (5)b + )-P-Rha-( 1 + 4)-P-Gal-( 1+ + )-a-Rha-( 1 + 3)-a-Gal-( 1+

+)-a-Rha-(1 + 3)-p-Gal-(l+

+ )-a-Rha-( 1 + 2 ) - p G a l - ( l+

0-Specific polysaccharides

Salmonella serogroups A, B, D I , Dz, El, Ed Salmonella serogroups Ez, E3 E. coli 069, Sh. dysenreriae type 1

-

Capsular polysaccharides

Klebsiella K73 Klebsiella K9, K12, K32, K41 Klebsiella K18, K36, K47, K81, K83 Klebsiella K56 Sir. pneumoniae

type 23F

2. Sequence D-Man-L-Rha (2) + )-a-Man-( 1 + 4)-a-Rha-( 1+

3. Sequence D-Gd-D-MaO (7) +)-a-Gal-(l-+6)-a-Man-(l+ +)-a-Gal-(l+6)-p-Man(l+ + )-&Gal-( 1 -+ 6)-P-Man-( 1 -* + )-P-Gal-(1 + 4)-p-Man-( 1+ + )-a-Gal-( 1 + 3)-a-Man-( 1 +

Salmonella serogroups A, B, Dl Salmonella serogroups DZ, El-4

Sh. boydii type 6 Salmonella serogroups D,, El, E4 Salmonella serogroups Ez, E3 Sh. dysenreriae type 9 -

Salmonella serogroups A, B, Dl -

Klebsiella K30

E. coli K30, Klebsiella K20, K28, K60, K61, DD-45 E. coli K29, Klebsiella K49 E. coli K30, Klebsiella K20, K59, K74

a The disaccharide fragments listed in this Table are not found in the polysaccharides of Gram-positive cell-walls. For references on the structures of 0-specific and capsular polysaccharides mentioned in this Table, see Refs. 98 and 276. The total number of isomeric, disaccharide fragments identified in different polymers is shown in parentheses.

The list of the most common monosaccharide components of bacterial polysaccharides, shown in Table VI, includes the sugars that were found at the nonreducing end of nine or more different disaccharide fragments of the polymers. The right-hand column of the Table, which shows the total number of such fragments, gives an estimate of the number of different glycosyltransferases that use the activated form of the monosaccharides as

308

VLADIMIR N. SHIBAEV TABLEVI

Number of Different Disaccharide Fragments of the Polysaccharides Having the Most Common Monosaccharides at the Nonreducing End" Disaccharide fragments

Glycosyl group D-Glucopyranosyl D-Galactopyranosyl L-Rhamnopyranosyl D-Glucopyranosyhronic acid 2-Acetamido-2-deoxyD-glucopyranosyl D-Mannopyranosyl 2-Acetamido-2-deoxyD-galactopyranosyl D-Galactofuranosyl L-Fucopyranosyl 2-Acetamido-2-deoxyD-tIIannOpyraIIOSyl D-Ribofuranosyl

Activated form

With a-linked sugar

With &linked sugar

Total number

UDPUDPdTDPUDP-

37 38 26 14

45 28 11 21

82 64 37 35

UDPGDP-

15 17

19 11

34 28

UDPXGDP-

14 1 10

11 13 1

25 14 11

UDPX-

3 0

7 9

10 9

Structures of bacterial polysaccharides collected in Refs. 98 and 276, with additions from subsequent publications, were analyzed.

glycosyl donors in the biosynthesis of polysaccharide chains.277The data on activated forms of monosaccharides, and the frequency of glycosylation with inversion and with retention of configuration at the glycosidic center, are also included. It may be seen that UDP-activated sugars strongly preponderate among the monosaccharides most frequently found in the polysaccharides, although L-rhamnose (dTDP-activated) and D-mannose (GDP-activated) are also in the top half of the list. In most cases, a- and P-linked glycosyl units are almost equally frequent in the polymers, although glycosylation with inversion of configuration seems to predominate for L-rhamnose and, especially, for L-fucose. Furanosyl residues are present exclusively in the p configuration, with only one exception, namely, for D-galactofuranose. Table VII illustrates the diversity of inter-monomeric linkages in bacterial polysaccharides. It shows how many different isomeric disaccharide frag(276) K. Jann and B. Jann, Methods Enzyrnol, 50 (1978) 251-272. (277) Strictly speaking, the glycosidic linkages in bacterial polysaccharides are not always formed with participation of glycosyltransferases (see Section V,l).

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

309

TABLE VII Total Number of Isomeric Disaccharide Fragments of the Polysaccharides Composed of the Most Common Monosaccharides' ~

Nonreducing group of the fragment D-GlC

D-Gal L-Rha D-GICA D-GIcNAc D-Man D-GalNAc a

~~

Reducing residue of the fragment D-Glc

D-Gd

L-Rha

D-GkA

D-GIcNAc

D-Man

13 11 9 I 6 4 2

11 9

10 9 6 5

10 9 4

7

6

I 2

-

4 2

3 4 3

5 4 3 3 4 2 1

4 3 7 2

3 2 2 4

5 7 3 3 3

2

1

D-GaINAc

2

See footnote a to Table VI.

ments composed of the seven most frequent sugars have been identified in these polymers. Almost all of the disaccharide sequences possible were found to be present: among 49 sequences listed in Table VII, only the Rha-GalNAc fragment has not yet been detected. For some sequences, such as Glc-Glc, Glc-GlcA, and Glc-Rha, almost all of the possible variants of isomeric disaccharide fragments have been discovered (the number of isomers theoretically possible is 16 when a hexose is at the reducing end, and 12 for a deoxy sugar, an acetamidodeoxy sugar, or a glycuronic acid in this position). In these cases, many different glycosyltransferases should exist that use similar partners of the glycosylation reaction, but catalyze formation of different types of inter-monomeric linkage. Progress in the purification of the membrane-bound glycosyltransferases responsible for the synthesis of bacterial polysaccharides is still very limited. The available data are discussed in the next Section in connection with particular biosynthetic systems.

V. ASSEMBLY OF POLYMERIC CHAINS

The aim of the present Section is to discuss existing information concerning sequences of glycosylation reactions during the assembly of polysaccharide chains. Data concerning identification of intermediates in the process, and on solubilization and purification of the enzymes involved, are also included.

VLADIMIR N. SHIBAEV

310

1. The Mechanisms of the Chain Assembly

Two mechanisms were demonstrated for the assembly of linear chains of bacterial p ~ l y s a c c h a r i d e s . Their ~ ~ ~ *general ~ ~ ~ features are presented in Scheme 2, using as an example a hypothetical polysaccharide (-B-A-)” composed of disaccharide repeating units. pi

A-p;fq A-pp-Y

B- A- pp- Y (B- A ) , , - P P - ~

B-A-B-A-Y

+-I

PP-y

(B- A), -X

1

b

SCHEME2

The term “monomeric mechanism” will be used for the mechanism depicted in the left-hand part of Scheme 2 (sequence a). In this case, the monosaccharide residues are transferred consecutively from the corresponding glycosyl donors (Z-A or Z’-B) onto a membrane-bound glycosyl acceptor. The acceptor is generally a monosaccharide residue, which may be a fragment of an oligosaccharide chain linked to a hydrophobic molecule embedded in a cell membrane. In many instances, the acceptor that is used for assembly of the polymeric chain (Y) is not identical to the final acceptor (X) of the chain, and further transfer of the chain from Y to X, or liberation of the polysaccharide molecule in the case of exocellular polysaccharides, is a necessary step in the biosynthesis. At least three different glycosyltransferases participate in assembly of the polymeric chain through this mechanism: ( i ) the initiation A-transferase, (278) P. W. Robbins, D. Bray, M. Dankert, and A. Wright, Science, 158 (1967) 1536-1542. (279) J. F. Robyt, Trends Biochem. Sci., 4 (1979) 41-49.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

311

which specifically recognizes the structure of the intermediate acceptor Y, ( i i ) a B-transferase specific for A as a terminal monosaccharide residue of the acceptor, and (iii) an A-transferase specific for B as a terminal monosaccharide residue of the acceptor. In this mechanism, the polymeric chain grows from the nonreducing end, that is,28o“tailwards.” A feature characteristic of the second mechanism for polysaccharidechain assembly (sequence b in Scheme 2), which may be called the “block mechanism” consists in intermediate formation of an oligosaccharide repeating-unit of a polymer, followed by its further polymerization. In this case, a phosphorylated acceptor is necessary for the first stage of the process, which constitutes transfer of glycosyl phosphate onto the phosphate group of the acceptor. Polyprenyl phosphates (p-Y) generally serve as intermediate acceptors for the polymeric-chain assembly, and only nucleoside glycosyl diphosphates (A-ppN) may function as glycosyl phosphate donors in the initiation reaction which results in liberation of nucleoside monophosphate and is readily reversible. The second stage of the process is completely analogous to the second step in the monomeric mechanism; a B-transferase specific for A as a terminal monosaccharide residue of the acceptor is requiredfor the reaction. In the block mechanism, an A-B glycosidic linkage is formed in a reaction catalyzed by a repeating-unit polymerase, and additional monosaccharide donors are not necessary for the reaction. In all cases investigated, the polymerization occurs in such manner that the growing polymeric chain is transferred onto a single repeating-unit serving as an acceptor; that is, the chain grows “headwards” (from the reducing end). Polyprenyl diphosphate is liberated in the polymerization reaction, and its dephosphorylation is necessary before entrance of the acceptor into the next turn of the cycle. This reaction is specifically inhibited by the polypeptide antibiotic bacitracin. The concept of “one glycosyltransferase-one linkage” (see Section IV) seems to be completely valid for the monomeric mechanism of the assembly of chains. For the block mechanism, it should be slightly modified: formation of the glycosidic linkage of the monosaccharide-initiator of the chain growth ( a in Scheme 2) requires the consecutive action of two enzymes (the glycosyl phosphatetransferase and the polymerase), instead of one glycosyltransferase. (280) According to the general terminology suggested by F. Lipmann for the description of biosynthetic processes?’’ the head of a molecule is a functional group linked to a fragment of a precursor that is necessary for activation of the biosynthetic reaction, and the opposite end of the molecule may be called the tail. In oligosaccharide chains, the reducing end of the chain may be regarded as the head of the molecule, and the nonreducing end of the chain as its tail. (281) F. Lipmann, Essays Biochem., 4 (1968) 1-23.

312

VLADIMIR N. SHIBAEV

The final stages of the biosynthetic process may include transfer of a polysaccharide chain from an intermediate acceptor to a final acceptor, or liberation of the free polysaccharide. Monosaccharide residues included in a polymeric chain may be subjected to further enzymic modifications, such as incorporation of 0-linked substituents (see Section III,6) or the epimerization at C-5 of glycuronic acid residues mentioned in Section III,l,c. In the case of branched polysaccharides, incorporation of side chains may take place through different mechanisms. In one of them, the assembly of the main chain is independent of the presence of side chains, and their incorporation into a polymeric molecule occurs as a modification of an initially formed, linear polysaccharide. Another situation is possible when incorporation of monosaccharide residues present in side chains is a necessary condition for elongation of the main chain, either through the monomeric or the block mechanism; that is, intermediate formation of a linear, polysaccharide chain does not occur. Both mechanisms of incorporation of side chains were demonstrated to take place. 2. 0-Specific Chains of Bacterial Lipopolysaccharides

In bacterial lipopolysaccharides, 0-specific chains composed of repeating, or modified repeating, units are linked to a unique oligosaccharide sequence of the core region which is connected to a lipid A fragment serving as a hydrophobic anchor embedded in the bacterial outer-membrane. Biosynthesis of 0-specific chains was found to occur independently on formation of other structural fragments of the lipopolysaccharide molecule. Both block and monomeric mechanisms were demonstrated for the biosynthesis of these polymers. a. Biosynthesis Through a Block Mechanism.-The best-studied example of biosynthesis of bacterial polysaccharides is certainly that of the 0-specific polysaccharides of Salmonella serogroups E and B having the structures9’ shown in formulas 10-12. Their main chains are composed of trisaccharide repeating-units that differ in the type of linkages between monomeric units of the main chain (compare, Table V) and in the nature and position of the monosaccharide side-chains. + 6)-&Man-( 1 + 4)-a-Rha-(1 + 3)-a-Gal-(1 +

6

t

R

10

a R = Ac (S. anatum, serogroup E,) b R = a-GIG(1 + (S. senfrenberg, serogroup E4)

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

313

+6)-P-Man-(l+4)-a-Rha-(1+3)-P-Gal-(l+ 4

t

R

11

R = H (S. anaturn €I5, serogroup E2) b R = a-Glc-(l+ (S. anaturn serogroup E3)

a

+2)-u-Man-(l+4)-a-Rha-(l+3)-a-Gal-(l+ 3 n

t

t

a-Glc-1

a-Abe-1 2

t

X 12

a n = 4, X = Ac ( S . ryphirnurium, serogroup B) b n = 6, X = H (S. bredeney, serogroup B)

These polysaccharides were the first examples where the block mechanism of polysaccharide-chain assembly was established, and the main features of the mechanism were first demonstrated in just this single case (for reviews, see Refs. 282-284). Bacterial polyprenyl phosphate serves as the intermediate glycosyl acceptor in the assembly of repeating units of the main chains of the polymers; the reaction sequence includes transfer of a-D-galactopyranosyl phosphate from UDP-Gal to the acceptor, followed by transfer of an L-rhamnopyranosyl group from dTDP-Rha and of a D-mannopyranosyl group from GDP-Man. Such a mechanism was demonstrated in Salmonella strains belonging to serogroups B (Refs. 82, 83, 285, and 286), EI-E3 (Refs. 60, 81,287, and 288), and E4 (Ref. 289), and also in a strain of Citrobacter that produces a similar p o l y s a ~ c h a r i d e . ~ ~ ~ M. J. Osborn, in L. I. Rothfield (Ed.), Structure and Funcrion ofBiologica1 Membranes, Academic Press, New York, 1971,pp. 343-377. A. Wright and S. Kanegasaki, Physiol. Reu., 51 (1971)748-784. H. Nikaido. in L. Leive (Ed.), Bacterial Membranes and Walls, Dekker, New York, 1973, pp. 131-208. I. M. Weiner, T. Higuchi, L. Rothfield, M. Salmarsh-Andrew, M. J. Osborn, and 9. L. Horecker, Roc. Natl. Acad. Sci. USA, 54 (1965)228-235. T. N. Druzhinina, L. M. Gogilashvili, V. N. Shibaev, S. Sh. Rozhnova, and V. A. Kilesso, Bioorg. Khim., 9 (1983)1074-1081. A. Wright, M.Dankert, and P. W. Robbins, Roc. Natl. Acad. Sci. USA, 54 (1965)235-241. R. Losick and P. W. Robbins, J. Mol. Biol., 30 (1967)445-455. V. N. Shibaev, T. N. Druzhinina, A. N. Popova, S. Sh. Rozhnova, and V. A. Kilesso, Biorg. Khim., 5 (1979)1071-1082. R. Yuan and B. L. Horecker, 1. Bacteriol., 98 (1968)2242-2248.

314

VLADIMIR N. SHIBAEV

The structures of polyprenyl diphosphate-linked intermediates of Salmonella 0-specific-polysaccharide biosynthesis were confirmed by chemical synthesis of their analogs derived from the plant polyprenols ficaprenol and moraprenol (structurally related to bacterial p ~ l y p r e n o l with ~ ~ ) the following study of their behavior as substrates of enzymic reactions. Synthetic polyprenyl a-D-galactopyranosyl d i p h ~ s p h a t e ~was ~ ' . found ~ ~ ~ to serve as an effective acceptor for the transfer of L-rhamnosyl g r o ~ p s . Two ~ ~ ~ - ~ ~ ~ synthetic, isomeric disaccharide derivatives,29213 and29614, were tested as acceptors for enzymic D-mannosyl transfer from GDP-Man, but only the former was found to be an efficient substrate.294 Finally, the synthetic moraprenyl trisaccharide diphosphate 15a entered the next stage of biosynthesis, the polymerization of repeating units catalyzed by an enzyme preparation from S. a n a t ~ m . ~ ~ ~ a-Rha-(1 + 3)-a-Gal-pp-Mpr 13

P-Rha-(l+ 3)-a-Gal-pp-Mpr 14

~-Man-(l+4)-a-Rha-(l+3)-a-Gal-pp-Pre 15 a

Pre=Mpr

b Pre=Bpr

The ability of the polymerase to use exogenous polyprenyl trisaccharide diphosphate as a substrate had been demonstrated earlier by Kanegasaki and Wright,298 who employed the derivative 15b prepared by enzymic (291) C. D. Warren and R. W. Jeanloz, Biochemistry, 11 (1972) 2565-2572. (292) L. L. Danilov, S. D. Maltsev, V. N. Shibaev, and N. K. Kochetkov, Carbohydr. Res., 88 (1981) 203-211. (293) Yu.Yu. Kusov, E.V. Kiseleva, L. L. Danilov, V. N. Shibaev, N. K. Kochetkov, S. Sh. Rozhnova, and V. A. Kilesso, Bioorg. Khim., 5 (1979) 1863-1872. (294) V. N. Shibaev, L. L. Danilov, T. N. Druzhinina, V. 1. Torgov, L. M. Gogilashvili, and N. S. Utkina, Bioorg. Khim.,8 (1982) 564-566. (295) S. Kanegasaki, A. Wright, and C. D. Warren, Eur. J. Biochem., 133 (1983) 77-81. (296) L. L. Danilov, N. S. Utkina, V. N. Shibaev, and N. K. Kochetkov, Bioorg. Khim., 7 (1981) 1718-1721. (297) V. N. Shibaev, T. N. Druzhinina, N. A. Kalinchuk, S. D. Maltsev, L. L. Danilov, V. 1. Torgov, N. K. Kochetkov, S. Sh. Rozhnova, and V. A. Kilesso, Bioorg. Khim., 9 (1983) 564-565. (298) S. Kanegasaki and A. Wright, froc. Natl. Acad. Sci. USA, 67 (1970) 951-958.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

315

synthesis. Pulse-chase experiment^^'^*^^^ showed growth of the polymeric chain from the reducing end in the reaction. The polymerases may catalyze the formation of new glycosidic linkages with both inversion and retention of configuration at the glycosidic center of the monosaccharide initiator. In the lysogenic transformation of S. anaturn with bacteriophage E ” , change of the structure of the 0-specific polysaccharide from 10a into lla occurs, and this conversion was ~ h o ~ to depend on production of three new proteins, directed by genetic information of the phage. These proteins include a repressor for the synthesis of enzyme(s) participating in polysaccharide acetylation, an inhibitor of the polymerase of the host responsible for the synthesis of a-(1+ 6)-glycosidic linkages, and a new polymerase that catalyzes formation of /3-(1+6) linkages. The factors that determine the degree of polymerization of the polysaccharides remain unclear. It seems that the process is not under strict control, as significant heterogeneity in chain length is observed for Salmonella 0-specific p o l y s a c ~ h a r i d e s . ~ In~some ~ - ~ ~cases, ~ the degree of polymerization was found to depend on the conditions of cultivation of the microorgani~m.~~’-~~~ Incorporation of abequosyl side-chains in 0-specific polysaccharides (12) of Salmonella serogroup B occurs at the stage of repeating-unit assembly. The polyprenyl trisaccharide diphosphate 16a serves as an acceptor for ~’ reaction was abequosyl transfer from its CDP d e r i ~ a t i v e . * ~A* ~similar demonstrated with an enzyme preparation from Citr~bacter.’~~ Polymerases ~ ” ~S.~ bredeney286were able to act on the derivafrom S. t y p h i r n u r i ~ r n ~and tives of linear trisaccharide 16a, but, in the former case, the efficiency of a-Man-(1 + 4)-a-Rha-(1 + 3)-a-Gal-pp-Pre 16

a Pre=Bpr b Pre= Mpr D. Bray and P. W. Robbins, Biochem. Biophys. Res. Commun., 28 (1967) 334-339. D. Bray and P. W. Robbins, J. Mol. Biol., 30 (1967) 457-475. R. Losick, 1. Mol. Biol., 42 (1969) 237-246. J. M. Ryan and H. E. Conrad, Arch. Biochem. Biophys., 162 (1974) 530-535. E. T. Palva and P. H. Makela, Eur. J. Biochem., 107 (1980) 137-143. R. C. Goldman and L. Leive, Eur. J. Biochem., 107 (1980) 145-153. S. Schlecht and 1. Fromme, Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg., Abt. 1 : Orig., Reihe A, 233 (1975) 199-222. (306) M. McConnel and A. Wright, J. Bacteriol., 137 (1979) 746-751. (307) M. J. Osborn and I. M. Weiner, J. Biol. Chem., 243 (1968) 2631-2639. (308) J. L. Kent and M. J. Osborn, Biochemistry, 7 (1968) 4409-4419.

(299) (300) (301) (302) (303) (304) (305)

n

~

316

VLADIMIR N. SHIBAEV

the polymerization increased significantly when the substrate contained abequosyl side-chains. 307*308The presence of these branches seems essential for the biosynthesis of 0-specific polysaccharides in uiuo?O9 as a mutant of S. typhimurium having a blocked synthesis of CDP-abequose does not produce the 0-specific polysaccharide. Incorporation of D-glucosyl branches that modify part of the D-galactosyl residues of the main chains in polymers lob, l l b , and 12 occurs with polyprenyl D-glucosyl monophosphate (2) as the D-glucosyl donor.6042,286,289.3 10 The branches linked through a-(1+ 4) linkages, as in polysaccharides l l b and 12a, seem to be introduced after, or simultaneously with, the polymerization reaction. The most convincing evidence was presented for the case of a biosynthetic system from Salmonella serogroup E3 strain:’ where formation of a D-ghcosylated polymer was demonstrated, whereas the enzymically prepared trisaccharide derivative 1Sb was not able to serve as a D-glucosyl acceptor. Absence of the reaction with the oligosaccharide intermediate may be explained by different anomeric configurations of the D-galactosyl residue in the polyprenol-linked derivative 15b and in polymer l l b . It was suggested6’ that D-glucosylation of the growing polymeric chain occurs after formation of the p-Gal-( 1 + 6)-Man linkage, and that all of the D-galactosyl residues in the resulting polymer are D-glucosylated, except that present at the reducing end of the chain and linked to a polyprenyl diphosphate residue. A similar mechanism of incorporation of D-glucosyl side-chains was suggested for the polysaccharide 12a from S. typhimurium. In this case, a partially D-glucosylated polymer having an unmodified D-galactosyl residue linked to polyprenyl phosphate was identified”’ in a mutant strain of the micro-organism, with blocked incorporation of the 0-specific chains into the lipopolysaccharide molecule. The enzymically prepared trisaccharide derivative 16a was found unable to undergo D-glucosylation, although some reaction was detected3’’ with a higher concentration of its synthetic analog 16b. In contrast, introduction of the a-(1+ 6)-linked D-glucosyl branches present in polysaccharides 10b and 12b was shown to occur readily at the level of polyprenyl oligosaccharide diphosphates. With enzymic systems from S. s e n f r e ~ z b e r gand ~~~ S. bredeney,286D-glucosylation was found to take place prior to the polymerization reaction. In the former case, participation (309) R. Yuasa, M. Levinthal. and H. Nikaido. 1. Bacterid, 100 (1969) 433-444. (310) H. Nikaido, K. Nikaido, T. Nakae, and P. H. Makela, 1. B i d Chem., 246 (1971) 3902-3911. (311) M. Takeshita and P. H. Makela, J. Bid. Chem., 246 (1971) 3920-3927. (312) T. N. Druzhinina, L. M. Gogilashvili, and V. N. Shibaev, in preparation.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

317

of the synthetic derivative 15b as the glucosyl acceptor,312and of analogs of 2 derived from moraprenol and other higher alcohols as ~-glucosyl was demonstrated. The synthetic moraprenyl diphosphate derivative (17) of a branched trisaccharide may be used as an acceptor for a-Rha-(1 + 3)-a-Gal-pp-Mpr 6

t

a-Glc-1 17

o-mannosyl transfer, and the resulting, D-glucosylated repeating-unit derivative was able to enter a polymerization reaction giving rise to fully D-glucosylated, polysaccharide chains.314 The last stage of biosynthesis of 0-specific polysaccharides consists in transfer of the polymeric chain from an intermediate acceptor, polyprenyl diphosphate, to a final acceptor, a D-glucosyl residue of the core region of the lipopolysaccharide. The reaction was demonstrated in the case of the S. fyphimuriurn lipopolysaccharide.83”5 Genetic data allow the suggestion of participation of at least two proteins in the r e a ~ t i o n . ”When ~ synthesis of these proteins or of the enzymes responsible for assembly of the core oligosaccharide region is blocked, the polysaccharide chain remains linked to polyprenyl diphosphate existing3” as so-called “0-hapten.” Purification of individual enzymes participating in the biosynthesis of Salmonella 0-specific polysaccharides has still not been achieved. The enzymes responsible for the assembly of oligosaccharide repeating-units, including those involved in the incorporation of D-glucosyl, but not abequosyl, branches, may be solubilized by treatment of the bacterial For the D-galactosyl cell-envelopes with non-ionic phosphatetransferase, partial solubilization was reported to occur after sonic treatment of the bacterial cells.319The first attempt at purification of this enzyme by the use of affinity chromatography was described.320Attempts to solubilize 0-specific polysaccharide polymerase were unsuccessful. (313) T. N. Druzhinina, L. L. Danilov, V. N. Shibaev, N. K. Kochetkov, A. N. Pankrushina, Yu. L. Sebjakin, L. V. Volkova, and R. P. Evstigneeva, Bioorg. Khim, 7 (1981) 760-767. (314) V. N. Shibaev, T. N. Druzhinina, L. M. Gogilashvili, N . K. Kochetkov, S. Sh. Rozhnova, and V. A. Kilesso, Dokl. Acad. Nauk SSSR, 270 (1983) 897-899. (315) J. L. Kent and M. J. Osborn, Biochemistry, 7 (1968) 4419-4422. (316) 9. A. D. Stocker and P. H. Makela, Roc. R. SOC.London, Ser. B, 202 (1978) 6-30. (317) J. L. Kent and M. J. Osborn, Biochemisfry, 7 (1968) 4396-4408. (318) V. N. Shibaev, Yu. Yu. Kusov, T. N. Druzhinina, N. A. Kalinchuk, N. K. Kochetkov, V. A. Kilesso, and S. Sh. Rozhnova, Bioorg. Khim., 4 (1978) 47-56. (319) K. Rundell and C. W. Shuster, J. Biol. Chem., 248 (1973) 5436-5442. (320) Yu. Yu. Kusov, V. N. Shibaev, N. A. Kalinchuk, V. V. Kupriyanov, L. M. Gogilashvili, and N. K. Kochetkov, Bioorg. Khim., 5 (1979) 438-448.

318

VLADIMIR N. SHIBAEV

Another example of 0-specific polysaccharides that are assembled through the block mechanism are the polymers from Salmonella serogroups C2 and C3. Their polysaccharide chains (18) are composed9' of linear, tetrasaccharide repeating-units having abequosyl and D-glucosyl branches, X

3 L

+4)-P-Rha-(l+2)-a-Man-(l+2)-a-Man-(l+3)-P-Gal-(l+ 3 3 4

t

t

R'

a-Abe-1

t

R2

18

R' = a-Glc2Ac-(1+ , R2 = H, X = Ac b R' = H, R2 = a-Glc2Ac-(l+ , X = H a

(S. newporr, serogroup C,) (S. kenrucky, serogroup C,)

and are modified by acetylation. The assembly of the main chains of the polymers was shown321to begin with transfer of the D-galactosyl phosphate moiety of UDP-Gal onto polyprenyl phosphate. The resulting a-D-Galpp-Bpr serves as the glycosyl acceptor for consecutive transfer of two D-mannosyl groups from GDP-Man and an L-rhamnosyl group from dTDP-Rha. The tetrasaccharide-unit derivative formed may be subjected to enzymic polymerization. Synthetic moraprenyl a-D-galactopyranosyl diphosphate, but not its p anomer, may substitute for the natural acceptor in these reactions.322Incorporation of abequosyl side-chains was not investigated, but D-glucosyl branches may be introduced into the repeating unit prior to the polymerization step.321

b. Biosynthesis Through a Monomeric Mechanism.-The monomeric mechanism of polymeric chain-assembly was demonstrated for 0-specific polysaccharides of E. coli 0 9 , which is323a D-mannan composed of pentasaccharide repeating-units (19), and of 08, a similar polymer having324the trisaccharide repeating-unit 20. +3)-a-Man-(l+3)-a-Man-(l+2)-a-Man-(l+2)-a-Man-(l+2)-a-Man-(l+ 19

+3)-a-Man-(1+2)-a-Man-(l+2)-a-Man-(l+ 20

(321) V. N. Shibaev, T. N. Druzhinina, A. N. Popova, S. Sh. Rozhnova, and V. A. Kilesso, Eur. J. Biochem., 101 (1979) 309-316. (322) L. M. Gogilashvili, T. N. Druzhinina, and V. N. Shibaev, Khimiya i Biokhimiya Ugleuodou (Abstr. VII, USSR Conference on Chemistry and Biochemistry of Carbohydrates), Putschino, 1982, p. 29. (323) P. Prehm, B. Jann, and K. Jann, Eur. J. Biochem., 67 (1976) 53-56. (324) K. Reske and K. Jann, Eur. J. Biochem., 31 (1972) 320-328.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

319

During a study of the biosynthesis of the former polysaccharide, GDPMan was identified as a D-mannosyl donor, and the absence of oligosaccharide intermediates325and the growth of the polymeric chain from the nonreducing end326were demonstrated. The acceptor for D-mannosyl transfer was found to be a ~-glucosyl-lipid,~~’ finally identified80.328 as polyprenyl D-glucosyl diphosphate (3). The polymer 19, present as the 0-hapten in cytoplasmic membrane, contained a D-glucose residue at the reducing end of the chain.326 Synthetic moraprenyl a-D-glucopyranosyl diphosphate serves as an acceptor for D-mannosyl transfer. Among the synthetic moraprenyl diphos21-24) tested as D-mannosyl accepphates of d i s a ~ c h a r i d e (compounds s~~~ An tors, the a-(1+ 3) derivative 23 was found to be the most analogous mechanism of polysaccharide-chain assembly was s h o ~ n ~ ~ ~ . to operate in the biosynthesis of the polysaccharide 20. a-Man-(1 + 6)-cu-Glc-pp-Mpr 21 a-Man-(I + 4)-a-Glc-pp-Mpr

22 a - M a n - ( l + 3)-a-Glc-pp-Mpr 23

a-Man-(1 + 2 ) - ~ G l c - p p - M p r 24

The biosynthesis of other 0-specific polysaccharides has still not been investigated. The only additional example of demonstration of such a process is in the formation of (1 + 3)-a-~-galactan,the 01 antigen of K l e b ~ i e l l a UDP-Gal .~~~ was identified as the glycosyl donor in the reaction, but further details of the mechanism remain unclear. (325) (326) (327) (328) (329) (330) (331) (332) (333)

H. J. Kopmann and K. Jann, Eur. J. Biochem., 60 (1975) 587-601. H. C. Flemming and K. Jann, Eur. J. Biochem., 83 (1978) 47-52. S. Kanegesaki and K. Jam, Eur. J. Biochem., 95 (1979) 287-293. K. Jann, G. Goldemann, C. Weisgerber, C. Wolf-Ullisch, and S. Kanegasaki, Eur. J. Biochern., 127 (1982) 157-164. V. I. Torgov, V. N. Shibaev, and N. K. Kochetkov, Bioorg. Khim., 10 (1984) 946-953. K. Jann, M. Pillat, C. Weisgerber, V. N, Shibaev, and V. I. Torgov, Eur. J. Biochem., 151 (1985) 393-397. H. C. Fleming and K. Jann, FEMS Microbiol. Lett., 4 (1978) 203-205. H. Goldeman, S. Kanegasaki, and K. Jann, FEMS Microbial. Lett., 5 (1979) 443-445. I. R. Poxton and 1. W. Sutherland, J. Gen. Microbiol., 96 (1976) 195-202.

320

VLADIMIR N. SHIBAEV

3. Exocellular Polysaccharides Most of the exocellular polysaccharides produced by bacteria are synthesized inside the bacterial cell, with the use of membrane-bound enzymes. Both types of chain assembly were observed for these polymers. In many cases, the mechanism of the assembly remains unidentified, and the nature of the glycosyl acceptors in the process is not clear. most extensively a. Biosynthesis Through a Block Mechanism.-The studied example of the biosynthesis of a capsular polysaccharide through this mechanism is334polymer 25 from a strain of Klebsiella uerogenes originally designated DD-45; the polymer is not identical to any of the type-specific, capsular polysaccharides of Klebsiella thus far identified. + 3)-a-Gal-(1 + 3)-a-Man-(1 + 3)-a-Gal-( 1 +

2

t

a-GkA-1 25

Formula 25 shows the biological repeating-unit of the polymer, as its biosynthesis was f o ~ n d * ~to* begin ~ ~ ’ with formation of a-D-Gal-pp-Bpr, which serves as an acceptor for the transfer of a D-mannosyl group from GDP-Man. The resulting disaccharide derivative (26) accepts from UDP-GlcA the a-D-glucosyluronicgroup present in side chains of the polysaccharide. Only after its transfer does incorporation of another D-galactosyl residue of the a-Man-(1 + 3)-a-Gal-pp-Bpr 26

main chain become possible. The resulting tetrasaccharide derivative enters a polymerization reaction, and a polyprenyl diphosphate-linked intermediate was isolated that contained an octasaccharide chain corresponding to two repeating units of the polymer. Further processing of the polyprenyl diphosphate-linked polysaccharide remains unclear; after splitting of the capsular polysaccharide with a specific endo-glycosidase, attempts to identify octasaccharide fragments linked to any acceptor were unsuccessful. Some results of studies on the biosynthesis of two other Klebsiella capsular polysaccharides have also been reported. In the case of type 2 specific p o l y ~ a c c h a r i d e(27), ~ ~ ~incorporation of the D-mannosyl group from GDPMan into glycosyl-lipids and the polymer, catalyzed by a cell-envelope (334) E. C. Yurewicz, M. A. Ghalambor, and E. C. Heath, J. Biol. Chem., 246 (1971) 5596-5606. (335) F. A. Troy, F. E. Frerman, and E. C. Heath, Merhods Enzymol., 28 (1972) 602-624. (336) L. C. Gahan, P. A. Sandford, and H. E. Conrad, Biochemistry, 6 (1967) 2755-2767.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

321

+ 3)-p-Glc-(1 +4)-p-Man-(1 + 4)-a-Glc-( 1 +

3

t

O-GlcA-1 27

fraction, occurs only in the presence333of UDP-Glc. It may be suggested that assembly of the repeating unit begins from one of the D-glucosyl residues of the main chain of the polymer. For the poly~accharide~~’ of type 8 (28), polyprenyl D-glucosyl diphosphate (3) was identified as the first intermediate of the biosynthetic pathway.79 Its further conversion into the disaccharide338and tri~accharide’~ + 3)-p-Gal-( 1+ 3)-a-Gal-(1 + 3)-p-Glc-(1 +

4 o

t

- GkA- 1 28

derivatives was demonstrated, but incorporation of a D-glucosyluronicacid residue was not achieved. Polymerization of the trisaccharide repeating units of the main chain was observed. The enzymes that catalyze the first and second steps of the process may be solubilized by treatment of the membrane preparation with l - b ~ t a n o l . ’ ~ ~ The extracellular polysaccharide of Rhizobium meliloti, having the structure339shown in formula 29 (some of the 6-hydroxyl groups of the D-glucosyl + 4)-p-Glc-(1 + 4)-p-Glc-(1 + 4)-p-Glc-(

1+ 3)-p-Gal-(1+

6

t

R

R = P-Glc-( 1 + 3)-&Glc-( 1 + 3)-p-Glc-(1 + 6)-P-Glc-( 1 + 6 4

H3C’

‘CO,H 29

residues are acetylated) is also synthesized through a block mechanism. The polyprenyl glycosyl diphosphates formed after incubation of UDP-Glc with enzyme preparation were identifiedss*266 as the derivatives of (Y-Dgalactopyranose, of the disaccharide ester 30, and of the octasaccharide (337) I. W. Sutherland, Biochemistry, 9 (1970) 2180-2185. (338) J. A. Lomax, I. R. Poxton, and I. W . Sutherland, FEES Leu., 34 (1973) 232-234. (339) P.-E. Jansson, L. Kenne, 9. Lindberg, J. Liinngren, J. Liunngren, U. Rudbn, and S. Svensson, 1. Am. Chem. SOC.,99 (1977) 3812-3815.

322

VLADIMIR N. SHIBAEV P-Glc-( 1+ 3)-cr-Gal-pp-Pre 30

corresponding to the unmodified repeating-unit of the polymer. Acetylation, and incorporation of pyruvic acetal groups, were shown to take place prior to polymerization. In Alculigenesfuecalis var. myxogenes, which produces a succinoglycan having a similar octasaccharide repeating-unit, the same mechanism seems to operate. When the organism was grown in the presence of penicillin or bacitracin, production of the repeating-unit oligosaccharide was observed.340 The industrially important, extracellular polysaccharide (31) of Xunthomonas ~ a r n p e s t r i s ' ~ is ' ~another ~ ~ ' ~ ~example ~~ of complete assembly of the repeating unit during the biosynthesis of a branched polysaccharide. + 4)-P-Glc-( 1 + 4)-P-GIc-(1+

3

t

Man-( 1+ 4)-P-GlcA-(1+ 2)-cr-Man6Ac-l 6 4

31

The structure shown represents the biological repeating-unit of the polysaccharide. Its assembly was found343to begin with formation of polyprenyl a-D-glucosyl diphosphate, with consecutive transfer of other monosaccharide residues to give, finally, the pentasaccharide derivative. Incorporation of pyruvic acetal groups occurs prior to polymerization of the repeating A series of polyprenyl diphosphates was formed when EDTA-treated cells of Acetobacter xylinum were incubated with glycosyl nucleotides. The most complicated of them is the heptasaccharide derivative3" (32),which is considered to be an intermediate in the biosynthesis of the exocellular Rha-( 1+ 6)-P-Glc-( 1+ 6)-a-Glc-( 1 + 4)-P-GlcA-(1+ 6)-P-Man-( 1 + 3)-P-Glc-(1+ 4)-cr-Glc-pp-Pre

32 (340) M. Himatsu, J. Abe, A. Amemura, and T. Harada, Curbohydr. Res., 66 (1978) 289-294. (341) P.-E. Jansson, L. Kenne, and B. Lindberg, Curbohydr. Res., 45 (1975) 275-282. (342) L. D. Melton, L. Mindt, D. A. Rees, and G. R. Sanderson, Curbohydr. Res., 46 (1976) 245-257. (343) L. Ielpi, R. 0. Couso, and M. A. Dankert, FEBS Left., 130 (1981) 253-256. (344) R. 0. Couso, L. Ielpi, R. C. Garcia, and M. A. Dankert, Eur. J. Biochem., 123 (1982) 617-627.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

323

heteropolysaccharide produced by this bacterium. Its assembly occurs through consecutive transfer of monosaccharide residues onto the polyprenyl D-glucosyl diphosphate initially formed77; in addition, the derivatives of the di~accharide,’~t r i ~ a c c h a r i d e ,t~e~t r~a s a c ~ h a r i d e ,and ~ ~ hexasachar ride^^ fragments of compound 32 were identified. It may be possible that the block mechanism of chain assembly also operates in the biosynthesis of colanic acid (33), the extracellular polysaccharide of E. coli.346*347 + ~ ) - P - F u c - ( ~ + ~ ) - u - F L+4)-P-Glc-(l+ Ic-(~ 4 20)

f

p-Gal-(1 +4)-P-GlcA-( 1 + 3)-P-Glc-l 4 3

t

Ac

\I I

,

C H,C/

‘C02H 33

After incubation of an enzyme preparation from the micro-organism with UDP-Glc and GDP-Fuc, formation of the hexasaccharide derivative 34 was observed.348It does not correspond in composition to the repeating unit of the polysaccharide, or to its main chain, and further enzymic reactions are necessary in order to complete their assembly. (Fuc,GIc,)-cy-Glc-pp-Bpr

34

b. Biosynthesis Through a Monomeric Mechanism.-The monomeric type of polymeric-chain assembly was observed for hyaluronic acid (35), the capsular polysaccharide of Streptococcus group A. Formation of the polymer from UDP-GlcNAc and UDP-GlcA was demonstrated some 25 years + 3)-P-GlcNAc-(1 + 4)-P-GlcA-( 1 +

35

ago349’350 as the first example of the biosynthesis of a bacterial polysaccharide in a cell-free system. No evidence for the intermediate formation of a disaccharide derivative was obtained, despite special attempts to identify (345) R. 0. Couso, L. Ielpi, R. C. Garcia, and M. A. Dankert, Arch. Biochem. Biophys., 203 (1980) 434-443. (346) I. W. Sutherland, Biochem. J., 115 (1969) 935-945. (347) P. J. Garegg, B. Lindberg,T. Onn,andT. Holme, ActaChem. Scand.,25 (1971) 1185-1194. (348) J. G. Johnson and D. 9. Wilson, J. Bacteriol., 129 (1977) 225-236. (349) A. Markovitz, J. A. Cifonelli, and A. Dorfman, 1. Biol. Chem., 234 (1959) 2343-2350. (350) A. Markovitz and A. Dorfman, J. Biol. Chem., 237 (1962) 273-279.

324

VLADIMIR N. SHIBAEV

it on three different occasion^.^^^-^^^ Growth of the polymeric chains occurs from the nonreducing end. It was shown that only elongation of the preexisting carbohydrate chains took place in a cell-free as polymeric product may be identified after incubation with a single glycosyl nucleotide precursor. The mechanism of chain initiation remains unclear, and the nature of an acceptor for transfer of glycosyl units was not established. Formation of trichloroacetic acid-insoluble polymer prior to liberation of hyaluronic acid was dem~nstrated.~’~ The monomeric mechanism of chain assembly is characteristic for homopolysaccharides, which constitute the most difficult case for biosynthetic studies, as accumulation of intermediates could not be induced by removal of one of the glycosyl donors required for chain elongation. Among these polymers, the most extensive information has been obtained for polymers of N-acetylneuraminic acid, namely, the capsular polysaccharides (36and 37)of E. coli K1 (Ref. 354) and Neisseria meningitidis type c (Ref. 359,respectively. CMP-NeuAc serves as the glycosyl donor in the formation -D

8)-(r-NeuAc-(2+ 36

+9)-o-NeuAc-(2+ 31

of the polysaccharide chain^.^"*^^^-^'^ In the E. coli system, intermediate conversion of the glycosyl nucleotide into polyprenyl N-acetylneuraminic monophosphate was demon~trated.’~”~~ A similar intermediate was not detected in the meningococcal systemY2”but this may be connected with difficulties of identification owing to its high instability. The membrane-bound acceptor for the transfer of NeuAc groups contains oligosaccharide chains composed of 160 monosaccharide units361that are linked to an unidentified fragment. It was suggested that a membrane-bound

-

(351) (352) (353) (354) (355) (356) (357) (358) (359) (360) (361)

N. Ishimoto and J. L. Strominger, Biochim. Biophys. Actu, 148 (1967) 296-297. A. C. Stoolmiller and A. Dorfman, J. Bid. Chem., 244 (1969) 236-246. K. Sugahara, N. B. Schwartz, and A. Dorfman, J. BioL Chem., 254 (1979) 6252-6261. E. J. McGuire and S. B. Binkley, Biochemistry, 3 (1964) 247-251. A. K. Bhattacharjee, H. J. Jennings, C. P. Kenny, A. Martin, and I. C. P. Smith, J. Bid. Chern, 250 (1975) 1926-1932. D. Aminoff and F. D. Kundig, Methods Enzymol., 8 (1966) 419-423. F. D. Kundig, D. Aminoff, and S. Roseman, J. Bid. Chem., 246 (1971) 2543-2550. I. K. Vijay and F. A. Troy, J. Bid. Chem., 250 (1975) 164-170. F. A. Troy. I. K. Vijay, M. A. McCloskey, and T. E. Rohr, Mefhods EnzymoL, 83 (1982) 540-549. F. A. Troy and M. A. McCloskey, J. Bid. Chem., 254 (1979) 7377-7387. T. E. Rohr and F. A. Troy, J. Biol. Chem., 255 (1980) 2332-2342.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

325

protein serves as the anchor for the but a phosphatidic acid residue has now been shown to be present at the reducing end of the polysaccharide 37 and of several related polymers.362 Oligosaccharides composed of N-acetylneuraminic acid residues may serve as acceptors for glycosyl transfer.360The system becomes fully dependent on the addition of exogenous acceptors if the micro-organisms are grown at low temperature. During the enzymic reaction, the polymeric chain grows from the nonreducing end,76,3s7*3s8 achieving a length of -200 monosaccharide residues.361The exocellular polysaccharide shows a much lower degree of polymerization. It may be connected with nonenzymic hydrolysis of glycosidic linkages of sialic acid, which occurs readily, even at pH 5. Other examples of exocellular homopolysaccharides whose biosynthetic process has been investigated include ~ - m a n n u r o n a n ,an ~ ~intermediate ~.~~~ in the biosynthesis of bacterial alginic acid (mentioned in Section III,l,c), and bacterial cellulose. Although the first demonstration of the formation of a cellulose-like polymer from UDP-Glc with an enzymic system from Acetobacter xylinum was described363 in 1958, the process is still far from being completely understood. At least three types of intermediate in the formation of cellulose fibers were identified. These included lipid-linked oligosa~charides,~~~*~~~ oligosaccharides linked to a water-soluble acceptor, presumably a protein,36s ~ ~ ” ~ ~by the bacterial cell and and a water-soluble p o l y ~ a c c h a r i d e ~excreted further converted into cellulose by extracellular enzymes; the structure of a (1 + 4)-/3-~-glucanwith every third residue of the main chain carrying D-glucosyl branches linked through &D-( 1+ 2) bonds was suggested for this intermediate.368 Cellulose synthesis from UDP-Glc is stimulated by cello-oligosac~ h a r i d e s ,thus ~ ~ allowing ~ , ~ ~ ~suggestion of a monomeric mechanism for the chain elongation. Polyprenyl a-D-glucosyl diphosphate7’ and the analogous c e l l o b i o ~ e ,already ~~ mentioned in connection with formation of the oligosaccharide derivative 32, may also be related to cellulose biosynthesis. (362) E. C. Gotschlich, B. A. Fraser, D. Nishimura, J. B. Robbins, and T.-Y. Liu, J. Biol. Chem., 256 (1982) 8915-8921. (363) L. Glaser, 1. Bid. Chem., 232 (1958) 627-636. (364) D. Cooper and R. S. J. Manley, Biochim. Biophys. Acta, 381 (1975) 97-108. (365) M. Swissa, Y. Aloni, H. Weinhouse, and M. Benziman, J. Bacreriol., 143 (1980) 11421150. (366) J. Kjosbakken and J. R. Colvin, Can. J. Microbiol., 21 (1975) 111-120. (367) J. R. Colvin and G. G. Leppard, Can. J. Microbiol., 23 (1977) 701-709. (368) J. R. Colvin, L. Chknk, L. C. Sowden, and M. Takai, Can. J. Biochem., 55 (1977) 1057- 1063. (369) D. Cooper and R. S. J. Manley, Biochim. Biophys. Acta, 381 (1975) 109-119.

326

VLADIMIR N. SHIBAEV

The situation is complicated by the fact that the efficiency of cellulose synthesis in the cell-free system is low, and the same enzyme preparation catalyzes the incorporation of D-glucosyl groups from UDP-Glc into alkaliA similar process was reported to occur with soluble ( 1 + 2)-P-~-glucans.~~' a membrane preparation of Rhizobium r n e l i l ~ t i . ~ ~ ' It has been discovered that the enzymic synthesis of cellulose is specifically activated by guanosine 5'-triphosphate in the presence of a protein factor and poly( e t h y l e n e g l y ~ o l )or~ ~calcium ~ ions.373This activation results in a dramatic increase in the rate of synthesis of the polymer. The enzyme, solubilized by treatment of membrane preparation with d i g i t ~ n i n ?retains ~~ its regulatory properties, and does not show any requirements in lipids for its activity. c. Biosynthesis by an Unidentified Mechanism of Chain Assembly.-In the early sixties, several papers were published on the biosynthesis of capsular polysaccharides in Streptococcus pneumoniae (for a review, see Ref. 374). Experiments which would allow distinguishing between the block and the monomeric mechanism of chain assembly were not performed at that time, and reinvestigation of the problem seems necessary. (38)was f o ~ n dto ~be ~efficiently ~ * ~ ~ ~ The type 3 capsular polysa~charide'~~ formed from UDP-Glc and UDP-GlcA. Under optimal conditions, >90% of the glycosyl groups of the glycosyl nucleotides was incorporated into polymer identified by immunochemical methods. + 3)-P-GlcA-( 1 + 4)-P-Glc-(1 +

38

The revised377structure of the type 14 capsular polysaccharide is shown in formula 39.The precursors of the polymer were identified378as UDP-Glc, UDP-Gal, and UDP-GlcNAc. Incorporation of a glycosyl group from (370) H. Sandermann and R. F. H. Decker, FEES Left., 107 (1979) 237-240. (371) R. A. Dedonder and W. Z. Hassid, Biochim. Biophys. Acta, 90 (1964) 239-248. (372) Y. Aloni, D. P. Delmer, and M. Benziman, Proc. Nafl. Acad. Sci. USA, 79 (1982) 6448-6452. (373) Y. Aloni, R. Cohen, M. Benziman, and D. P. Delmer, J. Biol. Chem., 258 (1983) 4419-4423. (374) G. T. Mills and E. E. B. Smith, Bull. SOC.Chim. Biol., 47 (1965) 1751-1765. (375) E. E. B. Smith, G . T. Mills, H. P. Bernheimer, and R. Austrian, J. Biol. Chem., 235 (1960) 1876-1880; E. E. B. Smith, G . T. Mills, and H. P. Bernheimer, ibid., 236 (1961) 2 179-2 182. (376) E. E. B. Smith and G . T. Mills, Merhods Enzymol., 8 (1966) 446-450. (377) B. Lindberg, J. Lonngren, and D. A. Powell, Carbohydr. Res., 58 (1977) 177-186. (378) J. Distler and S. Roseman, Roc. Natl. Acad. Sci. USA, 51 (1964) 897-905; J. Distler, B. Kaufman, and S. Roseman, Methods Enzymol., 8 (1966) 450-455.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

327

UDP-Glc, or UDP-Gal, or both, into the polymer is necessary, prior to the incorporation of 2-acetamido-2-deoxy-~-glucose. Some data concerning the biosynthesis of S. pneumoniae types 1 and 8 capsular polysaccharides were mentioned in the review,374but experimental details were not reported. The polysaccharide chain of the capsular polysaccharide of E. coli K7 was found379to be formed from UDP-Glc and UDP-ManNAcA, but the mechanism of the assembly has not yet been clarified. 4. Polysaccharides of Gram-positive Cell-Walls

Carbohydrate chains of teichuronic acids and neutral polysaccharides linked to the carbohydrate chains of peptidoglycans are fragments of macromolecules of Gram-positive cell-wall. Only two examples of the biosynthesis of these polymers have been studied in detail. Evidence for both block and monomeric mechanisms of the chain assembly was obtained. a. Biosynthesis Through a Block Mechanism.-Biosynthesis of the teichuronic acid present in the cell wall of Bacillus lichenifomis was first studied3*' in 1966. At that time, participation of UDP-GlcA and UDPGalNAc as glycosyl donors for polymeric chain-formation was shown. Subsequent investigation of the processg0 has revealed the features characteristic of the block mechanism of chain assembly, namely, growth of the polymeric chain from the reducing end, and participation of polyprenyl oligosaccharide diphosphate intermediates. The first reaction of the cycle consists in transfer of a 2-acetamido-2deoxy-a-D-galactopyranosyl phosphate residue from UDP-GalNAc onto polyprenyl phosphate. The resulting a-GalNAc-pp-Pre serves as the glycosy1 acceptor for transfer of D-glUCOSylUrOniC acid groups from UDP-GlcA, giving the disaccharide intermediate that is subjected to enzymic polymerization. The observed sequence of biosynthetic reactions is well compatible with structure 40, initially suggested for the polymer,3*' but not with structure 41 obtained in a subsequent rein~estigation.~'~ Differences in the bacterial

(379) (380) (381) (382)

N. Ichihpra, N. Ishimoto, and E. Ito, FEES Lerr., 40 (1974) 309-311. R. C. Hughes, Biochem. J., 101 (1966) 692-697. R. C. Hughes and P. F. Thurman, Biochem. J., 117 (1970) 441-449. M . R. Lifely, E. Tarelli, and J. Baddiley, Biochem. J., 191 (1980) 305-318.

VLADIMIR N. SHIBAEV

328

+ 4)-cr-GlcA-( 1 + 3)-cr-GalNAc-( 1 +

40 + 4)-p-GlcA-( 1 + 4)-/3-GlcA-( 1 + 3)-/3-GalNAc-( 1 + 6)-a-GalNAc-( 1 +

41

strains used for the structural and the biosynthetic studies may be the reason for this discrepancy. It seems possible that a block mechanism also operates in the assembly of the chain of polysaccharide 42, present in the cell wall of Bacillus cereus.124 In this case, formation of the polymer from a-GlcNAc-pp-Bpr, UDPManNAc, UDP-GalNAc, and UDP-Glc was ob~erved.'~ + 3)-n-GalNAc-( 1 + 4)-P-ManNAc-( 1 + 3)-a-GlcNAc-( 1 +

6

t

a-Glc-1

3

r p-GlcNAc-1

6

t

/3-GlcNAc-l 6

t

P-GlcNAc-1 42

b. Biosynthesis Through a Monomeric Mechanism.-Formation of the carbohydrate chain of the teichuronic acid (43) from Micrococcus l ~ t e u s ~ ' ~ is an example of the monomeric mechanism of chain assembly. The trisaccharide derivative 44, which corresponds to the structure of the unique oligosaccharide sequence of the "linkage region," was to serve as + 4)-P-ManNAcA-( 1 + 6)-a-Glc-( 1 +

43

ManNAcA-ManNAcA-GlcNAc-p-Re

44

the glycosyl acceptor in the chain elongation. Its synthesis requires participation of UDP-GlcNAc and U D P - M ~ ~ N A C A . ~Growth ~ ' * ~ ' ~of the polysaccharide chain (43) of teichuronic acid occurs by transfer of glycosyl groups from UDP-Glc and UDP-ManNAcA onto the nonreducing end of the (383) S. Hase and Y.Matsushima, J. 1128. (384) N. J. Stark, G. N. Levy, T. E. 3466-3472. (385) R. L. Page and J. S. Anderson, (386) T. E. Rohr, G. N. Levy, N. J. 3460-3465.

Biochem. (Tokyo), 68 (1970) 723-730; 72 (1972) 1117Rohr, and J. S . Anderson, J. Bid. Chem., 252 (1977)

J. Biol. Chem., 247 (1972) 2471-2479. Stark, and J. S. Anderson, J. Bid. Chem., 252 (1977)

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

329

chain,384and the tetrasaccharide and pentasaccharide derivatives obtained from 44 by the addition of D-glucosyl and 2-acetamido-2-deoxy-~mannosyluronic groups were identified. The glycosyltransferases participating in the process may be separated by differential extraction with a nonionic detergent. The resulting polymer consists of approximately 40 disaccharide units,384and it was confirmed that its structure was identical to that of the natural polysaccharide by use of %n.m.r. spectro~copy.~~’ Elongation of pre-existing polysaccharide chains linked to peptidoglycan may occur with a “wall plus membrane” preparation:88 6- 12 disaccharide units being added during this process. c. Unidentified Mechanisms of Chain Assembly.-Incorporation of monosaccharide residues into the polysaccharide chain of the lipomannan from Micrococcus luteus was observed. The polysaccharide, which is a component of the bacterial, mesosomal membrane, is a branched, 0succinylated polymer having a di- 0-acylglycerol residue at the potentially reducing end of the chain.389-391Polyprenyl D-mannosyl monophosphate was identified as the D-mannosyl and only single, terminal D-mannosyl groups are transferred in the reaction. The process was the first example of observation of the participation of polyprenyl glycosyl monophosphates in the glycosylation reactions. Several studies concerning D-mannosyl transfer from GDP-Man and Man-p-Pre onto unidentified acceptors in Mycobacteria have been p ~ b l i s h e d . ~ The ’ . ~ ~enzymic ~ system may use methyl a-D-mannopyranoside as the exogenous acceptor for D-mannosyl transfer.394In this case, formation of a - ( 1 + 2) linkages was observed; they are present in 3-O-methyL~mannose-containing polysaccharides and a D-arabino-D-mannan characteristic of mycobacterial cell-wall. The situation that exists in the case of the biosynthesis of neutral polysaccharide chains of streptococcal cell-wall is still very uncertain. In S. sanguis, assembly of the trisaccharide derivative (45, X = an unidentified, hydrophobic group) from UDP-GlcNAc, dTDP-Rha, and UDP-Glc on an

(387) S. D. Johnson, K. P. Lacher, and J. S. Anderson, Biochemistry, 20 (1981) 4781-4785. (388) C. I. Traxler, A. S. Goustin, and J. S. Anderson, J. Eacferiol.. 150 (1982) 649-656. (389) P. Owen and M. J. R. Salton, Biochem. Eiophys. Res. Commun., 63 (1975) 875-880; Eiochim. Biophys. Acra, 406 (1975) 214-234. (390) D. A. Powell, M. Duckworth, and J. Baddiley, Biochem. J., 151 (1975) 387-397. (391) D. D. Pless, A. S. Schmit, and W. J. Lennarz, J. Eiol. Chem., 250 (1975) 1319-1327. (392) M. Scher and W. J. Lennarz, J. Biol. Chem., 244 (1969) 2777-2189; Mefhods Enzymol., 28 (1972) 563-571. (393) J. C. Schultz and K. Takayama, Eiochirn. Biophys. Acfa, 381 (1975) 175-184. (394) J. C. Schultz and K. Takayama, Eiochim. Biophys. Acfa, 428 (1976) 563-572.

330

VLADIMIR N. SHIBAEV Glc-Rha-GlcNAc-p-Pre I

x 45

endogenous acceptor present in a membrane preparation was shown.395Its composition does not correspond to that of the cell-wall polysaccharide from the micro-organism, which is3963 :4 : 1 Glc : Rha :GlcNAc; glycerol phosphate is also present in variable proportions. It may be suggested that the derivative 45 includes a fragment of the linkage region between the polysaccharide chain and the carbohydrate chain of a peptidoglycan, and that further addition of D-glucosyl and L-rhamnosyl units is necessary for elongation of the polymer chain. from UDP-GlcNAc Formation of 2-acetamido-2-deoxy-~-glucosyl-lipid was also with a membrane preparation from S.pyogenes group A. When both UDP-GlcNAc and dTDP-Rha were present, lipid-linked oligosaccharide derivatives were found that contained GlcNAc-Rha linkage, as was shown by identification of the corresponding N-deacylated disaccharide in the products of acid hydrolysis. Formation of lipid-linked oligosaccharides that contain D-glucosyl, Dgalactosyl, and L-rhamnosyl residues from the corresponding glycosyl nucleotides was demonstrated with a membrane preparation from Lactobacillus p l a n t a r ~ r n their ~ ~ ~ ;function remains to be established. When UDP-GlcNAc was incubated with membranes from Bacillus subtilis, synthesis of polyprenyl diphosphate-linked tetra- and hexa-saccharides composed of 2-acetamido-2-deoxy-~-glucosyl residues was observed,399and the process may be similar to the polymerization of peptidoglycan precursors discussed in the next Section.

5. Carbohydrate Chains of Bacterial Peptidoglycans Formation of the three-dimensional network of bacterial peptidoglycan includes synthesis both of glycosidic and peptide linkages, but only the former type of reaction is discussed in this Section. The assembly of the carbohydrate chains of a peptidoglycan has been shown to occur through a block mechanism. The initial reaction consists in transfer of N-acetylmuramyl-pentapeptide phosphate from the corresponding UDP derivative (395) (396) (397) (398) (399)

T. H. Chiu and C. Saralkar, J. Bacreriol., 133 (1978) 185-195. L. L. Emdur, C. Saralkar, J. C. McHugh, and T. H.Chiu, J. Bacteriol., 120 (1974) 724-732. V. M. Reusch and C. Panos, J. Bacreriol., 129 (1977) 1407-1414. K.J. Thorne, J. Bacreriol., 116 (1973) 235-244. C. E. Bettinger, A. N. Chatterjee, and F. E. Young, J. Bid. Chem., 252 (1977) 4118-4124.

BIOSYNTHESIS O F BACTERIAL POLYSACCHARIDE CHAINS

331

onto polyprenyl phosphate, followed by transfer of the 2-acetamido-2deoxy-D-glucosyl group from U D P - G ~ C N A C . ~ ~ - ~ ~ * ~ ~ ~ - ~ ~ ~ The resulting polyprenyl disaccharide pentapeptide diphosphate (46; the structure of the peptide chain shown is that for Staphylococcus aureus) may be further modified by amidation403of the carboxyl group of the D-glutamic P-GlcNAc-( 1 + 4)-a-MurAc-pp-Bpr

I

L-Ala

I

D-GIu

D-Ala

I

D-Ala 46

acid residue, or incorporation of additional amino The disaccharide-peptide units are further p ~ l y m e r i z e din ~ ~such ~ , ~a ~manner ~ that the carbohydrate chain grows from the reducing An intermediate having a polysaccharide chain composed of 12 disaccharide units was isolated from Bacillus r n e g a ~ e r i u r nits ~ ~incorporation ~; into the peptidoglycan molecule seems to occur through transpeptidation reactions. A similar intermediate having higher degree of polymerization was identified4l4 in Micrococcus luteus. The membrane-bound enzymes participating in the assembly of the peptidoglycan carbohydrate chains were solubilized, and partially purified. The (400) J. L. Strominger, M. Matsuhashi, J. S. Anderson, C. P. Dietrich, P. M. Meadow, W. Katz, G. Siewert, and J. M. Gilbert, Methods Enzymol., 8 (1966) 473-486. (401) W. G. Struve, R. K. Sinha, and F. C. Neuhaus, Biochemistry, 5 (1966) 82-93. (402) K. Izaki, M. Matsuhashi, and J. L. Strominger, J. Biol. Chem., 243 (1968) 3180-3192. (403) G. Siewert and J. L. Strominger, J. B i d . Chem., 243 (1968) 783-790. (404) M. Matsuhashi, C. P. Dietrich, and J. L. Strominger, J. Biol. Chem., 242 (1967) 3191-3206. (405) W. Katz, M. Matsuhashi, C. P. Dietrich, and J. L. Strominger, J. B i d . Chem., 242 (1967) 3207-3217. (406) W. S. L. Roberts, J. L. Strominger, and D. SOU, J. Biol. Chem., 243 (1968) 749-756. (407) J.-F. Petit, J . L. Strominger, and D. Soll, J. B i d . Chem., 243 (1968) 757-767. (408) R. Plapp and J. L. Strominger, J. Biol. Chem., 245 (1970) 3667-3674. (409) Y. van Heijenoort, M. Derrien, and J. van Heijenoort, FEBS Lett., 89 (1978) 141-144. (410) Y. van Heijenoort and J. van Heijenoort, FEBS Lett., 110 (1980) 241-244. (411) J. B. Ward and H. R. Perkins, Biochern. J., 135 (1973) 721-728. (412) A. Weston, J. B. Ward, and H. R. Perkins, J. Gen. Microbiol., 99 (1977) 171-181. (413) E. Fuchs-Cleveland and C. Gilvard, ?'roc. Natl. Acad. Sci. USA, 73 (1976) 4200-4204. (414) S. J. Thorpe and H. R. Perkins, FEBS Lett., 105 (1979) 151-154.

332

VLADIMIR N. SHIBAEV

most extensive studies were performed with the first enzyme of the cycle, namely N-acetylmuramyl-pentapeptidephosphatetransferase. Initially, the use of urea, dilute alkali, or anionic detergent was suggested415for solubilization of the enzyme from S. aureus membrane. Nonionic detergents were effective for this purpose with the enzyme from the . ~ ~ ~freezing * ~ ~ ~ same micro-organism and from Micrococcus l ~ t e ~ sRepeated and thawing of a membrane preparation was used in the case of Escherichia

~01i.~~~ Addition of polar, and, in some cases, neutral, lipids is necessary for activity of the solubilized enzyme?16-418Their function seems to consist in provision of a nonpolar micro-environment for the enzyme, and this was demonstrated by experiments with a fluorescent-labeled Studies on the influence of and of addition of l - b ~ t a n o l on ~ ~ the ' rate of the enzymic reaction confirmed this conclusion. and the interThe kinetic mechanism of the reaction was action of the enzyme with a series of analogs of UDP-N-acetylmuramylor in the amino acids of the pentapeptide modified in the uracil peptide was investigated. Particularly, incorporation of spin or of a fluorophore into the polyprenyl monosaccharidepentapeptide diphosphate was achieved through the enzymic reaction. The second enzyme of the cycle, N-acetylglucosaminetransferase, was extracted from Bacillus megaterium membranes with lithium and extensively The activity of the enzyme is also stimulated by addition of lipids. The combined action of LEI, cholate, and EDTA was effective in solubilization of the membranes from B. megaterium. An enzyme that catalyzes the polymerization of the disaccharide-pentapeptide units linked to polyprenyl diphosphate, with formation of glycosidic bonds, was purified (415) M. G. Heydanek and F. C. Neuhaus, Biochemistry, 8 (1969) 1474-1481. (416) J. N. Umbreit and J. L. Strominger, Proc. Natl. Acad. Sci. USA, 69 (1972) 1972-1974. (417) D. D. Pless and F. C. Neuhaus, J. Biol. Chem., 248 (1973) 1568-1576. (418) A. Geis and R. Plapp, Biochim. Biophys. Acta, 527 (1978) 414-424. (419) W. A. Weppner and F. C. Neuhaus, J. Biol. Chem., 253 (1978) 472-478. (420) W. A. Weppner and F. C. Neuhaus, Biochim. Biophys. Acra, 552 (1979) 418-427. (421) P. P. Lee, W. A. Weppner, and F. C. Neuhaus, Biochim. Biophys. Acta, 597 (1980) 603-613. (422) M. G. Heydanek, W. G. Struve, and F. C. Neuhaus, Biochemistry, 8 (1969) 1214-1221. (423) P. A. Stickgold and F. C. Neuhaus, J. Bid. Chem., 242 (1967) 1331-1337. (424) W. P. Hammes and F. C. Neuhaus, J. Bid. Chem., 249 (1974) 3140-3150. (425) W. P. Hammes and F. C. Neuhaus, J. Bacteriol, 120 (1974) 210-218. (426) L. S. Johnston and F. C. Neuhaus, Biochemistry, 14 (1975) 2754-2760. (427) W. A. Weppner and F. C. Neuhaus, J. Bid. Chem., 252 (1977) 2296-2303. (428) A. Taku, H. L. Gardner, and D. P. Fan, J. Biol. Chem., 250 (1975) 3375-3380. (429) A. Taku and D. P. Fan, J. Biol. Chem., 251 (1976) 1889-1896; 6154-6156.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

333

from this e ~ t r a c t . ~ ~A' *similar ~ ~ l procedure was found effective for solubiliPenicillinzation and purification of an analogous protein from E. binding proteins lA, l B , and 3 (purified from E. coli cell-envelope after extraction with nonionic detergents) were found to catalyze formation both of glycosidic and peptide linkages; the reaction results in synthesis of cross-linked peptidoglycan from polyprenyl-linked intermediate^.^^^-^^* 6. Structure of Polysaccharide Chains, and Mechanisms of Their Assembly

The material presented in previous sub-sections clearly shows that both of the possible mechanisms of polysaccharide chain-assembly may operate in the biosynthesis of bacterial polysaccharides. There is no clearcut, mechanistic difference in the biosynthesis of 0-specific chains of lipopolysaccharides, exocellular polysaccharides, and carbohydrate chains of Grampositive, cell-wall polymers; for every class of polymer, the existence of both mechanisms of chain assembly was demonstrated. It seemed of interest to check whether the type of biosynthetic mechanism involved may be related to the structure of the polymeric chain formed. In the block mechanism of chain assembly, formation of polyprenyl glycosyl diphosphates through transfer of glycosyl phosphate is a necessary step of the process. Thus far, only UDP-activated monosaccharides have been found to participate in this reaction (see Section 11,3). Consequently, the presence of a monosaccharide residue of this group in the main chain of a polymer may be regarded as a necessary condition for assembly of the chain through the block mechanism. The currently known structures of bacterial polysaccharides may be separated into several, biosynthetic types according to the nature of the activated forms of the monosaccharide residues in the main chain (for example, the UGT type means that UDP-, GDP-, and dTDP-activated sugars are present in the main chain). Table VIII shows the total number '(430) A. Taku and D. P. Fan, J. Bid. Chem., 254 (1979) 3991-3999. (431) A. Taku, M. Stuckey, and D. P. Fan, J. Biol. Chem., 257 (1982) 5018-5022. (432) A. Taku, T. M. Hirsch, and D. P. Fan, J. Biol. Chem., 255 (1980) 2848-2854. (433) J. Nakagawa, S. Tamaki, and M. Matsuhashi, Agric. Bid. Chem., 43 (1979) 1379-1380. (434) F. Ishino, K. Mitsui, S. Tamaki, and M. Matsuhashi, Biochem. Biophys. Res. Commun., 97 (1980) 287-293. (435) H. Suzuki, Y. van Heijenoort, T. Tamura, J. Mizoguchi, Y. Hirota, and J. van Heijenoort, FEBS Leu., 110 (1980) 245-249. (436) F. Ishino and M. Matsuhashi, Biochem. Biophys. Res. Commun.,101 (1981) 905-911. (437) J. Nakagawa and M. Matsuhashi, Biochem. Biophys. Res. Commun., 105 (1982) 15461553. (438) S. Tomioka, F. Ishino, S. Tamaki, and M. Matsuhashi, Biochem. Biophys. Res. Commun., 106 (1982) 1175-1182.

VLADIMIR N. SHIBAEV

334

TABLEVIlI Biosynthetic Classification of Bacterial Polysaccharides Total number of different structures knownh

Biosynthetic type of the main chaina

0-Specific polysaccharides'

-

EXopoIy saccharides

lin.

br.

UGT

3

UG

1

UT

9

10 13 11

TC

-

U

6

G T C

4

1

1

2

2

-

-

-

1

1

3

-

uc

lin.

Gram-positive cell-walId

br.

lin

.

br.

Mechanisms of the chain assembly demonstrated block block

2 27 18

2 3

block and monomeric monomeric monomeric

a Abbreviations for biosynthetic types are composed from abbreviations of nucleoside See footnote a to Table residues of activated forms of the monosaccharide components. Including other bacterial amphiVI: /in. and br. mean linear and branched polysaccharides. Including amphiphiles of Gram-positive cellphiles of Gram-negative, outer membranes. membranes.

of different polysaccharide structures of each type so far reported to be present in polymers of bacterial cell-surfaces. It may be seen that polymers of the U, UG, and UT types are the most usual; the UGT and G types are also relatively frequent, whereas the UG, TG, C, and T types are represented by only a few examples. About a quarter of the currently established structures cannot be so classified, as their main chains either contain a monosaccharide residue having an unidentified, activated form, or a phosphoric diester linkage. The block mechanism of chain assembly is characteristic for polymeric chains of the UGT type (see Salmonella 0-specific polysaccharides 10-12 and 18) and the UG type (see capsular polysaccharides 25, 27, and 33), with UDP-activated sugars serving as initiators of chain growth. It seems rather safe to suggest that the biosynthesis of other polymers of these types occurs through a block mechanism as well. Predominance of the monomeric mechanism of chain assembly is characteristic for polysaccharides of the G type (see structures 19 and 20) and the C type (see structures 36 and 37). Here again, the existence of a similar mechanism of chain assembly for other polymers of these types seems very probable.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

335

Among polysaccharide structures of the U type, which are the most ubiquitous, both mechanisms of chain assembly have been demonstrated. In this group, the block mechanism is certainly characteristic for polymers having repeating units containing more than three monosaccharide residues (see structures 28, 29, and 31), whereas, for homopolysaccharides, the monomeric mechanism of assembly seems usual. When a polymer is composed of disaccharide repeating-units, it seems not possible to predict a mechanism of chain assembly; the block mechanism was shown to operate for the carbohydrate chain of peptidoglycan 46, and for the Bacillus licheniformis teichuronic acid (40) but the structurally related polymers 35 and 43 are assembled through a monomeric mechanism. The suggested classification of polysaccharide structures seems to be useful for prediction of the mechanisms of chain assembly. Investigation of these mechanisms for other types of polysaccharide chains, especially for the very frequent UT type, should be particularly interesting.

VI. ENZYMIC SYNTHESIS OF BACTERIALPOLYSACCHARIDES FROM MODIFIEDPRECURSORS Preparation of modified, bacterial polysaccharides having monosaccharide analogs inserted into the polymeric chain is of interest for study of the structure-properties relationship in these biopolymers. Incorporation of chemically prepared, modified, biosynthetic precursors of the polymers in enzymic reactions seems a promising approach for achieving this aim. Such an approach, which may be termed “chemical-enzymic synthesis,” has now been studied by our g r o ~ p , using ~ ~ ~ 0-specific - ~ ~ ~polysaccharides (10-12) of Salmonella serogroups B and E as an example. To clarify the scope and the limitations of this approach, it was first of all necessary to ascertain whether precursors having modified monosaccharide residues may be accepted as substrates by the enzymes responsible both for assembly of the oligosaccharide repeating-units of the polymers and for their polymerization. Biosynthesis of repeating units of the main chain of these polysaccharides (see Section V,2,a) includes the consecutive transfer of a-D-galactopyranosyl phosphate from UDP-Gal, the L-rhamnopyranosyl group from dTDP-Rha, and the D-mannopyranosyl group from GDP-Man onto a poly(439) N. K. Kochetkov, V. N. Shibaev, T. N. Druzhinina, L. M. Gogilashvili, L. L. Danilov, V. I. Torgov, S. D. Maltsev, and N. S. Utkina, Dokl. Akad. Nauk SSSR, 262 (1982) 1393- 1397. (440) V. N. Shibaev, Pure Appl. Chem., 50 (1978) 1421-1436. (441) V. N. Shibaev, Lecture at 1st Soviet-Swedish Symposium on Carbohydrates, Moscow, 1983.

336

VLADlMlR N. SHIBAEV

prenyl phosphate acceptor. A number of analogs of natural polyprenyl glycosyl diphosphates and glycosyl nucleotides were synthesized chemically, and studied as putative precursors of the modified trisaccharide derivatives that correspond to structures 15 or 16, with alteration of one of the monosaccharide residues. Two groups of substrate analogs were studied more extensively. The first one included analogs of a-D-Gal-pp-Mpr that contained a-D-fucopyranosyl,"2 a-~-glucopyranosyl,~~~ 4-deoxy-a-~-xylo-hexopyranosyl,~~ a-D-talopyranosyl,44Za-D-mannopyranosyl,442and p- ~-galactopyranosyl~~ groups instead of the a-D-galactopyranosyl group. A series of analogs of GDP-Man that were examined as substitutes for the natural glycosyl nucleotide included the derivatives containing the a-D-rhamnopyranosyl,444 2-deoxy-aa-D-talopyranosyl,4S 3-deoxy-a-~-arabino-hexopyranosyl,~ D-arabino-hexopyranosyl,w and a-D-glucopyranosyl groups. In addition, the possibility of incorporation, into the trisaccharide derivatives, of the monosaccharide residues from UDP-a-~-talopyranose"~(used as an analog of UDP-Gal) and dTDP-P-L-mannopyranose446(used instead of dTDPRha) was studied. It turned out that the specificity of the glycosyltransferases towards the structure of the monosaccharide residues of both the glycosyl donors and acceptors is surprisingly broad, and a series of modified polyprenyl trisaccharide diphosphates may be prepared with the use of precursor analogs.322.439-441.447,44S The enzymes from S. anaturn (serogroup E,), S. newington (serogroup EJ,S. senftenberg (serogroup E4),and S. typhirnuriurn (serogroup B) were similar in their requirements for substrate structure. The results of the specificity studies of these enzymes are summarized in Table IX. It may be seen that only several elements of the trisaccharide structure seem to be strictly controlled by the specificity of the enzymes. These include the anomeric configuration of the terminal monosaccharide residue of the glycosyl acceptor and the configuration of C-4 of the D-mannopyranosyl (442) L. L. Danilov, S. D. Maltsev, and N. K. Kochetkov, Bioorg. Khim., 8 (1982) 109-113. (443) S. D. Maltsev, N. N. Yurchenko, L. L. Danilov, and V. N. Shibaev, Bioorg. Khim., 9 (1983) 1097-1100. (444) 5. KuEar, J. Zamock9, J. Zemek and 5. Bauer, Chem. Zuesti, 32 (1978) 414-419. (445) V. N. Shibaev, G . I. Eliseeva, M. A. Kraevskaya, and N. K. Kochetkov, Bioorg. Khim., 7 (1981) 376-380. (446) V. N. Shibaev, Yu. Yu. Kusov, V. A. Petrenko, and N. K. Kochetkov, Izu. Akad. Nauk SSSJ?, Ser. Khim., (1976) 2588-2591. (447) V. N. Shibaev, T. N. Druzhinina, N. K. Kochetkov, KuEar, and Bauer, Bioorg. Khim., 4 (1978) 410-414. (448) V. N. Shibaev, L. L. Danilov, T. N. Druzhinina, L. M. Gogilashvili, S. D. Maltsev, and N. K. Kochetkov, FEES Leu., 139 (1982) 177-180.

s.

s.

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

331

TABLEIX Specificity of the Enzymes of Biosynthesis of Polyprenyl Diphosphate Trisaccharides in Salmonella Serogroups B and E towards the Structure of the Monosaccharide Residues of Substrates Modifications" The enzyme Galactosyl phosphatetransferase Rhamnosyltransferase Mannosyltransferase

Sugar unit

The substrate

UDP-Gal a-Gal-pp-Mpr dTDP-Rha Rha-a-Gal-pp-Mpr

Gal Gal Rha Gal Rha Man

GDP-Man

Acceptable

Nonacceptable

-

E2, E4 E', E4 E' E4

H6,H4,E2 O6

H6,H4, E4, E2 O6

H6,H', H2, E2

a See Ref. 101 for abbreviations of modifications. The abbreviation O6 stands for exchange of the 6-hydroxyl group for the hydrogen atom of an L-rhamnose unit.

and D-galactopyranosyl residues. The latter seems to be especially important. UDP-Glc, a common glycosyl nucleotide, cannot serve as a substrate for the D-galactosyl phosphatetransferase. Moreover, a-D-Glc-pp-Pre, which is the expected product of the transfer of a-D-glucopyranosyl phosphate from UDP-Glc, was unable to accept an L-rhamnosyl group in the following reaction.us It is interesting that recognition of the correct configuration of C-4 of the glycosyl residue of the acceptor with the rhamnosyltransferase does not depend on specific interactions involving the axial hydroxyl group at C-4, as the analogous 4-deoxy derivative may serve as a substrate for the rea~tion.~~~'~~~ To achieve incorporation into the trisaccharide unit of a D-glucosyl instead of a D-galactosyl residue, it was found necessary to prepare the ~ ~ to ~ .use ~ ~ it as the disaccharide derivative 47 by chemical s y n t h e s i ~ ,and a-Rha-( 1 + 3)-a-Glc-pp-Mpr 47

substrate for the D-mannosyltransferase. Only in this case does modification of the D-galactopyranosyl residue by epimerization at C-4 become tolerable for the enzyme.322*439 Incorporation of the correct monosaccharide residues into the polysaccharide repeating-units under physiological conditions seems to depend not (449) V. I. Torgov, 0. V. Kudashova, V. N. Shibaev, and N. K. Kochetkov, Bioorg. Khim., 8 (1982) 114-119.

338

VLADIMIR N. SHIBAEV

only on the specificity of the glycosyltransferases that control the most crucial structural points, but also on the specificity of the glycosyl nucleotideforming enzymes, discussed in Sections I1 and 111, which produce only limited series of the glycosyl donors. The polymerases responsible for conversion of the polyprenyl trisaccharide diphosphates 15 and 16 into 0-specific polysaccharides were also found to accept modified precursors as substrates. Almost all of the analogs of 15 and 16 prepared from modified polyprenyl glycosyl diphosphates or glycosyl nucleotides through the enzymic reactions already discussed were found to enter the polymerization reaction, giving rise to modified, 0-specific p o l y s a c ~ h a r i d e s . The ~ ~ ~only ~ ~ ~ exceptions ~*~~ were the cases when a group participating in formation of a new glycosidic linkage during polymerization was subjected to modification. Thus, with the polymerase preparation from S. anaturn, which catalyzes formation of a-(1 + 6)-glycosidic linkages, an analog of polysaccharide 10a containing D-rhamnose instead of D-mannose obviously cannot be obtained, whereas preparation was achieved for modified polysaccharides wherein 3-deoxy-~-arabinohexose or D-glucose was substituted for D-mannose, L-mannose for L-rhamnose, and 4-deoxy-~xylo-hexose, D-glucose, D-fucose, or D-talose for D-galactose residues. The configuration of the glycosidic linkages formed was shown to be the same as in the natural polymer. Similar results in the preparation of analogs of polysaccharide I l a were obtained with the polymerase from S. newingtonYu'catalyzing formation of p-( 1 + 6)-glycosidic linkages. When S. typhirnuriurn polymerase, forming the a-(1+ 2) linkage, was tested with the modified polyprenyl trisaccharide diphosphates, the series of polysaccharides shown in formulas 48 and 49 were obtained. They represent analogs of the main chain of polysaccharide 12. A polymer wherein D-glucosyl are present instead of D-mannosyl residues cannot be thus prepared. + 2 ) - a - ~ - M a n -1(+ 4)-a-~-Rha-( 1 + 3)-a-Sug-( 1 +

48

SUg = D-FUC,D-Tal, D-Glc, or 4-deoxy-~-xy/o-hexose + 2)-a-Sug-( 1 + 4 ) - a - ~ - R h a1 - (+ 3 ) - a - ~ - G a l -1(+

49

Sug = D-Rha or 3-deoxy-~-urubino-hexose

It may be concluded that polymerases possess a rather broad specificity towards the structure of the monosaccharide residues that participate in formation of a new glycosidic linkage. Chemical synthesis of polyprenyl

BIOSYNTHESIS OF BACTERIAL POLYSACCHARIDE CHAINS

339

phosphate derivatives of different trisaccharides may probably open the possibility of preparation of even more extended series of modified bacterial polysaccharides. The chemical-enzymic approach to the synthesis of modified polysaccharides presents a good prospect for the preparation of small quantities of these polymers, which may prove very useful for immunochemical studies. The approach is certainly not limited by the specific case of Salmonella polysaccharides 10-12, and may well be extended to other polymers. The first results from this group3** show that several analogs of 0-specific polysaccharides (18) of Salmonella serogroups C , and C3 may be prepared through this approach. ACKNOWLEDGMENTS T h e author is very grateful to hofessor N. K. Kochetkov and Dr. T. N. Druzhinina for reading the Chapter and for making helpful suggestions. The assistance of Mrs. N. A. Kalinchuk and Mrs. N. S. Utkina in the preparation of the manuscript is also acknowledged and greatly appreciated.

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ADVANCES IN CARBOHYDRATE CHEMISTRY A N D BIOCHEMISTRY. VGL. 44

LIPID-LINKED SUGARS AS INTERMEDIATES IN THE BIOSYNTHESIS OF COMPLEX CARBOHYDRATES IN PLANTS BY RAFAELPONTLEZICA,*GUSTAVO R. DALEO,?

* Instituio

de Investigaciones Biolbgicas y t Depariamenio de Biologia, Faculiad de Ciencias Exacias y Naturales, Universidad Nacional de Mar del Plaia, 7600 Mar del Plaia, Argentina

AND

PRAKASH M. DEY

Department of Biochemisiry, Royal Holloway and Bedford New College, University of London, Egham Hill, Egham, Surrey 7W20 OEX, Uniied Kingdom

I. INTRODUCTION 1. Historical

With the discovery of glycosyl esters of nucleotides, the so-called “sugar nucleotides” by Leloir and coworkers at the end of the forties,’ a new period in the knowledge of saccharide synthesis was initiated. The requirement of a glycosyl donor containing an activated sugar then became clear, and sugar nucleotides play this role in most reactions within the cell. A limited number of reactions are known to use a-D-glucosyl phosphate or sucrose as an a-D-glucosyl donor.’ The energy for the polymerization of the saccharides is provided by the attachment of the monomer units to an activating group through a high-energy linkage. The addition of the activated glycosyl units to the nonreducing end of an acceptor is one of the general mechanisms for the polymerization of saccharides. The acceptor molecule (primer) is an oligomer or polymer whose nonreducing ends are the sites where new glycosyl groups are added. Studies on the biogenesis of several components of bacterial cell-walls led to the discovery, in 1965, of lipophilic compounds containing sugar (1) L. F. Leloir, Biochem. J., 91 (1964) 1-8. (2) W. Z. Hassid, in R. Piras and H. G . Pontis (Eds.), Biochemistry of the Glycosidic Linkage, Academic Press, New York, 1972, pp. 315-335.

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RAFAEL PONT LEZICA er al.

residue^.^-^ These glycolipids are required for the transfer of sugars from the sugar nucleotides, at the cytoplasmic side of the membrane, to the glycan polymers on the other side. An important feature of these intermediates is that the transfer potential of the glycosyl-phosphate bond seen in nucleoside glycosyl diphosphates is also present in the lipid monophosphate or diphosphate sugars. In some bacterial systems (the synthesis of peptidoglycan or 0-antigen lipopolysaccharide), the glycosyl units transferred to the lipid acceptor, a polyprenyl phosphate, may be sequentially added to the nonreducing end, forming a lipid-linked oligosaccharide. This is later transferred to the reducing end of a growing chain, liberating the polyprenyl diphosphate. Other reactions related to the synthesis of bacterial cell-walls (modification of the 0-antigen or D-mannan) also proceed through polyprenyl phosphate intermediates, but only one sugar is transferred at a time to the nonreducing end of the chain. Earlier review articles included comprehensive discussions of these pathways:-' Similar studies were undertaken on animal systems, especially on the glycosylation of one class of glycoprotein having the oligosaccharide Nglycosylically linked from 2-acetamido-2-deoxy-~-glucose (GlcNAc) to the amide nitrogen atom of L-asparagine in the peptide? Many of these glycoproteins have a core structure composed of D-mannose and GlcNAc, and it is this core region that is synthesized on a polyprenyl diphosphate carrier attached to membranes, at the cytoplasmic side, by successive addition of glycosyl groups from sugar nucleotides to the reducing end. When completed, the oligosaccharide is transferred en bloc to the protein acceptor on the other side of the membrane. The reader may, with advantage, consult other, more-detailed reviews on this

(3) J. S. Anderson, M. Matsuhashi, M. A. Haskin, and J. L. Strominger, Proc. Narl. Acad. Sci. USA, 53 (1965) 881-889. (4) I. M. Weiner, T. Higuchi, L. Rothfield, M. S. Andrew, M. J. Osborn, and B. L. Horecker, Proc. Narl. Acad. Sci. USA, 54 (1965) 228-235. (5) A. Wright, M. Dankert, and P. W. Robbins, Prac. Narl. Acad. Sci. USA, 54 (1965) 235-24 1. (6) M. J. Osborn, Annu. Rev. Biochem., 38 (1969) 501-538. (7) W. J. Lennarz and M. G . Scher, Biochim. Biophys. Acra, 265 (1972) 417-441. (8) F. W. Hemming, MTP Inr. Rev. Sci., Biochem. Lipids, (1974) 39-97. (9) R. Kornfeld and S. Kornfeld, Annu. Rev. Biochem., 45 (1976) 217-237. (10) N. H. Behrens, in Y. C. Lee and E. E. Smith (Eds.), Biology and Chemistry ofEukaryotic Cell Surfaces, Academic Press, New York, 1974, pp. 159-178. (11) C. J. Waechter and W. J. Lennarz, Annu. Rev. Biochem., 45 (1976) 95-112. (12) F. W. Hemming, Biochem. Rev., 5 (1977) 1223-1231. (13) A. J. Parodi and L. F. Leloir, Biochim. Biophys. Acra, 559 (1979) 1-37. (14) R. J. Staneloni and L. F. Leloir, Crit. Rev. Biochem., 12 (1982) 289-326.

LIPID-LINKED SUGARS AS INTERMEDIATES IN PLANTS

343

Plants appear to be especially interesting systems as, like bacteria,14athey have cell walls formed by extracellular polysaccharides and glycoproteins, and, like animal cells, glycoproteins containing L-asparagine-linked oligosaccharides. On the other hand, the widespread occurrence of polyprenols in plants, and the fact that many glycosyltransferases involved in the synthesis of extracellular saccharides are membrane-bound, led to the concept of lipid-linked sugars as potential intermediates in those reactions. Previous reviews of this field centered attention on glycoprotein synThe present article not only covers the better-known area of glycoprotein synthesis, but emphasizes those fields in which active research is needed in order to fill in wide gaps in our knowledge of the synthesis of complex glycans. It is important to point out that the term “plant” is used herein in its widest connotation, to include algae, fungi, and seed plants. 2. General Techniques Used

For the study of polyprenyl glycosyl phosphates as intermediates in the synthesis of complex glycans, several techniques have been developed, and these have been described elsewhere in Two important features should be emphasized. First, the very small amounts of polyprenyl phosphates that are present in most tissues; for this reason, the use of radioactive techniques for the detection of products is obligatory. Second, on account of the hydrophobic nature of these compounds, and as the enzymes involved in the reactions are membrane-bound, the use of detergents and organic solvents becomes necessary. Those derivatives containing one sugar residue, or a few, are soluble in such conventional, lipid solvents as 2 : 1 or 3 :2 (v/v) chloroform-methanol, or water-saturated butanol. For animal tissues, this partition was performed by using 3 :2 : 1 (v/v) chloroform-methanol-4 m M MgClz. Three phases are formed by low-speed centrifugation: ( a ) the aqueous phase containing sugar nucleotides and hydrophilic molecules, (b) an interphase containing proteins and the lipid-oligosaccharides, and (c) the organic phase containing the lipid-linked sugars. This procedure is not convenient with plant material, which contains low proportions of protein, and is therefore (14a) (15) (16) (17)

V. N. Shibaev, This Volume, pp. 277-339. R. Pont Lezica, Biochem. Soc. Trans., 7 (1979) 334-337. A. D. Elbein, Annu. Rev. Plant Physiol., 30 (1979) 239-272. A. D. Elbein, in W. Tanner and F. Loewus (Eds.), Encyclopedia of Plunr Physiology, New Series, Vol. 138, Springer Verlag, Berlin, Heidelberg, 1981, pp. 166-193. (18) N. H. Behrens and E. TBbora, Merbods Enzymol., 50 (1978) 402-437. (19) C. T. Brett, Tech. Carbobydr. Merab., B306 (1981) 1-8. (20) C. T. Brett, Tech. Carbobydr. Metab., 8305 (1981) 1-14.

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changed to the following: to 50 to 100 pL of the incubation mixture is added 2: 1 (v/v) chloroform-methanol (2 mL) and they are well mixed; only one phase is formed, and the proteins and lipid-oligosaccharides adhere to the tube walls. The liquid is decanted into a clean tube, and is washed free of contaminants by means of Folch’s upper phase.21The (mare hydrophilic) lipid-linked oligosaccharides are then extracted by a more polar solvent, such as 1 : 1 :0.3 (v/v) chloroform-methanol-water.22 Nevertheless, the solubility properties of these compounds cannot be used as identification criteria, because many other glycolipids may be found in the organic phase. Plant preparations synthesize neutral glycolipids that can be separated by different procedures. Polyprenyl glycosyl phosphates are very labile to mild acid, liberating their sugar moiety to the water phase. Glycosyl derivatives of sterols and ceramides are stable, and may be separated by this procedure. On the other hand, polyprenyl glycosyl phosphates are stable to mild alkali, and can be distinguished from acylated glycoglycerides by treatment with 0.1 M NaOH for 15 min at 37”. Another procedure used to separate polyprenyl-linked sugars from other glycolipids is based on the acidic properties of polyprenyl phosphate derivatives. Paper chromatography with DEAE-cellulose, and water-saturated butanol as the solvent, has been used.23Columns of DEAE-cellulose in the acetate form,24 commonly employed, are developed with 99% methanol, 2: 1 (v/v) chloroform-methanol, or 1: 1:0.3 (v/v) chloroform-methanol-water. When the last solvent is used, in small columns, only the polyprenyl glycosyl diphosphates are retained.** It is difficult to identify the lipid moiety as a polyprenol, because of the small amounts present in tissues. Nevertheless, some information can be obtained by using glycosylated lipids labelled in the sugar moiety. The first indication of the presence of polyprenyl glycosyl phosphates comes from study of the properties just mentioned, namely, lability to mild acid, stability to mild alkali, and acidic properties. Information on the polyprenyl nature of the lipid may be obtained by labelling the lipid with radioactive 2,4dideoxy-3-C-methyl-~-glycero-pentono-1,5-lactone (mevalonic “acid”). (21) J. Folch, M. Lees, and G . H. Sloane-Stanley, J. Biol. Chem., 226 (1957) 497-509. (22) N. H. Behrens, A. J. Parodi, and L. F. Leloir, Roc. Nad Acad. Sci. USA,68 (1971) 2857-2860. (23) R. Pont Lezica, C. T. Brett, P. Romero Martinez, and M. A. Dankert, Biochem. Biophys. Res. Commun., 66 (1975) 980-987. (24) G . Rouser. G. Kritchesky, and A. Yamamoto, in G . U. Marinetti (Ed.), Lipid Chromatographic Analysis, Vol. 1, Dekker, New York, 1967, pp. 99-162. (25) L. F. Leloir, R. J. Staneloni, H. Carminatti, and N. H. Behrens, Biochem. Biophys. Res. Commun., 52 (1973) 1285-1292.

LIPID-LINKED SUGARS AS INTERMEDIATES IN PLANTS

345

The main problem in this approach is the very low permeability of mevalonic acid to membranes, resulting in very low incorporation. Positive results have been obtained by the use of cell-free systems incubated with [‘“CImevalonic [‘“C]isopentenyl diphosphate,2s or [32P]orthophoshate.'^ Incubation of these radioactive lipids with glycosyl nucleotides labelled in the glycosyl group with a different isotope, followed by extraction and cochromatography in different solvent systems, may indicate that both compounds are present in the same molecule. When the lipid moiety becomes labelled from mevalonic acid or isopentenyl diphosphate, chromatography on DEAE-cellulose columns should be performed, in order to avoid confusion with steryl glycosides. Some additional information on the saturation of the a-isoprene residue and the length of the polyprenol chain may be obtained from experiments with unlabelled polyprenyl phosphates and polyprenyl glycosyl phosphates labelled in the glycosyl group. Many polyprenyl phosphates found in plants have an unsaturated a-residue. The phosphate bound to an allylic alcohol is extremely labile to mild acid (pH 2.0), releasing the phosphate. The free alcohol cannot serve as glycosyl acceptor when incubated with glycosyl nucleotides and the cell-free s y ~ t e m . ~Allylic-polyprenyl ~ * ~ ~ * ~ ~ ~glycosyl ~ ~ phosphates are degraded by treatment with 50% phenol or catalytic hydrogenation, which releases the sugar moiety. On the other hand, nonallylic derivatives are stable to these treatment^.^^,^'*^^'^ Finally, the chain length of the polyprenyl glycosyl phosphate may be estimated by gel filtration in columns of Sephadex G-75;this method requires the use of aqueous Columns of Sephadex LH-20have also been solutions with used s a t i s f a c t ~ r i l y ~with ~ . ~ organic ~ solvents. Reversed-phase, thin-layer chromatography may be used to estimate the chain length of the free p o ~ y p r e n o ~ . ~ *All * ~of ~ *the ~ * aforementioned methods require the use, as markers, of polyprenyl glycosyl phosphates or polyprenols of known molecular weight. Complete characterization of the polyprenol requires sufficient pure material for spectroscopic (26) H. Kauss, FEBS Lett., 5 (1969) 81-84. (27) E. Hopp, P. Romero, G . R. Daleo, and R. Pont Lezica, Plant Physiol., 59 Suppl. (1977) 82. (28) G. R. Daleo and R. Pont Lezica, FEBS Lett., 74 (1977) 247-250. (29) S. S. Alam and F. W. Hemming, Phyrochemisrry, 12 (1973) 1641-1649. (30) C. T. Brett and L. F. Leloir, Biochem. J., 161 (1977) 93-101. (31) R. C. Garcia, E. Recondo, and M. Dankert, Eur. J. Biochem., 43 (1974) 93-105. (32) P. J. Dunphy, J. D. Kerr, J. F. Pennock, K. J. Whittle, and J. Feeney, Biochim. Biophys. Acra, 136 (1967) 136-147. (33) D. P. Delmer, C. Kulow, and M. C. Ericson, Plant Physiol., 61 (1978) 25-29.

TABLEI Structure of Plant Polyprenols Characterized No. of double bonds Source

No. of isoprene units

E

Z

a-Residue

References

Aglaonema robellinii Beta vulgaris Betula vemcosa Aesculus hippocastanum Saccharomyces cerevisiae 0 Phytophtora cactorum

10-13 10-13 6-9 11-13 13-17 13-17

3 3 2

6-9 6-9 3-6 7-9 8-12 8-12

allylic allylic allylic allylic saturated saturated

32 34 35,36 37 32,38 39

Aspergillus niger Ficus elastica Hevea brasiliensis Aspergillus fumigatus Juniperus communis Pinus sylvestris Nicotiana tabacum Arum maculatum

19-23 10-13 10-13 19-23 14-21 13-17 9 10

2 3 3 2 3

14-18 6-9 6-9 14-18 10-17

n.d.b

mainly

9 10

0 0

saturated allylic allylic saturated allylic allylic allylic allylic

40 41 32 42 43 44,45 46,47 48

Polyprenol

Aglaprenol Betaprenol Betulaprenol Castaprenol Dolichol Hexahydro-exomethylenepolyprenol Ficaprenol Heveaprenol Hexahydropolyprenol Juniprenol Pinoprenol Solanesol Spadicol

~ ~ ~ ~ _ _ _ _ _ _ _ _ _ _

See Table I1 for other sources.

n.d., not determined.

LIPID-LINKED SUGARS AS INTERMEDIATES I N PLANTS

347

11. LIPID-LINKED SUGARS

1. The Lipid Moiety

a. Occurrence and Structural Aspects.-A great variety of polyprenols occurs naturally in plants; they have a carbon skeleton made up of isoprene residues linked head to tail and contain a primary alcohol group. The general structure is shown in formula 1, and the number (x) of isoprene residues is variable from 6 to 21, depending on the source. OH JX

L

1

Table I summarizes the plant polyprenols that have been isolated and whose sources and some structural aspects have been characterized. These polyprenols occur in different species as mixtures, differing from each other in one isoprene unit. One or two members of the family are very abundant and normally account for 80% of the total polyprenol content of the plant. Most long-chain polyprenols have 2, E double bonds, the E-isoprene units being adjacent to the w-residue and to a variable number of 2 residues, depending on the plant (see Table I).

(34) (35) (36) (37)

A. R. Wellburn and F. W. Hemming, J. Cbromatogr., 23 (1966) 51-55. B. 0. Lindgren, Acta Cbem. Scand., 19 (1965) 1317-1323. A. R. Wellburn and F. W. Hemming, Nafure (London), 212 (1966) 1364-1366. A. R. Wellburn, J. Stevenson, F. W. Hemming, and R. A. Morton, Biocbern. J., 102 (1967) 313-324.

(38) (39) (40) (41)

P. Jung and W. Tanner, Eur. J. Biocbem., 37 (1973) 1-6. J. B. Richards and F. W. Hemming, Biochem. J., 128 (1972) 1345-1352. R. M. Barr and F. W. Hemming, Biocbern. J., 126 (1972) 1193-1202. K. J. Stone, A. R. Wellburn, F. W. Hemming, and J. F. Pennock, Biochem. J., 102

(1967) 325-330. (42) K.J. Stone, P. H. W. Buttenvorth, and F. W. Hemming, Biocbem. J., 102 (1967) 443-455. (43) W. Sasak, T. Mankowski, T. Chojnacki, and W. D. Daniewski, FEES Lert., 64 (1976) 55-58. (44) K. Hannus and G . Pensar, Pbyrocbemistry, 13 (1974) 2563-2566. (45) D. F. Zinkel and B. B. Evans, Pbytocbemistry, 11 (1972) 3387-3389. (46) R. L. Rowland, P. H.Latimer, and H.A. Giles, J. Am. Chem. Soc., 78 (1956) 4680-4685. (47) M. Kofler, A. Langemann, R. Ruegg, U.Gloor, U. Schweiter, J. Wursch, 0. Wiss, and 0. Isler, Helu. Cbim. Acra, 42 (1959) 2252-2254. (48) F. W. Hemming, R. A. Morton, and J. F. Pennock, Proc. R Soc. London, Ser. B, 158 (1963) 291-293.

TABLEI 1 Plant Sources from Which Dolichol Has Been Isolated and Characterized Division Chlorophyta (algae) Eumycophyta (fungi)

Spermatophyta (higher plants)

Genus, species

a-Residue

No. of isoprene units

Prororheca zopfii Saceharomyces cerevisiae

saturated saturated

18-21 13-17

c.h., reversed-phase t.1.c.’ ozonolysis, g.f.,b I.c., n.m.r., m.s., t.1.c.

Fusarium solani Gossypium hirmtum Glycine max Phaseolus vulgaris Pisum sativum

saturated n.d.‘ saturated saturated saturated

18-21

I.C.

Vigna sinemis Zea mays

n.d. n.d.

Methods used for identification References

n.d. 17-18 18 18-21 18-21

n.d.

a c.h., Catalytic hydrogenation; t.l.c., thin-layer chromatography. g.f., gel filtration; I.c., resonance spectroscopy; m.s., mass spectrometry. n.d., not determined.

c.h., t.l.c., g.f. c.h., m.s. c.h., reversed-phase t.l.c., g.f. t.1.c.

53 32,38, 54,s 56 57 30,23, 58 33 23, 30, 28 59 60

liquid chromatography; n.m.r., nuclear magnetic

LIPID-LINKED SUGARS AS INTERMEDIATES IN PLANTS

349

The polyprenols obtained from tobacco and Arum maculatum (solanesol and spadicol), which are all E, are the exceptions. Another interesting fact is that, in most seed-plants, the polyprenols are allylic, as is bacterial undecaprenol. Saturated or slightly modified polyprenols, such as animal dolichol, seem to be present only in fungi. Any of those plant polyprenols, in a phosphorylated form, can be potential sugar acceptors for transfer reactions. Experiments were performed with phosphorylated allylic polyprenols and membrane preparations from Phaseolus aureus. D-Mannose from GDP-D-mannose was incorporated into the exogenous, polyprenyl p h o ~ p h a t e s , 2 ~but * ~ ~it -was ~ ~ not possible to establish unequivocally the role of these polyprenols as lipid intermediates. The first evidence showing that plant polyprenols involved in glycosyltransfer reactions are not allylic, but a-saturated, like animal dolichol (2), came from Pont Lezica and c o ~ o r k e r sThe . ~ ~ authors postulated that the presence of sugar acceptors having the properties of a-saturated polyprenyl phosphates may be a general feature of eukaryotic cells, in contrast to the a-unsaturated polyprenyl phosphates characteristic of prokaryotic cells.

02

S. S. Alam and F. W. Hemming, FEES Lerr., 19 (1971) 60-62. C. L. Villemez and A. F. Clark, Biochem. Biophys. Res. Commun, 36 (1969) 57-63. C. L. Villemez, Biochem. Biophys. Res. Commun.,40 (1970) 636-641. A. F. Clark and C. L. Villemez, FEES Lerr., 32 (1973) 84-86. H. E. Hopp, G. R. Daleo, P. A. Romero, and R. Pont Lezica, Planr. Physiol., 61 (1978) 248-251. A. J. Parodi, Eur. J. Biochem, 75 (1977) 171-180. F. Reuvers, P. Boer, and F. W. Hemming, Biochem. J., 169 (1978) 505-508. C. L. Soliday and P. E. Kolattukudy, Arch. Biochem. Biophys., 197 (1979) 367-378. W. T. Forsee and A. D. Elbein, 1. Biol. Chem., 248 (1973) 2858-2867. C. M. Chadwick and D. H. Northcote, Biochem. J., 190 (1980) 255-261. L. Lehle, F. Fartaczek, W. Tanner, and H. Kauss, Arch. Biochem. Biophys., 175 (1976) 419-426. J. R. Green and D. H. Northcote, Biochem. J., 178 (1979) 661-671.

350

RAFAEL PONT LEZICA etal.

Further work in plant systems confirmed, by different methods (biosynthesis from m e v a l ~ n a t e , ~mass ” ~ ~~ p e c t r o m e t r yand , ~ ~the techniques already mentioned3’), that the polyprenyl phosphate involved in glycosyl-transfer reactions is dolichyl phosphate. Table I1 shows different sources from which dolichyl phosphate has been identified in plants. At present, it seems clear that, in plants, as in other eukaryotic organisms, dolichyl phosphate is the lipid involved when glycosyl-transfer reactions occur. However, it has been reported that the lipid carrier for 2-acetamido-2deoxy-D-glucose is different from that for D-mannose in mung beans and cotton fibers.61Because the polyprenols involved in those reactions are all a-saturated, it has been suggested that different chain-lengths of dolichyl phosphate serve as carriers for different sugars.16 This point should be further explored in order that the regulation of the pathway may be understood. b. Biosynthesis and Metabolism of Dolicho1.-Most of the knowledge on the biosynthesis and metabolism of dolichol has come from studies on animal systems. It is synthesized from acetyl-CoA through mevalonate (MVA) to form isopentenyl diphosphate (IPP) and 3,3-dimethylallyl diphosphate (DMAPP). The addition of an IPP residue to a molecule of DMAPP or to a growing isoprenoid chain having an allylic structure is a feature of the polymerization pattern. At each condensation step, two isomers could be obtained having a 2 or E configuration.62 It is generally accepted that a single prenyltransferase (EC 2.5.1.1) could give one isomer. Dolichols have E and 2 double bonds, and therefore, a minimum of two prenyltransferases is required for the polymerization of the chain (see Scheme 1). Little is known as to the biosynthesis and metabolism of plant dolichols. Nevertheless, this is an interesting field if the variety of polyprenols synthesized by plants (see Table I and Scheme l), as compared with animals, is considered. The early knowledge on the stereochemistry of E, 2-polyprenol biosynthesis was obtained by Gough and Hemming63with a plant system. They found that the 4-S hydrogen atom of mevalonate is retained when a Z-double bond is formed, whereas this hydrogen atom is lost in the biosynthesis of an E-double bond. The same authors found similar results for rat-liver dolich01.~~ With this technique, [3H]dolichol was found in rat-liver 4-( S)-C3H]mevalmitochondria when the animals were injected (61) (62) (63) (64) (65)

M. C. Ericson, J. T. Gafford, and A. D. Elbein, Plant Physiol., 61 (1978) 274-277. E. D. Beytia and J. W. Porter, Annu. Rev. Biochern., 45 (1976) 113-142. D. P. Gough and F. W. Hemming, Biochem. J., 117 (1970) 309-317. D. P. Gough and F. W. Hemming, Biochem. J., 118 (1970) 163-166. H. G. Martin and K. J. I. Thorne, Biochem. J., 138 (1974) 277-280.

LIPID-LINKED SUGARS AS INTERMEDIATES IN PLANTS

351

Acetyl-Co A

1

Mevalonote

/

Isopentenyl diphosphote 1-,3,3-Dimethylallyl diphosphate

J

Rubber ( p o l y - Z - isoprene)

Poly-E,Z-isoprene-PP (a-unsatura ted)

4 2,3

- Dehydro -Dot-

PP

I Do1-

PP

I 1

Geronyl diphosphate -+

cc

Farnesyl diphosphate

I

MonoterPenes Essential oils

+~

!

~

~

Abscisic acid Phytol

Geronylgeranyl diphosphate +Carotenes Gibberellins Ubiquinone Plasioquinone

Gutta percha (poly-E- isoprene)

11

D o l - f a t t y acids

SCHEME 1.-The Biosynthesis and Metabolism of Dolichyl Phosphate and Other Plant Isoprenoids. ~ n a t e .Dolichols ~~ from different animal sources have shown the ( S ) configuration at C-3 (see 2).66 No information is available regarding the absolute configuration of plant dolichols. The in nitro synthesis of dolichyl phosphate was also achieved for the first time with plant systems, namely, from isopentenyl diphosphate (from garden peasz8) or mevalonate (from algae53). These early experiments, as well as others using chicken and rabbit liver by the same authors, indicated that membrane fractions are required and that the mitochondria-rich fractions show the highest biosynthetic activity.67These results were in agreement with those from previous ~ o r k , 6 *thus * ~ ~indicating that free dolichol is present in higher concentrations in mitochondria. Work with rat liver, using improved techniques, showed that the 2-prenyltransferase(s) adding (66) W. L. Adair, Jr., and S. Robertson, Biochem. J., 189 (1980) 441-445. (67) G. R. Daleo, H. E. Hopp, P. A. Romero, and R. Pont Lezica, FEBS Lett., 81 (1977) 411-414. (68) J. Burgos and R. A. Morton, Biochem. J., 82 (1962) 454-456. (69) P. H. W. Butterworth and F. W. Hemming, Arch. Biochem. Biophys., 128 (1968) 503-508.

~

s

352

RAFAEL PONT LEZICA et a/.

isopentenyl diphosphate residues to farnesyl diphosphate are located mainly in the endoplasmic reticulum-rich fraction, and only a minor part in mito~hrondria.~'.~~ On the other hand, the biosynthesized dolichol is rapidly transferred and accumulated in a mitochondria-lysosome fraction. A careful separation of these organelles in a ficoll gradient showed that most of the dolichol is accumulated in the lysosomal fra~tion.~' Grange and Adair73isolated 2,3-dehydrodolichyl phosphate from in v i m studies on hen oviduct. It may be derived from 2,3-dehydrodolichyl diphosphate, a likely intermediate in the biosynthesis of dolichyl phosphate. Many questions still remain unanswered, such as: is the substrate for the aisoprene reductase, the diphosphate or the monophosphate of 2,3-dehydrodolichol? Are the unsaturated polyprenols a reserve pool for the synthesis of dolichol in plants? What is the function of unsaturated, plant polyprenols? How is the synthesis of saturated and unsaturated polyprenols regulated? Dolichyl phosphate phosphatase has been described in animal t i s s ~ e s , ~ ~ - ~ ~ and it is possibly responsible for the free dolichol found in tissues. This free dolichol can be rephosphorylated by a dolichol kinase using cytidine triphosphate (CTP) as phosphoric donor,77*78 or acylated by a dolichol a~yltransferase~~ (see Scheme 1). 2. The Saccharide Derivatives a. Lipid-linked Monosaccharides.-Dolichol-linked monosaccharides have been isolated from different sources, including fungi, algae, and higher plants (see Table 111). Two kinds of reaction lead to the synthesis of dolichol-linked monosaccharides. NDP-sugar+ Dol-P

* NDP+glycosyl-P-Do1

NDP-sugar+ Dol-P -+ NMP+glycosyl-PP-Do1

(1)

(2)

Reaction (1) is catalyzed by membrane-bound glycosyltransferases requiring Mg2+,and is inhibited by an excess of the nucleoside diphosphate (NDP) or (ethylenedinitri1o)tetraacetic acid (EDTA). Although the (70) (71) (72) (73) (74) (75) (76)

T. K. Wong and W. J. Lennarz, J. Bid. Chem., 257 (1982) 6619-6624. W. L. Adair and R. K. Keller, 1. Bid. Chem., 257 (1982) 8990-8996. T. K. Wong, G. L. Decker, and W. J. Lennarz, J. Bid. Chem., 257 (1982) 6614-6618. D. K. Grange and L. Adair, Biochem. Biophys. Res. Commun., 79 (1977) 734-740. G. S. Adrian and R. W. Keenan, Biochim. Biophys. Acra, 575 (1979) 431-438. J. F. Wedgwood and J. L. Strominger, J. Bid. Chem., 255 (1980) 1120-1123. V. Idoyaga-Vargas, E. Belocopitow, A. Mentaberry, and H. Caminatti, FEBS Lett.,

112 (1980) 63-66. (77) C. M. Allen, J. R. Kalin, J. Sach, and D. Veruzzo, Biochemisfry, 17 (1978) 5020-5026. (78) W. A. Burton, M. G. Scher, and C. J. Waechter, J. Bid. Chem., 254 (1979) 7129-7136. (79) R. W. Keenan and M. E. Kruczek, Biochemistry, 15 (1976) 1586-1591.

LIPID-LINKED SUGARS AS INTERMEDIATES IN PLANTS

353

TABLEIll

Lipid-linked Monosaccharides Isolated from Plant Systems Sugar moiety D-Glucose

Anomeric configuration

Phosphate linkage

n.d." n.d. n.d. n.d.

n.d. n.d.' n.d.' n.d. n.d. n.d. n.d.

P P P P P P P PP PP P P P P

n.d.

P

P P

D-Mannose

P n.d. n.d. n.d. n.d. n.d.

P 2-Acetarnido2-d eox y D-ghICOSe 6-Deoxy-~galactose D-Galacturonic acid 2-Deoxy-~arabinohexose

n.d. n.d. n.d.' n.d.' ad.'

P P P P PP PP PP

n.d.

n.d. n.d. n.d.

Source Glycine max (seeds) Saccharomyces cereuisiae Gossypium hirsurum (fibers) Zea mays (roots) Pisum sariuum (epicotyls) Prototheca zopjii Vigna sinensis (hypocotyls) Prototheca zopjii Pisum satiuum (seedlings) Vigna sinensis (seedlings) Codium fragile Licopersicum esculenrum (roots) Pisum satiuum (seedlings, cotyledons) Phaseolus uulgaris (seeds, cotyledons) Fusarium solani Gossypium hirsurum (fibers) Prototheca zopjii Ricinus communis (endospenn) Acer pseudoplatanus (cell cultures) Hansenula holstii Saccharomyces cereuisiae Aspergillus niger Neurospora crassa Pisum safiuum (seedlings) Saccharomyces cereuisiae Vigna sinensis (seedlings) Zea mays (roots)

P P P

Lemna minor Saccharomyces cereuisiae Prototheca zopjii

References 30 54 57, 80 60 81 82 83 82 84 29, 59, 85 29 29 30, 86 33, 87 56 57, 88 89 90 91 92 93, 94 95, 96 91 30 55, 88 59. 85 60

98 99 100

n.d., Not determined. Vigna sinensis is the actual name for Phaseolus aureus. 'The anorneric configuration of the pyrophosphate derivatives should be a,as the configuration of the sugar nucleotide is maintained.

354

RAFAEL PONT LEZICA

et

al.

anomeric configuration of the lipid-linked sugars has not been studied in all systems, those so far examined show the formation of P-glycosyl-P-Do1 This indicates in plants (see Table 111), as well as in animal systems.101~102 that the reaction occurs with inversion of the configuration. Either D-glucose or D-mannose is the sugar most frequently involved in these reactions. An L-fucosyl-lipid has been isolated, although its role is not yet clear.60 Lipid-linked 2-deoxy-~-arabino-hexose(2-dGlc) has been formed by plant systems, but it does not seem to be a natural sugar. The evidence obtained indicate^^^^"' that the D-mannosyltransferase (EC 2.4.1.83) involved in the formation of Man-P-Do1 is responsible for the formation of 2-dGlc-P-Dol. This sugar is an analog of D-glucose and D-mannose, but the D-glucosyltransferase (EC 2.4.1.78) is not able to form a 2-deoxy-~-arabino-hexosederivative."' D-Xylose has been reported to be incorporated into a dolichyl derivative by hen-oviduct mi~rosomes,''~but no report exists of such in plant systems. (80) W. T. Forsee and A. D. Elbein, Biochem. Biophys. Res. Commun., 49 (1972) 930-939. (81) R. Pont Lezica, P. A. Romero, and M. A. Dankert, Plant Physiol., 58 (1976) 675-680. (82) H. E. Hopp, P. A. Romero, G. R. Daleo, and R. Pont Lezica, Eur. J. Biochem., 84 (1978) 561-571. (83) D. J. Bowles, L. Lehle, and H. Krauss, Planta, 134 (1977) 177-181. (84) P. A. Romero and R. Pont Lezica, Acra Physiol. Latinoam., 26 (1976) 364-370. (85) W. T. Forsee, G. Volkovich, and A. D. Elbein, Arch. Biochem. Biophys., 174 (1976) 469-478. (86) L. Beevers and R. M. Mense, Plant Physiol., 60 (1977) 703-708. (87) M. C. Ericson and D. P. Delmer, Plant Physiol., 59 (1977) 341-347. (88) F. Reuvers, C. Habets-Willems, A. Reinking, and P. Boer, Biochim. Biophys. Acta, 486 (1977) 541-552. (89) P. A. Romero, H. E. Hopp, and R. Pont Lezica, Biochim. Biophys. Acta, 586 (1979) 545-559. (90) R. B. Mellor and J. M. Lord, Planta, 146 (1979) 91-99. (91) M. M. Smith, M. Axelos, and C. Piaud-Lenoel, Biochimie, 58 (1976) 1195-1211. (92) R. K. Bretthauer, S. Wu, and W. E. Irwin, Biochim. Biophys. Acta, 304 (1973) 736-747. (93) L. Lehle and W. Tanner, Eur. J. Biochem., 83 (1978) 563-570. (94) W. Tanner, Biochem. Biophys. Res. Commun., 35 (1969) 144-150. (95) R. M. Barr and F. W. Hemming, Biochem. J., 126 (1972) 1203-1208. (96) R. C. P. Letoublon, J. Compte, and R. Got, Eur. J. Biochem., 40 (1973) 95-101. (97) M. H. Gold and H. J. Hahn, Biochemistry, 15 (1976) 1808-1813. (98) G. R. Daleo and P. Kindel, Fed. Proc. Fed. Am. Soc. Exp. Biol.,38 (1979) 790. (99) L. Lehle and R. T. Schwarz, Eur. J. Biochem., 67 (1976) 239-245. (100) R. Datema, R. T. Schwarz, L. A. Rivas, and R. Pont Lezica, Plant Physiol., 71 (1983) 76-81. (101) A. Herscovics, B. Bugge, and R. W. Jeanloz, J. Biol. Chem., 252 (1977) 2271-2277. (102) A. Herscovics, C. D. Warren, and R. W. Jeanloz, J. Biol. Chem., 250 (1975) 8079-8084. (103) C. J. Waechter, J. J. Lucas, and W. J. Lennarz, Biochem. Biophys. Res. Commun., 56 (1974) 343-350.

LIPID-LINKED SUGARS AS INTERMEDIATES IN PLANTS

355

D-Xylose is a normal component of plant cell-walls, and it will be interesting to study the possibility of a lipid-mediated pathway for such products. The synthesis of Gal-P-Do1 has been reported, using Acetobacter xylinum enzymes and exogenous Dol-P. The bacterium uses undecaprenyl-P as .~~~ substrate in uiuo.'04For other systems, the reports are c o n t r o ~ e r s i a lUsing a Chlamydomonas preparation, some incorporation from UDP-D-galactose into a polyprenyl type of glycolipid was supposedly obtained but study of the product indicated that the sugar incorporated was D-glucose, not Dgalactose. Presumably, a 4-epimerase had converted UDP-D-galactose into UDP-~-glucose.'~~ In reaction (2), a glycosyl 1-P is transferred from the glycosyl nucleotide to the lipid-P, and a diphosphate derivative is formed. In this case, the a configuration present in the glycosyl nucleotide is presumably retained in the lipid derivative. Most of the reports indicated that 2-acetamido-2-deoxyD-glucose is the sugar involved in this type of reaction. However, similar reactions involving D-glucosyl 1-P have been found in higher plants,84 algae,82and bacteria.lo6The formation of 2-acetamido-2-deoxy-~-mannosylPP-Do1 by pig-liver microsomes has been described.Io7 Such a compound has not been found in plant systems, and even its role in animal tissues is still completely unknown. b. Lipid-1inkedlOligosaccharides.-Oligosaccharides linked to dolichol by a diphosphate link have been isolated'08-'2' from different plant-sources (see Table IV). Most of the oligosaccharides listed are intermediates in the biosynthesis of a unique oligosaccharide containing Glc, Man,GlcNAc2. (104) (105) (106) (107) (108) (109) (110) (1 11) (112) (113) (114) (115) (116) (117) (118) (119)

(120) (121)

P. Romero, R. C. Garcia, and M. Dankert, Mol. Cell. Biochem., 16 (1977) 205-212. W. C. Lang, Plant Cell Physiol., 23 (1982) 1259-1266. M. Dankert, R. Garcia, and E. Recondo, in Ref. 2, pp. 199-206. G. Palamarczyk and F. W. Hemming, Biochem. J., 148 (1975) 245-251. K. Nakayama, Y. Araki, and E. Ito, FEBS Lett., 72 (1976) 287-290. L. Lehle and W. Tanner, Biochim. Biophys. Acta, 539 (1978) 218-229. A. J. Parodi, Eur. J. Biochem., 83 (1978) 253-259. L. Lehle and W. Tanner, Biochim. Biophys. Acta, 399 (1975) 364-374. W. T. Forsee and A. D. Elbein, J. Biol. Chem., 250 (1975) 9283-9293. H. Hori and A. D. Elbein, Plant Physiol., 70 (1982) 12-20. R. J. Staneloni, M. E. Tolmasky, C. Petriella, R. A. Ugalde, and L. F. Leloir, Biochem. J., 191 (1980) 257-260. H. Hori, D. W. James, and A. D. Elbein, Arch. Biochem. Biophys., 215 (1982) 12-21. L. Lehle, I. Schulz, and W. Tanner, Arch. Microbiol., 127 (1980) 231-237. A. J. Parodi, Arch. Biochem. Biophys., 210 (1981) 372-382. L. Lehle, Eur. J. Biochem., 109 (1980) 589-601. R. J. Staneloni, M. E. Tohasky, C. Petriella, and L. F. Leloir, Plant Physiol., 68 (1981) 1175-1179. L. Lehle, FEBS Lett., 123 (1981) 63-66. R. Pont Lezica, P. A. Romero, and H. E. Hopp, Planta, 140 (1978) 177-183.

RAFAEL PONT LEZICA er a/.

356

TABLEIV Lipid-linked Oligosaccharides Isolated from Plants Oligosaccharide moiety (GalNAc)2

Source Pisum sarivum (seedlings,

cotyledons)

Man-(GlcNAc), (Man),-(GlcNAc),

(Man),-(GlcNAc), (Glc)y-(Man),-(GlcNAc), (GI&-( Man),-(GlcNAc),

(Man),-(neutral),-(amino sugar), (Glc),

References

Vigna sinensis" (seedlings) Prororheca zopfi Saccharomyces cerevisiae Vigna sinensis" (seedlings) Saccharomyces cerevisiae Prororheca zopfi Saccharomyces cerevisiae Gossypium hirsutum (seed fibers) Vigna sinensis" (seedlings) Saccharomyces cerevisiae Medicago sariva (roots) Glycine max (cell suspension) Saccharomyces cerevisiae Pisum sativum (cotyledons) Nicoriana rabacum (cell culture) Spinacia oleracea (leaves) Acer pseudoplatanus Prororheca zopfi Pisum sativum

30, 86 59.85 89 88 59, 85 93, 108 89 109-111 112 113 54 114 115 116-118 119 120 120 91 82 121

Vigna sinensis is the current name for Phaseolus aureus.

This lipid-linked oligosaccharide is present in fungi, seed plants, and animals, and serves as precursor for yeast mannans116-118 or L-asparaginelinked glycans.l3-I6 The structure of Glc3Man9GlcNAcz-PP-Dolhas been extensively studied in animal systems, and it seems to be a common feature for all eukaryotic cells.'zz Fig. 1(A) shows the structure widely accepted for the intermediate. Nevertheless, some modifications have been proposed for the oligosaccharide found in higher plants113s11s [see Fig. l(B)]. Further studies are needed in order to elucidate whether oligosaccharides from higher-plant sources fit with the general structure shown in Fig. l(A).

3. Turnover No experimental evidence is as yet available that would throw light on the turnover of lipid intermediates. Nevertheless, the cycle of dolichol in (122) R. C. Hughes and T. D. Butters, Trends Biochem. Sci., 6 (1981) 228-230.

a-D-Gkp-(l+ 2)-[a-~-Glcp-(1 + 3)1,-[a-~-Manp-(l+ 2)],-a-~-Manp 1

1 3 p-D-Manp-(l+ 4)-p-D-GlcpNAo( 1 + 4)-p-~-GlcpNAc-l-P PP-Do1 6

t 1 a-D-Manp-(1 + 2 ) - a - ~ - M a n p - ( l +3 ) - a - ~ - M a n p 6

t 1

a-D-Manp-(l

+ Z)-a-D-Manp

(A) a-o-Glcp-(l+2)-[a-D-G~cp-(l+3)],-(a-~-Manp-(l +2)]30r4-a-~-Manp 1

1 3 8-D-Manp-(l+4)-p-~-GlcpNAc-( 1 + 4 ) - p - ~ - G l c p N A c - l +PP-Do1 1 A

R1or R2

(B) I

6

t 1

I

6

t

R2= [a-D-Manp(l+ Z)],,,,-a-~-Manp 1

R' = [a-D-Manp-(1 +2)]oo,l-a-~-Manp-(l+ 3 ) - a - ~ - M a n p 6

t

1 a-D-Manp

FIG.1.-Structures

of the Oligosaccharide-linked Lipids (A) Present in Animal Cells and (B)Proposed for Plant cell^."^*"^

358

RAFAEL PONT LEZICA et al.

plant and animal systems indicates that the lipid can be recycled. Because the total amount of dolichyl phosphate in tissues is very low, and the synthesis of final product in uiuo is quite rapid, the turnover of dolichyl phosphate should be very efficient, but published work on this subject is still lacking. 111. COMPLEX CARBOHYDRATES

1. Polysaccharides

The cell wall of fungi is a complex, multilayered structure formed by at least two major polysaccharides. These major polysaccharides have been used as a taxonomic criterion for fungi, as some of them contain chitinglucans, and others, glu~omannans.'~~ The primary cell-wall of plants is formed by microfibrils of cellulose in a complex matrix of polysaccharides, proteins, and glycoproteins. The structure and relationship of the different components is under continuous study and revision, and most of them can now be identified and their biosynthesis ~ t u d i e d . ' ~ ~ - ' ~ ~ In fungi, and in higher plants, the cell wall is a dynamic structure, changing during growth and differentiation, or in response to environmental factors. The main polysaccharides found in plant cell-walls are listed in this Section. The biosynthesis of those which are synthesized by way of lipid intermediates (see Table V) will be analyzed later. (123) S. Bartnicki-Garcia, Annu. Rev. Microbiol., 22 (1968) 87-108. (124) A. Darvill, M. McNeil, P. Albersheim, and D. P. Delmer, in N. E. Tolbert (Ed.), The Biochemistry of Plants, Vol. 1, Academic Press, New York, 1980, pp. 75-92. (125) G. 0 . Aspinall, in J. Preiss (Ed.), The Biochemistry of Plants, Vol. 3, Academic Press, New York, 1980, pp. 473-500. (126) M. C. Ericson and A. D. Elbein, in Ref. 125, pp. 589-615. (127) W. Odzuck and H. Kauss, Phytochernistry, 1 1 (1972) 2489-2494. (128) N. Panayotatos and C. L. Villemez, Biochem. J., 133 (1973) 263-271. (129) H. Meier and J. S. G. Reid, Planta, 133 (1977) 243-248. (130) C. L. Villemez, A. L. Swanson, and W. 2. Hassid, Arch. Biochem. Biophys., 116 (1966) 446-452. (131) L. Glaser, J. Biol. Chem., 232 (1958) 627-636. (132) J. Kjosbakken and J. R. Colvin, in F. Loewus (Ed.), Biogenesis of Plant Cell Wall Polysuccharides, Academic Press, New York, 1973, pp. 361-371. (133) A. D. Elbein and W. T. Forsee, in Ref. 132, pp. 259-295. (134) G. Franz, Appl. Polym. Symp., 28 (1976) 611-621. (135) G. Shore and G. A. MacLachlan, Biochim. Biophys. Acru, 329 (1973) 271-282. (136) G. Shore, Y. Raymond, and G. A. MacLachlan, Plunt Physiol., 56 (1975) 34-38. (137) C. T. Brett and D. H. Northcote, Biochem. J., 148 (1975) 107-117. (138) W. Van der Woude, C. A. Lembi, and D. J. Morrt, Plant Physiol., 54 (1974) 333-340.

TABLEV Polysaccbarides from Plant Cell-walls Polymer

Arabinan Galactan Galactomannan Galacturonan (1-* 4)-P-Glucan

(1 + 3)-P-Glucan

Glucomannan Mannan

PuI1uIa n Xylan

Xyloglucan

Precursor

Product linkage

Lipid intermediates

Gal-( 1+ 4),( 1+ 3)

n.d.b suggested

127 128

n.d. a-GalA-( 1 + 4) a-GalA-( 1 + 4) B-Glc-( 1 -* 4) P-Glc-( 1+ 4) p-( 1 + 4)-glucoprotein P-Glc-(1+4) @-Glc-(1+ 3),( 1+ 4) j3-Glc-(l-* 4) P-Glc-( 1-P 3) B-Glc-(l+ 3) B-Glc-(1-* 3) P-Glc-( 1+ 3) p-Glc-( 1 + 3) p-Man-( 1 + 4)-P-Glc-( 1+ 4)

n.d. n.d. suggested Yes n.d. n.d. n.d. suggested Yes suggested n.d. n.d. discarded suggested suggested

129 130 98 131,132 133 134 135, 136 137 82 138 139 133 140 137 141, 142 143

yeast mannan p-Man-(l+ 4) p-Man-( 1 + 4) p-Man-(1+4)

Yes Yes n.d. n.d.

a-Glc-( 1+ 3),( 1-* 6) Xyl-(1+ 4)

suggested n.d. n.d. discarded n.d.

Source

Vigna sinensis" Vigna sinensis" Trigonellafoenumgraecum (in uiuo) UDP-GalA Vigna sinensis' Lemna minor UDP-GalA UDP-Glc Acetobacter xylinum Vigna sinensis" GDP-Glc Vigna sinensis" UDP-Glc UDP-Glc-GDP-Glc Pisum satioum UDP-Glc Pisum satiuum UDP-Glc-GDP-Clc Prototheca zopfi UDP-Glc Allium cepa UDP-Glc Glycine mar Gossypium hirsutum UDP-Glc Petunia hybrida UDP-Glc Pisum satioum UDP-Glc UDP-Glc-GDP-Man Pisum satioum Vigna sinensis" GDP-Man Saccharomyces cereviriae Acer pseudoplatanus GDP-Man Vigna sinensis" GDP-Man ODP-Man Orchis mono Glc Aureobasidium pullulans Vigna sinensis" UDP-Xyl Zea mays Acer pseudoplatanus UDP-Glc-UDP-Xyl Vigna sinensis" UDP- Ara UDP-Gal Gal

'

Vigna sinensis is the current name for Phaseolus aureus. n.d., Not determined:

References

110,117,144 91

145 146 147 127 148 149, 150 151

360

RAFAEL FQNT LEZICA et4L

The mechanism of synthesis of polysaccharides is a controversial issue. After discovery, by Cardini's group, that starch may be polymerized on a that all nascent, polysaccharide chains it was might be covalently associated with a protein. Connected with the formation of glycoprotein is the involvement of lipid intermediates. We shall analyze the biosynthetic pathways of polysaccharides where partial or complete evidence of this kind of mechanism has been educed. a. Cellulose.-Although cellulose is the most abundant carbohydrate on earth, being the main structural material of the plant cell-wall, our knowlis minuscule. Moreover, results have been edge of its biosynthesi~'~~ obtained that support contradictory opinions as to the mechanism of synthesis. Among the controversial aspects are the criteria for considering true cellulose synthesis (solubility, size, crystalline structure, assembly in a structural network); whether one enzymic system, or more, is involved; whether the chains grow at the reducing or nonreducing end, and whether or not a primer is required for cellulose biosynthesis. We shall center our interest on the aspects related to the mechanism of chain initiation. This is where the search for lipid intermediates has been partially successful. C o l ~ i n ' ~was ' the first to postulate a lipid-bound D-glucose as an intermediate in the biosynthesis of bacterial cellulose. Lipid-sugar derivatives, tentatively identified as lipid-diphosphate-D-glucose, lipid-diphosphatecellobiose, and, perhaps, higher polymers, were detected in this system.'*' These lipid-sugar compounds, which were acid- and alkali-labile, seemed to be formed prior to cellulose, and their formation was inhibited by adding C. T. Brett, Plant Physiol., 62 (1978) 377-382. J. P. F. G. Helsper, Plant4 144 (1979) 443-450. A. D. Elbein, J. Eiol. Chem., 244 (1969) 1608-1616. M. B. Hinman and C. L. Villemez, P/unr Physiol., 56 (1975) 608-612. J. S. Heller and C. L. Villemez, Eiochem. J., 129 (1972) 645-653. E. Bause and L. Lehle, Eur. J. Eiochem, 101 (1979) 531-540. J. S. Heller and C. L. Villemez, Eiochem. J., 128 (1972) 243-252. G. Franz, Phytochemistry, 12 (1973) 2369-2373. B. J. Catley and W. McDowell, Carbohydr. Res., 103 (1982) 65-75. R. W. Bailey and W. 2. Hassid, Proc. Nut/. Acud. Sci USA, 56 (1966) 1586-1593. G. Dalessandro and D. H. Northcote, Plunra, 151 (1981) 54-60. G. Dalessandro and D. H. Northcote, Planmu, 151 (1981) 61-67. C. L. Villemez and M. Hinman, PTunr Physiol.. 56 (1975) Suppl. 15. N. Lavintman and C. E. Cardini, FEES Lerr.. 29 (1973) 43-46. N. Lavintman, J. Tandecan, M. Carceller, S. Mendiara, and C. E. Cardini, Eur. J. Eiochem., 50 (1974) 145-155. (1530) W. J. Whelan, Trends Biochern Sci, 1 (1976) 13-15. (154) D. P. Delmer. Adu. Curbohydr. Chem. Eiochem., 41 (1983) 105-153. (155) J. R. Colvin, Nature (London), 183 (1959) 1135-1136. (139) (140) (141) (142) (143) (144) (145) (146) (147) (148) (149) (150) (151) (152) (153)

LIPID-LINKED SUGARS AS INTERMEDIATES IN PLANTS

..

6I-PP

(Glcl, - PP- Do1

Protein

4-

’ UDP

1’

(Glc1,-

GDP-GIC

361

Protein

GDP

Cellulose

SCHEME 2.-Proposed

Pathway for the Biosynthesis of Cellulose in Algae.82

either UMP or UDP. Although other author^^'"^ confirmed the formation of lipid derivatives, these have not been clearly identified as intermediates. Glucolipid formation was detected in several plant systems in uitro. Some of them were not characterized, but others were identified as steryl Dglucosides not involved in cellulose biosynthesis. Enzyme preparations from cotton fibers catalyzed the formation of polyprenyl glucosyl phosphates,s7s80 but unequivocal evidence for their role as intermediates was not presented. Brett and N ~ r t h c o t e ’reported ~~ formation of D-glucoiipid from UDP-Dglucose with membrane fractions from pea roots. One to three D-glucosyl residues bound to lipid through a phosphoric ester were obtained, as well as protein-bound oligosaccharides and P-D-glucans. All products contained p-(1+3) and P-(1+4) linkages, but in different ratios, and alternative pathways, with the D-glucolipid playing an intermediate role, were postulated. More-conclusive results were obtained by working with the green alga Prototheca z ~ p f i i . In ~ ~this ” ~system ~ (see Scheme 2), dolichyl-diphosphateoligosaccharides having (1 + 4)-linked D-glucose were formed from UDPD-glucose. One D-glucosyl group, or more, was then transferred from Glc-P-Do1 to the oligosaccharide. The carbohydrate moiety was now (156) H. E. Hopp, P. A. Romero, G. R. Daleo, and R. Pont Lezica, in L. A. Appelqvist and C. Liljenberg (Eds.), Advances in Biochemisiry and Physiology of Plant Lipids, ElsevierNorth Holland Medical Ress, Amsterdam, 1979, pp. 313-318.

362

RAFAEL PONT LEZICA et a/.

transferred to a protein acceptor, in a reaction that can be inhibited by co~marin.'~'It is not known whether the D-glucosyl groups added by way of Glc-P-Do1 have a distinct feature (for example, a different configuration) that could serve as a signal for the transfer, and it would be of interest to study this problem. The aforementioned reactions take place in the endoplasmic reticulum; the glucoprotein can serve in the Golgi apparatus as a primer for cellulose synthase (EC 2.4.1.29), which adds glucosyl groups from GDP-D-glucose, to form an alkali-insoluble polymer.'56 So far, the results obtained with higher plants do not allow generalization of the scheme. Moreover, kinetic experiments performed on cotton cellulose indicated that the primary and secondary cell-wall celluloses would be formed by different, simultaneously operating me~hanisrns.'~~ b. Yeast Mannan.-The structure and biosynthesis of yeast mannan have been the subjects of extensive research. Strong evidence supports the structure proposed by Nakajima and B a l l o ~ , having ' ~ ~ two types of saccharide bound to protein in Saccharomyces cerevisiae D-mannan (see Scheme 3). One of them is composed of short chains of D-mannose glycosidically (1 + 4)-linked to 0 - 3 of L-serine or L-threonine. The other type is composed of an inner core containing two L-asparagine-linked 2-acetamido-2-deoxyD-glucosyl and -12 D-mannosyl residues, and an outer chain of 100-150 D-mannosyl residues. Man-P-Do1 is the precursor of the D-mannosyl groups joined to L-serine or L-threonine.'62 So far, direct 0-glycosylation seems to be restricted to fungi. It has also been reported in Neurospora crassa9' and in Fusarium , ~ it~ has not been shown to occur in animal system^.'^^"^^ solani f. p i ~ ibut Upon incubation of membrane fractions of S. cerevisiae with D-['*C]mannose-P-Do1 plus unlabelled GDP-D-mannose, the oligosaccharides released by p-elimination were radioactive, with the label at the reducing end. GPD-D-mannose is the substrate for further glycosylation of the residues linked to L-serine or L-threonine.I6*Both kinds of glycosyl-transfer reaction are differentially affected by aging of the membrane preparations, and they also have different ion requirements: transfer of D-mannose to

(157) (158) (159) (160) (161) (162) (163) (164)

H. E. Hopp, P. A. Romero, and R. Pont Lezica, FEES Lett, 86 (1978) 259-262. M. Marx-Figini, J. Polym. Sci., Part C, 28 (1969) 57-67. T. Nakajima and C. E. Ballou, J. Biol. Chem., 249 (1974) 7685-7694. A. J. Parodi, J. Biol. Chem., 254 (1979) 8343-8352. P. W. Robins, S. C. Hubbard, S. J. Turco, and D. F. Wirth, Cell, 12 (1977) 893-900. C. B. Sharma, P. Babczinski, L. Lehle, and W. Tanner, Eur. J. Biochem., 46 (1974) 35-41. P. Babczinski. FEES Letr., 117 (1980) 207-211. J. A. Hanover, W. J. Lennarz, and J. D. Young, J. Biol. Chem., 255 (1980) 6713-6716.

LIPID-LINKED SUGARS AS INTERMEDIATES IN PLANTS

363

UDP- GIc NAC UDP

uMp');""c-~FTDoI UDP-GIcNAc

\

GlcNAc,-PP-Do1

y,ycno L",.. "Yr -l*I",,

Dol-P UDP-Glc

\

\

GDP

Man-GlcNAc,- PP-Do1 Man-P-Do1 Man

F

GIc3Mon,GIcNAcp-PP-Dol Asn-Protein

-4

Glc3Man9GlcNAcp- Asn-Protein

1

Processing

and from Dol-P requires Mg2+ or Mn2+, whereas the elongation reaction occurs 0nly'62*'65in the presence of Mn2+. Using synthetic .peptides, Bause and Lehle'# studied the structural requirements for 0-glycosylation with Man-P-Dol. Their results indicated that D-mannosyl transfer to an L-serine or L-threonine residue requires at least a tripeptide, with the hydroxyamino acid at a nonterminal position. When longer peptide chains are used as acceptors, D-mannosyl incorporation is significantly enhanced; further elongation to D-manno-oligosaccharides seems, in contrast, to be independent of peptide structure. Because no marker sequence could be deduced for the formation of the glycosidic linkage, it was assumed that accessibility, not recognition of a specific sequence, might be the key to this reaction. The P-D-mannosyl group is transferred from P-D-mannosyl-P-Do1 with inversion of its configuration, to form an a-D-mannosylpeptide. The inner core of the N-glycosylically linked polysaccharides is formed by way of an en bloc transfer from Dol-PP-oligosaccharide, in a reaction (165) P. Babczinski and W. Tanner, Biochem. Biophys. Res. Commun., 54 (1973) 1119-1124.

364

RAFAEL PONT LEZICA etal.

sequence that so far shows no difference from the general, protein-glycosylation pathway in animal In a series of studies by P a r ~ d i ' ~and ~~''~ Lehle,"' evidence was presented showing the occurrence of a Dol-PP derivative containing two 2-acetamido-2-deoxy-~-glucosyl, 9 D-mannosyl, and 3 D-glucosyl residues. The size, monosaccharide composition, and pattern obtained upon acetolysisof the oligosaccharide and paper chromatography were the same for the wild-type and mutant cells as regards the structure of the outer chain, as well as for the main, corresponding compound isolated from animal tissues.'66 However, a detailed structural analysis of the yeast Dol-PP-bound oligosaccharide must be performed in order to show complete identity. Formation of the Dol-PP-trisaccharide has been found to follow the general pathway shown'6p111 in Scheme 3, that is, GlcNAc-1-P, GlcNAc, and Man are sequentially transferred to Dol-P from the corresponding glycosyl nucleotides. The glycosyl donor for further elongation has not been conclusively identified, but evidence indicating that, from Man,GlcNAc2 to Man,GlcNAc2, D-mannosyl groups are transferred from Man-P-Do1 has been presented."' Glc-P-Do1 is the donor of the three D-glucosyl groups in the completion of the oligosaccharide-PP-dol.'18~160 This oligosaccharide, containing the full complement of monosaccharides, is then transferred to endogenous p r ~ t e i n , "also ~ following the pathway of protein glycosylation in animal cells.161 When synthetic peptides were used as acceptors for N-glycosylation from GlcNAc2-PP-Dol,the necessity for the tripeptide sequence Asn-X-Ser/Thr could be demonstrated. Moreover, the rate of glycosylation was affected by the chain length of the peptides. Other factors, such as hydrophobicity and steric hindrance, have different effects on the acceptor properties of the peptides, as judged by the results of (2,4-dinitrophenyl)ationand dansylation of them.'34 The transfer of the oligosaccharide to protein is followed in uiuo by the excision of the D-glucosyl residues; the nine D-mannosyl residues present in the transferred oligosaccharide probably remain in the inner core of the D-mannan. However, the possibility that some D-mannosyl residues could be almost simultaneously removed and added cannot be pre~luded."~*'~' Finally, terminal, a - ~ -1(+ 3)-linked D-mannosyl groups are added to the trimmed, protein-bound oligosaccharide."' The outer chain of the Dmannan is formed after the inner-core oligosaccharide has been transferred to protein and processed, apparently by direct transfer of D-mannosyl groups from GDP-D-mannose.16'The similarity of the structures of the outer chain (166) E. Li, I. Tabas, and S. Kornfeld, J. Bid. Chem, 253 (1978) 7762-7770. (167) A. J. Parodi, J. Biol. Chem., 254 (1979) 10,051-10,060. (168) L. Lehle and W. Tanner, Biochim. Eiophys. Acfa, 350 (1974) 225-235.

LIPID-LINKED SUGARS AS INTERMEDIATES IN PLANTS

365

and the inner core raised the possibility that both syntheses are catalyzed by the same D-mannosyltransferase. However, biochemical and genetic evidence shows major diff erences between them. The D-mannosyltransferase involved in the synthesis of the outer chain has an absolute requirement for Mn2+ and is activated when enzyme preparations are stored at 2", whereas the transferase responsible for the formation of Man-P-Do1 and Dol-PP-oligosaccharides is inactivated on storage, and requires Mg2+ or Mn2+,the former being the more effective. In addition, both sets of enzymes can be separated by ion-exchange chromatography on DEAE-cellulose, and in uitro conditions that stimulate the synthesis of Man-P-Do1do not enhance D-mannosyl incorporation into the outer chain,160ruling out the involvement of lipid intermediates in the synthesis of the outer chain. Diversity of D-mannosyltransferases catalyzing the formation of different types of glycosidic bonds of the outer chain have been demonstrated by Nakajima and Bal10u'~~and by FarkaS and using exogenous acceptors. Differences were found in acceptor specificity, pH-activity curves, and rates of thermal inactivation. The outer chain contains D-mannosyl phosphate and D-mannobiosyl phosphate groups attached by a phosphoric diester linkage to oligosaccharide side-chains. Karson and Bal10u'~' reported the presence, in a homogenate of mechanically broken yeast-cells, of a D-mannosyl phosphate transferase activity. It catalyzes the transfer of D-mannosyl phosphate residues from GDP-D-mannose to reduced a - ~ -1(+ 2)-mannotetraose to yield D-mannosyl (D-mannotetraosyl phosphate). This membrane-bound activity has been solubilized by means of a detergent, and purified; it requires Mn2+or Co2+ions for activity, and is stimulated by various detergents. The enzyme cannot act on large D-mannan molecules lacking phosphate, and it has a much lower activity with acceptors modified by the a - ~ - ( l + 3)mannosyltransferase, suggesting that the phosphate branches are introduced during the synthesis of the outer chain, and not as a subsequent modification. The study of the biosynthesis of D-mannoproteins in other fungi is less developed. It has already been mentioned that the participation of Man-PDo1 as the glycosyl donor in 0-glycosylation was demonstrated in Neurospora crassa9' and in Fusariurn solani f. In the latter, the lipid acceptor was identified as C90-to C1,,-dolichols. In Hunsenula species, O-glycosylation follows the same pathway; that is D-mannose linked to L-serine or L-threonine is incorporated by way of Man-P-Dol, and further elongation (169) T. Nakajima and C. E. Ballou, Proc. Natl. Acad. Sci. US4-72 (1975) 3912-3916. (170) V. Farkas, V. M. Vagabou, and 5. Bauer, Biochim. Biophys. Acta, 428 (1976) 573-582. (171) E. M. Karson and C. E. Ballou, 1. Biol. Chem., 253 (1978) 6484-6492.

366

RAFAEL PONT LEZICA

et

al.

proceeds by way of G D P - ~ - m a n n o s e . ~The ~ * formation '~~ of a D-mannosyllipid in Cryprococcus luurentii in the presence of GDP-D-mannose has been reported,'73 but its role in protein glycosylation has not been demonstrated.

c. Others.-Aspergillus niger galactomannan is composed of approximately equal numbers of D-galactosyl and D-mannosyl residues.'74 A peliminable glycopeptide is formed in this species by way of a Man-Ppolyprenol?6 Occurrence of a similar reaction has been reported by Barr and Hemming,4°995using a well characterized, endogenous polyprenyl phosphate. Information about the synthesis of D-mannan in plants is even more fragmentary. In suspension-cultured cells of Acer pseudoplatanus (sycamore), participation of a polyprenyl (D-mannosyl phosphate) in the synthesis of D-mannan or glucomannan has been shown." During the study of the synthesis of the D-mannan of the alga Codium, complete similarity between the mechanism of biopolymerization of this polysaccharide and ~ , the '~~ that of cellulose in Vuloniu and higher plants was f o ~ n d . ' ~ If mechanism of biosynthesis of cellulose found in P, zopfii is general, a pathway involving lipid intermediates in the biosynthesis of D-mannan in Codium could be expected to occur, and it would be interesting to study this. Panayotatos and Villemez'28raised the possibility of involvement of lipid intermediates in the formation of a (1 + 4)-~-galactanin Vigna sinensis (Phaseolus aureus). The suggestion was made after observing that D['4C]galactose was added at the reducing end of the growing polysaccharide, as in 0-antigen biosynthesis, but no further experimental evidence has been produced to support the hypothesis. It would be even more speculative to suggest such a mechanism for the formation of D-galactomannan in Trigonellafoenum-graecum, on the basis that it is synthesized in vesicles of rough endoplasmic reticulum, perhaps as a glycoprotein in a nascent state.'29 During a study of the biosynthesis of P-D-glucan in fractions isolated from onion stem (ANium cepu), using UDP-D-glucose, Van der Woude and stated that the glycolipids formed could be intermediates in the synthesis of the (1+ 3)-p- and (1+ 4)-P-~-glucansformed; but the glycolipid fraction was not characterized, nor was a precursor-product relationship shown experimentally. The possible role of lipid intermediates in the synthesis of P-D-glucans by a membrane fraction from pollen tubes (172) R. K. Bretthauer and S. Wu, Arch. Biochem Biophys., 167 (1975) 151-160. (173) H. Ankel, E. Ankel, J. S. Schutzbach, and J. C. Garancis, J. Biol. Chem., 245 (1970) 3945-3955. (174) P. C. Bardalaye and J. H. Nordin, J. Biol. Chem., 252 (1977) 2584-2591. (175) M. Marx-Figini and M. C. de Matus, An. Asoc. Quim. Argent., 64 (1976) 225-237. (176) M. Marx-Figini and M. C. de Matus, Makromol. Chem., 179 (1978) 1541-1548.

LIPID-LINKED SUGARS AS INTERMEDIATES IN PLANTS

367

of Petunia hybrida has been in~estigated.'~' Based on kinetic evidence, the possibility was discarded, as a higher initial rate of incorporation in the final products, rather than in the polyprenyl (D-glucosyl phosphate), was shown, as well as a lower rate of change in the 3H : I4C ratio for the putative intermediates in double-labelling experiments. Pisum dyctiosome preparations form polyprenyl (oligo-p-D-glucosyl diphosphate)s having ( 1 + 3) and (1 + 4) linkages in the ratio of 3 : 1, but a precursor role in the biosynthesis of P-D-glucans has not been d e m o n ~ t r a t e d . ' ~ ~ The formation of lipid-linked saccharides during the biosynthesis of pullulan in the fungus Aureobasidium pullulans has been This extracellular, neutral o-glucan is composed of a repeating unit of a poly[( 1 + 4)-a-~-maltotriose]linked by a - ~ 1+ ( 6) bonds. The acidic glycolipids were shown to yield 6-O-a-~-glucosyl-60-a-D-glucosyl-D-glucose upon acid hydrolysis. The authors proposed these glycolipids as possible intermediates in the biosynthesis of pullulan, but conclusive evidence is lacking. When particulate enzyme preparations from Lemna minor were incubated with UDP-[U-'4C]galacturonic acid (UDP-GalA), radioactive label was incorporated into pectic acid and into material soluble in organic solvents. This radioactive material had the solubility properties, and the chromatographic behavior on paper and DEAE-cellulose, of a lipid derivative of GalA; however, confirmation awaits further characterization. Its formation is enhanced by UMP, and by addition of a polar, lipid fraction prepared from Lemna minor.98 Transfer of radioactivity from this material to pectic acid has been obtained.177 2. Peptidopolysaccharides

Besides cell-wall polysaccharides, plants contain glycoproteins, as do animal cells, and it is in the biosynthesis of glycoprotein that evidence for involvement of lipid-linked sugars has accumulated. Most of the data have been obtained for animal cells, and the subject is well covered in several 1.13.178 Since 1964, when glycoproteins were first detected in plants,'79 their wide distribution has become evident, and reviews are We shall describe aspects of this subject not as yet covered. Similarly to animal glycoproteins, plant glycoproteins having different roles have been isolated and studied: enzymes, lectins, toxins, and storage (177) G. R. Daleo and P. K. Kindel, Rep. Annu. Meet. SAIB, 16th, Bermejo, Mendoza, Argentina (1980). (178) R. T. Schwarz and R. Datema, Adu. Carbohydr. Chem. Biochem., 40 (1982) 287-379. (179) H . Lis, N. Sharon, and E. Katchalski, Biochim. Biophys. Acra, 83 (1964) 376-378. (180) N. Sharon and H . Lis, Biochem. SOC.Trans., 7 (1979) 783-799.

368

RAFAEL PONT LEZICA etal.

and structural compounds, as well as others for which no function has yet been found. Information on the occurrence and structure of glycoproteins in animals has been steadily increasing, but much less is known about plant glycoproteins. Glycoproteins containing the “complex” type of oligosaccharides have not been reported in plants. Most of the glycoproteins studied have either an N-glycosyliclinkage between 2-acetamido-2-deoxy-~-glucose and the amide nitrogen atom of an L-asparagine residue of the protein, with an oligosaccharide chain of the “high mannose” type, or a glycosidiclinkage between hydroxy-L-proline and a sugar, usually L-arabinose. The latter structure seems to be unique to the plant kingdom, where it is the most common one. The general scheme for the glycosylation of N-glycosylically linked glycoproteins seems to be similar to that found in animal systems and yeast, which is summarized in Scheme 3. As was shown in Section 11, the formation of practically all of the lipid-linked mono- and oligo-saccharides present in animal tissues has also been obtained by using plant enzymes. However, in only a few cases has the nature of the glycoprotein under consideration been established. Ericson and Delmer” studied the biosynthesis of glycoproteins in developing cotyledons of Phaseolus vulgaris, where large quantities of the reserve protein vicilin (currently referred to as phaseolin) is synthesized.They showed that incorporation of D-[ ‘‘C]mannose from GDP~-[‘~C]mannose into D-mannolipid and glycoprotein occurs in a kinetic sequence, which is consistent with a precursor-product relationship between them.87Similar results were obtained by using UDP-D-[“C]GlcNAc as the substrate. Evidence showing that D-GlcNAcand D-mannose were contained in the same, lipid-bound oligosaccharides was presented, as well as for the similarity between glycopeptides isolated from the radioactive product and from vicilin after pronase treatment.I8’ The same workers produced evidence18’ showing that the endogenous acceptor-proteins for the in vitro transfer of D-GlcNAc from UDP-D-GlcNAc were mainly phaseolin and phytohemagglutinin. Because both proteins contain typical, L-asparaginelinked, “high D-mannose” types of oligosaccharide groups, concomitant incorporation of D-mannose by transfer of “core” oligosaccharides from lipid camers was expected. However, only a trace of incorporation of D-mannose into phaseolin could be shown.’82 It was suggested, therefore, that the bulk of the transfer of D-GlcNAc to phaseolin and phytohemagglutinin is not “core” glycosylation. This suggestion was confirmed when it was shown that most of the D-GIcNAc was incorporated into terminal linkage to phaseolin and phytohemagglutinin without the involvement of (181) M. C. Ericson and D. P. Delmer, Plant Physiol., 61 (1978) 819-823. (182) H. M. Davies and D. P. Delmer, Planra, 146 (1979) 513-520.

LIPID-LINKED SUGARS AS INTERMEDIATES IN PLANTS

369

lipid-linked intermediates, simultaneously with "core" glycosylation of a class of proteins of heterogeneous molecular weights by way of the lipid p a t h ~ a y . " These ~ results are similar in one respect to those obtained by Sevier and S h a n n ~ n , " who ~ found that preparations from horseradish (Amoracia rusticana) root would attach D-GlcNAc groups from UDPD-GlcNAc to exogenously supplied, horseradish peroxidase from which -12% of the D-GlcNAc residues had been removed by the action of a mixture of glycosidases covalently bound to Sepharose. This was interpreted as a transfer of D-GlcNAc from UDP-D-GlcNAc to the nonreducing end of oligosaccharides already bound to the protein, which is not unexpected, as peroxidase possesses terminal D-GlcNAc residues. Studies on glycosylation of legumin in cotyledons of Pisum satiuum L. produced results that are consistent with the usual N-glycosylation scheme, involving lipid intermediates. However, direct evidence on precursor-product relationships is la~king.''~-"~ Evidence indicating that formation of glycoproteins in castor bean (Ricinus cornrnunis) endosperm proceeds by way of lipid-linked intermediates has been published?' Some properties of a D-mannosyltransferase involved have been studied, as well as its developmental changes, which closely parallel changes in the capacity for organelle biosynthesis. For this reason, and because the main, biosynthetic activity in germinating endosperm is the production of gluconeogenic enzymes and the organelles that store them, the product glycoproteins are assumed to be glyoxysomal components. Marriot and Tanner"' obtained very similar results in the same system, which suggests that the glycosylation of the glyoxysomal glycoproteins may take place on the endoplasmic reticulum, as the various glycosyltransferases studied are all located in this fraction. Particulate preparations from Pisum satiuum were found to be able to incorporate glucose from UDP-~-['~C]glucoseinto an oligosaccharide linked to a derivative of dolichol, presumably a diphosphate, and to a membrane-bound glucoprotein. When D-[ ''C]glucose-P-Dol plus nonlabelled UDP-D-glucose were incubated with pea extracts, radioactivity was also incorporated into oligosaccharide-linked lipids and proteins, in a time-course suggesting sequential transfer of the label. SDS-poly(acrylamide) gel electrophoresis of the products after a long incubation time resulted in several radioactive peaks, one of them having the same mobility (183) (184) (185) (186) (187) (188)

H. M. Davies and D. P. Delmer, PIanr PhysioL, 68 (1981) 284-291. E. D. Sevier and L. M. Shannon, Biochim. Biophys. Acra, 497 (1977) 578-585. J. Nagahashi and L. Beevers, Planr Physiol., 61 (1978) 451-459. J. Nagahashi. S. K. Browder, and L. Beevers, Plant Physiol., 65 (1980) 648-657. S. K. Browder and L. Beevers, Planr PhysioL, 65 (1980) 924-930. K. M. Mamot and W. Tanner, Planr Physiol., 64 (1979) 445-449.

370

RAFAEL PONT LEZICA etal.

as the &subunit of pea lectin. Moreover, radioactive material binds to Sephadex in the presence of Ca2+ and Mg2+, and is released by washing with 0.3 M D-glucose, just as for pea lectin, but conclusive evidence for a covalent bond between sugar and protein was not presented.12’ The formation of D-glucose-P-Dol, and protein D-glucosylation activity, were found to be associated with the endoplasmic reticulum, in agreement with a cotranslational, glycosylation pathway.’89 The biosynthesis in yeast of two enzymes that are D-mannoproteins has been studied. A membrane-associated isozyme of invertase (EC 3.2.1.26) has been shown to be a precursor of the external in~ertase.”~ Its molecular weight, as determined by SDS-poly(acry1amide) gel electrophoresis, is 50,000, that is, smaller than that of the external invertase, and it correlates well with the presence of only the inner-core sugars of the final form. It is strictly bound to membranes, possibly those of the endoplasmic reticulum, and it can be completely split’” by endo-P-N-acetylglucosaminidaseH (EC 3.2.1.30). The addition of tunicamycin, which specifically inhibits formation of D-GlcNAc-PP-Dol, inhibits synthesis of external invertase, as well as further formation of the membrane-associated form, which completely disappears after addition of the antibiotic.’” In these aspects, the synthesis of this extracellular enzyme follows the pathway for secreted glycoproteins in animal systems. The other enzyme, carboxypeptidase Y (EC 3.4.12.-),is a D-mannoprotein for which a structure of four L-asparagine-linked oligosaccharides of the general formula (Man),S(GlcNAc)2 was proposed. It is localized in the yeast vacuole, and synthesized by way of a larger, precursor protein. The enzyme synthesized in the presence of tunicamycin has the molecular weight expected for a molecule devoid of sugars. The antibiotic also lessens the proportion of the carbohydrate-free protein present in the cells, but this is not due to an increased breakdown. The processing of the precursor in vivo and in uitro is not affected by the absence of the carbohydrate portion either, indicating that the lowered proportion must be due to an inhibition A possible explanation suggests that nonof the rate of bio~ynthesis.’~~ glycosylated protein accumulates in the endoplasmic reticulum, and its release into the lumen proceeds more slowly, although, when translocation has occurred, the protein is processed at the normal rate. A nonglycosylated carboxypeptidase Y, similar to that synthesized with tunicamycin, is formed by a 2-amino-2-deoxy-~-glucoseauxotroph starved for this amino sugar; attempts to achieve its in vitro glycosylation failed.19* However, direct (189) H.E. Hopp, P. Romero, and R. Pont Lezica, Plant Cell PhysioL, 20 (1979) 1063-1069. (190) P. Babczinski and W. Tanner, Biochim. Biophys. Acra, 538 (1978) 426-434. (191) P. Babninski, Biochim. Biophys. Acra, 614 (1980) 121-133. (192) A. Hasilik and W. Tanner, Eur. J. Biochem., 91 (1978) 567-575.

LIPID-LINKED SUGARS AS INTERMEDIATES IN PLANTS

371

enzymic transfer of the sugar moiety from a ~-[~~C]manno-oligosaccharidelipid to a chemically unfolded form of ribonuclease A has been reported; it is catalyzed by endoplasmic reticulum membranes isolated from the endosperm tissue of castor bean (Ricinus communis) seedlings.'" The glycosylation of an uncharacterized protein that seems to be an intermediate in the synthesis of cell-wall D-mannan in algae has been described. Particulate preparations from Prototheca zopji catalyze the incorinto glycoporation of D-mannose and 2-acetamido-2-deoxy-~-glucose lipids, characterized as D-mannose-P-Dol, N,N'-diacetylchitobiose-PPDol, and various oligosaccharide-linked lipids, with a variable number of residues. The D-mannose units plus two 2-acetamido-2-deoxy-~-glucosyl oligosaccharide moiety of the latter is transferred to a polymer sensible to pronase digestion, producing an N-glycosylic linkage to an L-asparagine residue. These reactions were found to occur in the rough endoplasmic reticulum. During longer incubation periods, a polymer insensitive to pronase hydrolysis, but precipitable by copper salts, such as cell-wall Dmannans, is formed. Evidence showing that polymer formation occurs in the Golgi vesicle, and that it is inhibited by bacitracin, was also pre~ented.'~ Evidence on the involvement of lipid intermediates in 0-glycosylation is scarce. In one case, introduction of 0-glycosylically linked units into proteins by way of dolichyl phosphate derivatives has been described. Microsomal preparations from Fusarium solani f. pisi that had been induced by cutin hydrolyzate to synthesize cutinase-catalyzed D-mannosyl transfer from GDP-D-mannose to P-D-mannosyl-€'-Do1 and to glycoprotein, but not into oligosaccharide-lipids. Partial evidence indicated that the glycoprotein acceptor may be cutinase, but exogenous cutinase was not glycosylated, even after denaturation or deglycosylation. The microsomal preparation also catalyzed the transfer of single D-mannosyl groups from exogenous D-mannosyl-P-Do1 to hydroxyl groups of endogenous proteins. This reaction does not require divalent cations, in contrast to that necessary for the introduction of the first glycosidically attached o-mannosyl group in the n-mannoproteins of Saccharomyces cerevisiae and Neurospora cra~sa.~' A particulate, D-galactosyltransferase system from Chlamydornonas reinhardii, which catalyzes the transfer of D-galactosyl group from UDP-Dgalactose to hydroxy-L-proline of a cell-wall glycoprotein, has been described.194Failure to detect D-galactosyl-P-polyprenols at any time during the incubation was interpreted by the author as an indication that Dgalactosylation of that protein might occur without the involvement of lipid intermediates.'" (193) R. B. Mellor, L. M. Roberts, and J. M. Lord, Biochem. J., 182 (1979) 629-631. (194) W. C. Lang, PIanr Physiol., 69 (1982) 678-681.

TABLEVI ClycoproteinsFound in Plants, Type of Linkage W e e n the Peptide and Saccharide Moieties, and Involvement of Lipid Intermediates in Their Biosyntbesis ~

Clycoprotein

Source KCIyeosylic N-Acetyl-B-D-glucosaminylL-asparagine

Clycosidic 4+?-~-ArabinosyloxyL-proline

4-B-D-GalactosyloxyL-proline 3-O-a-~-Galactosyl-~-serine

3-0-~-Xylosyl-~-threonine 3-O-~-Mannosyl-~threonine-serine Unknown ~

~~~~~

Lipid intermediates

Ananas comosus Ficus sp. Amomcia rusticana Saccharomyces cerevisiae Vigna sinensis Pisum sativum Glycine max Glycine max Phaseoh limensis Saccharomyces cereuisiae Rototheca zopjii

Bromelain (E)" Ficin (E) Peroxidase (E) Carboxypeptidase Y(E) Vicilin (SP)' Legumin (SP) 7s protein (SP) Agglutinin ( L ) ~ Lectin Mannan (CWP)e Mannoprotein (CWP)

n.d.b n.d. n.d. suggested controversial suggested n.d. n.d. n.d. Yes Yes

Acer pseudoplatanus Lycopersicum esculentum Green algae Solanum tuberosum Acer pseudoplatanus Chlamydomonas Lycopersicum esculentum Solanum tuberosum Zea mays Saccharomyces cerevisiae Fusarium solani Pisum sativum Saccharomyces cerevisiae

Arabinogalactan (CWP) Extensin (CWP) Cell wall

n.d. n.d. n.d. n.d. n.d. discarded n.d. n.d. n.d. Yes Y e Yes Yes

~

E, enzyme. n.d., not determined. SP. storage protein.

~~

Lectin (L) Arabinogalactan (CWP) Cell wall Extensin (CWP) Lectin (L) Unknown Mannan (CWP) Unknown Lectin (L) Invertase (E) ~

L, lectin. ' CWP, cell wall protein.

References

195 1% 197 192 87,181,183 185, 187 198 199 200 109 89 201 202 203 204 194,201 105, 205 206 204 207 165 56 121 190, 191

LIPID-LINKED SUGARS AS INTERMEDIATES IN PLANTS

373

Table VI summarizes the current information on the structure and biosynthesis of characterized plant glycoproteins, classified on the basis of their carbohydrate-amino acid linking-group. In conclusion, the experimental evidence indicates that the mechanism of protein N-glycosylation in plants is very similar to that in animals, as a cotranslational event, occurring at the same subcellular location, and sensitive to the same antibiotics. Knowledge regarding the process of 0-glycosylation is much less developed, and in only a few cases has the role of lipid intermediates been established. However, we adhere to the view that lipid intermediates are the best donors for the transmembrane glycosylation, regardless of which amino acid is to be glycosylated, or whether the polysaccharide chain is homo- or hetero-polymeric. Therefore, careful and systematic search for them may be worth while, even though detection is difficult, owing to the insensitivity of the methods available so far. 3. Glycosaminoglycans

Glycosaminoglycans are widely distributed among animals, mainly in the connective tissues. Different types of polysaccharides containing amino sugars have been found, including hyaluronic acid, chondroitin sulfates, heparin, and dermatan. On the other hand, glycosaminoglycans are poorly represented in the plant kingdom: only chitin, a &D-( 1 -* 4)-linked polymer of 2-acetamido-2-deoxy-~-glucose, is present in most filamentous fungi and some members of the Chlorophycea (algae). No data are available on the occurrence of chitin, or other amino sugar-containing polysaccharide, in the numerous other species of algae, liverworts, mosses, ferns, or higher plants. It is interesting that carbohydrates are of special significance in (195) (196) (197) (198)

Y. Yasuda, N. Takahashi, and T. Murachi, Biochemistry, 9 (1970) 25-32. B. Friedenson and L. E. Liener, Biochim. Biophys. Acra, 342 (1974) 209-212. J. Clarke and L. M. Shannon, Biochim. Biophys. Acra, 427 (1976) 428-442. F. Yamauchi, V. H. Thanh, M. Kawase, and K. Shibasaki, Agric. Biol. Chem, 40 (1976) 691-696.

(199) (200) (201) (202) (203) (204)

H. Lis, N. Sharon, and E. Katchealski, Biochim. Biophys. Acta, 192 (1969) 364-366. A. Misaki and 1. J. Goldstein, J. Biol. Chem., 252 (1977) 6995-6999. D. G. Pope, Plant Physiol., 59 (1977) 894-900. D. T. A. Lamport, Nature (London), 216 (1967) 1322-1324. D. T. A. Lamport and D. H. Miller, PIanr Physiol., 48 (1971) 454-456. A. K. Allen, N. N. Desai, A. Neuberger, and J. M. Creeth, Biochem. J., 171 (1978)

665-674. (205) D. H. Miller, D. T. A. Lamport, and N. Miller, Science, 176 (1972) 918-920. (206) D. T. A. Lamport, L. Katona, and S. Roerig, Biochern J., 133 (1973) 125-131. (207) J. R. Green and D. H. Northcote, Biochem. J., 170 (1978) 599-608.

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RAFAEL PONT LEZICA et al.

higher plants, and comprise SO-80% of their dry matter, but no amino sugars seem to be present, except for a few isolated cases. Kent and Whitehouse2" proposed that this is the result of biological economy in species where the supply of nitrogen is a critical, nutritional factor; in such cases, use of nitrogen is restricted to vital processes.208 Chitin is classically a linear polymer of P-D-(1 + 4)-linked 2-acetamido-2deoxy-D-glucosyl residues. However, after comparing chitin isolated from different sources, it became evident that substantial variations in solubility, molecular weight, acetyl values, and specific rotation occur among samples. It would appear that chitin is not a single, polymeric entity, but rather, a family of closely related products derived from natural chitin-protein complexes.209 Biosynthesis of chitin was first described with a cell-free preparation from Neurosporu c r ~ s s u The . ~ ~ incorporation ~ of D-GlcNAc groups from UDPGlcNAc into a polymer indistinguishable from authentic chitin obeys the following general equation. U D P - D - G ~ C N A C ++~)-P-D-GIcNAc], [(~ + [ ( I +~)-P-D-GIcNAc].+,+ U D P

The reaction is catalyzed by a single enzyme, namely, UDP-2-acetamido-2(chitin deoxy-D-glucose: chitin 2-acetamido-2-deoxy-~-glucosyltransferase synthase, EC 2.4.1.16). Particulate preparations of chitin synthase obtained from different fungi2" did not show significant differences. The enzyme exists in the cells as inactive zymogen, and can be converted into the active form by limited proteolysis.212The zymogenic character of chitin synthase was also confirmed in solubilized preparation^,^'^.^'^ as well as in preparations from other sources.211 Perhaps the most remarkable property of fungal, chitin synthase is that the presence of an endogenous primer is not absolutely necessary for the reaction to proceed. Solubilized preparations of chitin synthase are able to synthesize chitin with no macromolecular primer p r e ~ e n t . ~ " * ~ This '~-~'~ property is difficult to understand when chitin synthase is compared with other glycosyltransferases. On the other hand, crustacean chitin synthase into an endogenous acceptor incorporates 2-acetamido-2-deoxy-~-glucose (208) P. W. Kent and M. W. Whitehouse, Biochemistry ofthe Aminosugars, Academic Press, New York, 1953. (209) P. R. Austin, C. J. Brine, J. E. Castle, and J. P. Zikakis, Science, 212 (1981) 749-753. (210) L. Glaser and D. H. Brown, J. Biol. Chem., 228 (1957) 729-742. (21 1) V. Farkas, Microbiol. Reu., 43 (1979) 117-144. (212) E. Cabib and F. A. Keller, J. Biol. Chem., 246 (1971) 167-173. (213) J. Ruiz-Herrera and S. Bartniki-Garcia, Science, 186 (1974) 357-359. (214) A. Duran and E. Cabib, J. Biol. Chem., 253 (1978) 4419-4425. (215) G. W. Gooday, J. Gen. Microbiol., 99 (1977) 1-11.

LIPID-LINKED SUGARS AS INTERMEDIATES I N PLANTS

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or macromolecular chitin.216 In this system, polyprenyl-oligosaccharides ranging from 2 to -8 glycosyl units sensitive to purified chitinase (EC 3.2.1.14) are formed. The results suggested that initiation of chitin synthesis in crustaceans may involve the glycosylation of a protein acceptor by a dolichol p a t h ~ a y . ~ ~ ’ .It~ is ’ * possible that similar reactions occur in fungi, but experimental evidence is lacking. 4. Other Carbohydrate Derivatives

In addition to the previously described polysaccharides, which are classified on a structural basis as glycans of various types (for example, D-glucans, D-mannans, and D-xylans), a wide variety of complex structures is present in plants. These are difficult to classify on a structural basis; they are commonly known as plant gums, and seed and bark mucilages. The plant gums2I9 are exudates formed, at the site of injury, for plant protection. They are complex polysaccharides having highly branched structures containing D-glucuronic or D-galacturonic acid and such neutral sugars as D-galactose, L-arabinose, L-rhamnose, D-mannose, D-fructose, or Dxylose. The acidic sugars are found naturally in the form of salts, and some of the sugar hydroxyl groups are often esterified with acetic acid. The general structure is composed of an interior, (1 + 3)-linked D-galactan chain (gum arabic), or D-glucurono-D-mannan (gum ghatti), with a variety of branched chains containing D-galacturonan and D-galacturono-L-rhamnan (as in gum tragacanth).220 Neutral mucilages have different structures, such as a linear chain of p-( 1 + 4)-linked D-galacto-D-mannan with a single a-D-galactosyl group (1 + 6)-linked to a D-mannosyl residue at regular intervals (leguminous seeds) or a (1 + 4 ) - p - ~ - g l u c o - ~ - m a n nchain a n having a single D-galactosyl group as a “side chain” (iris seeds221and lily bulbsZZ2).Acidic mucilages frequently contain a linear chain of a-D-galactosyluronic-( 1 + 2 ) - ~ rhamnose and one or two D-galactosyl groups (1+3)-linked to the Lrhamnosyl residues.223Despite structural differences, these complex carbohydrates have a common feature, namely, a linear chain with lateral branches (216) (217) (218) (219) (220)

M. N. Horst, J. Biol. Chem., 256 (1981) 1412-1419. M. N. Horst, Fed. Roc. Fed. Am. SOC.Exp. Biol., 40 (1981) 1597. M. N. Horst, Arch. Biochem. Biophys., 223 (1983) 254-263. G. 0. Aspinall, Adu. Carbohydr. Chem. Biochem., 24 (1969) 333-339. G. 0. Aspinall, in W. Pigman and D. Horton (Eds.). 7’he Carbohydrates: Chemistry and Biochemistry, Vol. IIB, Academic Press, New York, 1970, pp. 515-536. (221) P. Andrews, L. Hough, and J. K. N. Jones, J. Chem. Soc., (1953) 1186-1192. (222) P. Andrews, L. Hough, and J. K. N. Jones, 1. Chem. Soc., (1956) 181-188. (223) R. E. Gill, E. L. Hirst, and J. K. N. Jones, J. Chem. SOC.,(1946) 1025-1029.

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RAFAEL PONT LEZICA eral.

ranging from 1 to 6-7 sugars, sometimes esterified with an organic acid. Little is known regarding the biosynthesis of plant gums and mucilages. On the other hand, microbial exopolysaccharides present the same general structure, composed of repeating units of 2 to 7 sugar residues to which are often attached acetyl and pyruvic acetal groups. Their synthesis from glycosyl nucleotides involves assembly on a polyprenyl phosphate, and extrusion from the cell.224-227 The structure of xanthan gum, an exopolysaccharide freely liberated into the culture medium by the Gram-negative bacterium Xanlhornonas carnpestris, may be compared with that of some plant gums. It is composed of a linear chain of P-D-(1+ 4)-linked D-glucosyl residues, with a side chain composed of P-D-mannosyl-(l+ 4)-/3-~-glucosyluronicacid-( 1+ 2)-~-mannoselinked (Y-D-(1+ 3) to every two D-glucosyl units. In addition, the external D-mannose is substituted at 0-4 and 0 - 6 by a pyruvic acid acetal group, and the internal D-mannose is acetylated228at 0-6. The biosynthesis of xanthan gum occurs in two steps; in the first, a pentasaccharide is assembled by sequential addition of glycosyl 'groups from the corresponding glycosyl nucleotide to a polyprenyl phosphate, and, in a second step, the pentasaccharide repeating-units are polym e r i ~ e d Pyruvation .~~~ and 0-acetylation also occur at the first stage, once the pentasaccharide is assembled on the lipid.230p231 The complete sequence is shown in Scheme 4. It therefore seems possible that the biosynthesis of plant gums and mucilages follows a mechanism similar to that of bacterial exopolysaccharides. Experimental evidence should be obtained for plant systems in order to test this hypothesis.

5. Regulation Mechanisms Little is known about the regulation mechanisms of the synthesis of complex carbohydrate in plants, through lipid intermediates. However, partial evidence indicates that lipid-mediated glycosylation in proteins could be a regulatory step. When glycosylation of carboxypeptidase Y is inhibited (224) 1. W. Sutherland, Trends Biochem. Sci., 4 (1979) 55-59. (225) R. 0. Couso, L. Ielpi, R. C. Garcia, and M.A. Dankert, Eur. J. Biochern., 123 (1982) 617-627. (226) M.E. Tolmasky, R.J. Staneloni, R. A. Ugalde, and L. F . Leloir, Arch. Biochem. Biophys., 203 (1980) 358-364. (227) M.E. Tolmasky, R. J. Staneloni, and L. F. Leloir, J. Biol. Chem., 257 (1982) 6751-6757. (228) P.-E. Jansson, L. Kenne, and B. Lindberg, Carbohydr. Res., 45 (1975) 275-282. (229) L. Ielpi, R. Couso, and M. A. Dankert, FEES Lerf., 130 (1981) 253-256. (230) L. Ielpi, R. 0. Couso, and M. A. Dankert, Biochem. Int., 6 (1983) 323-333. (231) L. Ielpi, R.0. Couso, and M.A. Dankert, Biochem. Biophys. Res. Commun., 102 (1981) 1400- 1408.

LIPID-LINKED SUGARS AS INTERMEDIATES IN PLANTS GDP-Man

377

GDP

Yl

Glc-Glc-PP-Lip

G Ic - G l c - P P - L i p

Man

\

/UDP-GlcA

,.---y& UDP

,/'

UMP

'\,

P-Lip

\,

G!c-Glc-PP-Lip

Glc-Glc - P P - L i p Mkn

A

pi

Glc-Glc-PP-Lip Ac-Mbn GIC~A

Pyr = Man

-GlcAc-Mbn

GILA I P y r = Man

I

Polymerization

Glc-GIC-Glc -GGl~-Glc-GlcI I Ac-don Ac-Man Ac-Man GI~A GI~A M a n P y r = Mdn Man

GILA

SCHEME4.-Proposed Pathway for the Biosynthesis of Bacterial Xanthan Gum (PEP, enolpyruvate p h o ~ p h a t e . ) ~ ~ ~ - ~ ~ '

in yeast, a decrease of 50 to 60% in the amount of enzyme occurs, indicating a regulatory link between the synthesis of the polypeptide chain and its g l y c ~ s y l a t i o n .In '~~ animal systems, the nonglycosylation of proteins appears to be subjected to a rapid intracellular d e g r a d a t i ~ n . ~Other ' ~ . ~ ~studies ~ suggested that the endogenous levels of dolichol derivatives and of transferring enzymes could, to a certain extent, regulate the glycosylation of proteins.I3 Another possible regulatory mechanism has been pointed out by Elbein for plant systems. His results implied either that the carrier lipid for different sugars are different polyprenyl phosphates, or that the glycosyltransferases have their own pools of lipid carriers, which do not mix.I6 The results, compared to those for other plant and animal systems, suggested that the polyprenyl phosphates are the same for different sugars in each system, thus ruling out the first hypothesis, and reinforcing the concept of different pools.3o (232) R. T. Schwarz, J. M. Rohrschneider, and M. F. G. Schmidt, J. Virol.,19 (1976) 782-791.

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The concentration of dolichyl phosphate in eukaryotic tissues is very low and is probably rate-limiting for the glycosylation processes. Variation of the concentration in the endoplasmic-reticulum membranes is a possible way of controlling the rate of glycosylation. It is important to point out that the early steps in the dolichol biosynthesis are common to such other prenyl derivatives in plants as steroids, essential oils, hormones, phytol, and carotenes (see Scheme l ) , and parameters affecting those reactions that may control the dolichol to dolichyl phosphate step could be another mechanism for regulation of the level of dolichyl phosphate. In those cases in which the initiation of polysaccharide chains is mediated by lipid-linked saccharides, it becomes clear that the level of dolichyl phosphate must be important for regulation. Because no experimental evidence is available at present, further work will be necessary in order to elucidate the regulation mechanisms. ASPECTS IV. FUNCTIONAL

Many biological functions have been attributed to complex carbohydrates, but the subject is still mainly a matter of speculation. It is in this direction that future research will put increasing effort, based on the progress in isolation and characterization methods, as well as in biosynthetic studies. We shall refer to some of the postulated functions, togetherwith supporting experimental evidence. 1. Biological Recognition

Perhaps the most interesting and stimulating function of a heteroglycan is based on its potential as a carrier of biological information. Taking into account the differences in anomeric configuration and position of glycosidic bonds, the monosaccharides may serve as letters of a code of biological specificity, comparable to that of proteins and nucleic acids. In addition, carbohydrates have the ability to interact with other carbohydrates and with proteins; a specific determinant or marker, formed by a saccharide sequence, has affinity with complementary structures in other molecules. This interaction of specific oligosaccharide sequences with complementary, carbohydrate-binding proteins is expressed by cell-molecule interaction, cell-cell interaction, and molecule-molecule interaction. Recognition systems in animal cells have been extensively reviewed. Some examples include the D-galactose- and L-fructose-recognition system of mammalian h e p a t ~ c y t e s , ~the ~ ”2-acetamido-2-deoxy-~-glucose-recogni~~~ (233) R. J. Stockert, D. J. Howard, A. G. Morel], and I. H. Scheinberg, J. Bid. Chem., 255 (1980) 9028-9029. (234) J. P. Prieels, S. V. Pizzo, L. R. Glasgow, J. C. Paulson, and R. L. Hill, Proc. Narl. Acad. Sci. USA, 75 (1978) 2215-2219.

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tion system of avian h e p a t ~ c y t e s ,the ~ ~ 'D-mannose 6-phosphate-recognition system in human fibroblasts,236and that for ~-mannose-2-acetamid0-2deoxy-D-glucose of the reticuloendothelial system.237 Interaction of molecules with these markers, and with receptors on the cell surface, is the first step in adsorptive e n d o c y t ~ s i s At . ~ ~a~ higher level of complexity, cell-surface sugars have been shown to serve as recognition markers in cell-virus, cell-bacteria, and cell-cell interactions,239 with the obvious involvement in pathogenesis, development, fertilization, and other cell functions. There are also numerous recognition-phenomena in plants, also considered to arise from specific union among chemical groupings on the surface of interacting cells.24oThe complementary molecules of the cell surfaces have been the subject of several biochemical studies. Those on the infection of legume roots by the nitrogen-fixing symbionts of the genus R h i ~ o b i u r n ~originated ~' the lectin-recognition hypothesis, according to which, host specificity is based on specific legume lectin-bacterial polysaccharide interaction. Although certain studies have clearly shown that successful infection requires several events of cellular recognition, one of the early steps, namely, bacterial attachment, has been studied in the greatest detail. Lectin-mediated, hapten-specific attachment of rhizobia to legume root-hairs has been demonstrated in several systems, and exhaustively revie~ed.~~~,~~' Studies on the white clover- Rhizobium trifolii interaction are the most advanced. Trifoliin A, a lectin present in clover-seedling roots, binds hapten reversibly to carbohydrate antigens cross-reactive on the capsular polysaccharide of R. trifolii and clover epidermal-cells.*" A specific hapten that inhibits binding of trifoliin A to both surfaces is 2-deoxy-~-arabinoh e ~ o s e It. ~has ~ ~also been shown that levels of trifoliin A on root hairs decline with increasing concentrations of nitrate, in parallel to root-nodule development,246and that lectin receptors are transient on R. frifolii, in a way coinciding with its capacity to be adsorbed to clover roots.247 (235) (236) (237) (238) (239) (240) (241) (242) (243) (244) (245) (246) (247)

J. Lunney and G. Ashwell, Proc. Natl. Acud. Sci. USA, 73 (1976) 341-343. H. D. Fischer, A. Gonzalez Noriega, and W. S. Sly, J. Biol. Chem., 255 (1980) 5069-5074. P. Stahl and P. H. Schlesinger, Trends Biochem. Sci., 5 (1980) 194-196. A. Hasilik, L. N. Rome, and E. F. Neufeld, Fed. Proc. Fed. Am. SOC.Exp. Biol., 38 (1979) 467. W. Frazier and L. Glaser, Annu. Rev. Biochem., 48 (1979) 491-523. F. Burnet, Nature (London), 232 (1971) 230-235. B. B. Bohlool and E. L. Schmidt, Science, 185 (1974) 269-271. F. B. Dazzo and G. L. Truchet, J. Membr. Biol., 73 (1983) 1-16. W. D. Bauer, Annu. Reo. Plunt Physiol., 32 (1981) 407-449. F. B. Dazzo and W. J. Brill, J. BucterioL, 137 (1979) 1362-1373. F. B. Dazzo and W. J. Brill, Appl. Enrironm. Microbiol., 33 (1977) 132-136. F. B. Dazzo and W. J. Brill, Plant Physiol., 62 (1978) 18-21. E. M. Hrabak, M. R. Urbano, and F. B. Dazzo, 1. Bacteriol., 148 (1981) 697-711.

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Similar results have been obtained in some aspects for the soybean-R. j a p ~ n i c u m ,pea~ ~ ~R.* l~e g~u~r n i n o s a r ~ mand ~ ~ alfalfa~ R. r n e l i l ~ t i ~sys~~’~~’ tems. Some points still remain unclear; for example, there is no general picture of the chemical nature of the saccharide-receptor binding the host lectin. It seems to be the capsular-extracellular polysaccharide for R. j a p o n i c ~ m , ~the ’ ~ lipopolysaccharide for R. m e l i l ~ t i , ~and ~ ’ extracellularcapsular polysaccharide and lipopolysaccharide, depending on the culture ~ ~ . peanut ~~~~~~~ age in the cases of R. trifolii, R. l e g u m i n o s a r ~ m , and r h i ~ o b i a .It~ ~is~ still early to accept or reject the lectin-recognition hypothesis, but a positive effect has been the stimulus for a considerable amount of experimentation which has produced some promising results. A similar role has been suggested for glycoproteins and l e ~ t i n s ~ in ’~’~~~ host-pathogen interactions. Particularly, the “all P - l e ~ t i n s ”which , ~ ~ ~ are widely distributed in a variety of plants, and recognize P-linked D-glucosyl residues, have been postulated as recognition molecules for the P-D-glucan elicitors involved in pathogenic interactions between fungi and plants.257 Along the same line, results have been obtained showing specific interaction ,~~~ of potato lectin with avirulent strains of Pseudomonas s o l a n a ~ e a r u mand with both compatible and noncompatible strains of Phytophthora infestans.259In both cases, binding was inhibited by chitin oligomers containing residues, the specific hapten the internal 2-acetamido-2-deoxy-~-glucosyl for potato l e ~ t i n . ~ The ~ ’ , lectin ~ ~ ~ has been located on the cell wall by use of fluorescent-lectin antiserum.260In addition, potato lectin showed a lytic activity on zoospores of P. infestans, and it produced precipitation of elicitors of terpenoid accumulation produced by the fungus.261 In the case of Agrobacterium tumefaciens, experimental results indicated that recognition

(248) W. Gade, M.A. Jack, J. B. Dahl, E. L. Schmidt, and F. Wolf, J. Bid. Chem., 256 (1981) 12,905- 12,910. (249) T. V. Bhuvaneswari, S. G. Pueppke, and W. D. Bauer, Plant PhysioL, 60 (1977) 486-491. (250) G . Kato, Y. Maruyama, and M. Nakamura, Plant Cell Physiol., 22 (1981) 759-771. (251) W. Kamberger, Arch. Microbiol., 121 (1979) 83-90. (252) A. J. Mort and W. D. Bauer, J. Bid. Chem., 257 (1982) 1870-1875. (253) A., Bhagwat and J. Thomas, J. Gen. Microbiol., 117 (1980) 119-125. (254) L. Sequeira, Annu. Rev. Phytopathol, 16 (1978) 453-481. (255) P. Albersheim and A. J. Anderson-Prouty, Annu. Reu. Plant Physiol., 26 (1975) 31-52. (256) M. A. Jermyn and Y. M.Yeow, Aust. J. Plant Physiol., 2 (1975) 501-531. (257) A. R. Ayers, B. Valent, J. Ebel, and P. Albersheim, Plant Physiol., 57 (1976) 766-774. (258) L. Sequeira and T. L. Graham, Physiol. PIant Pathol., 1 1 (1977) 43-54. (259) N. Furuichi, K. Tomiyama, and N. Doke, Physiol. Plant Pathol., 16 (1980) 249-256. (260) S. Leach, M.A. Cantrell, and L. Sequeira, Phytopathol. News, 12 (1978) 197-203. (261) N. A. Garas and J. KuL, Physiol. PIant Purhol., 18 (1981) 227-237.

LIPID-LINKED SUGARS AS INTERMEDIATES IN PLANTS

38 1

in induction of crown-gall tumor may involve a lipopolysaccharide-galacturonan interaction.262 Based on the observation of high lectin activity in sieve-tube sap from Robinia p ~ e u d o a c a c i aand ~ ~in ~ phloem exudate from Cucurbita maxima,264 a protecting role against bacterial or fungal infection has been proposed for lectins in the sugar-rich phloem. Information is still fragmentary, and the results are to some extent contradictory and difficult to understand, but further experimental inquiry to test the hypothesis will undoubtedly produce rewarding results. Complex carbohydrates seem to be involved in another recognitionsystem, that of pollen compatibility. In an incompatible response, germination of the pollen tube is inhibited on, or near, the stigma papilla surface, and a (1 +. 3)-P-~-glucan(callose) is formed, adjacent to rejected pollen; however, its formation is also part of the general, wound response.265It has been suggested that the arabinogalactan-protein component of the stigma surface may be involved in capture and adhesion of pollen.266It has been isolated from stigma of Gladiolus and Lilium, and related arabinogalactans are present in style extracts of other specie^,^^^‘^^^ but their role has not been established. The mature stigma of Brassica oleracea contains large quantities of a glycoprotein in the earlier stages of development.270Pretreatment of pollen with this glycoprotein in vitro prevented its germination on three classes of compatible stigmas, whereas it did not interfere with the germination of pollen of other genotypes on compatible ~tigmas.~” As in other recognition systems, the precise nature of the molecules involved is just beginning to be unravelled, and further work will be necessary in order that their role may be unequivocally understood.

(262) J. A. Lippincott and B. B. Lippincott, in B. Solheim and J. Raa (Eds.), Cell Wall

(263) (264) (265) (266) (267) (268) (269) (270)

Biochemistry Related to Specificity in Host- Plant Pathogen Interactions, Norway Universitets Forlaget, Oslo, pp. 439-451. C. Gietl, H. Kauss, and H. Ziegler, Planta, 144 (1979) 367-372. D. D. Sabnis and J. W. Hart, Planta, 142 (1978) 97-101. H. I. M. V. Vithanage, P. A. Gleeson, and A. E. Clarke, Planta, 148 (1980) 498-509. A. E. Clarke and R. B. b o x , Q.Reu. Biol., 53 (1978) 3-28. A. E. Clarke, R. L. Anderson, and B. A. Stone, Phytochernistry, 18 (1979) 521-540. P. A. Gleeson and A. E. Clarke, Biochem. J., 181 (1979) 607-621. G. 0. Aspinall and K. G. Rosell, Phytochernistry, 17 (1978) 919-921. 1. N. Roberts, A. D. Stead, D. J. Ockendon, and H. G. Dickinson, Planta, 146 (1979)

179-1 83. (271) T. E. Ferrari, D. Bruns, and D. H. Wallace, Plant Physiol., 67 (1981) 270-277.

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RAFAEL PONT LEZICA e t a / .

2. Glycoenzymes Several glycoproteins with enzymic properties have been isolated from plants. Most of them are either hydrolases or oxidoreductases. 191~194-196*272,273 In almost all cases studied, the carbohydrate moiety is not required for catalytic activity, which is not significantly diminished after removal of the s ~ g a r s . On ~ ~the ~ -other ~ ~ hand, ~ the carbohydrate portion has been shown to increase the stability of the enzyme against proteoly~is,2~' on storage,278 Based on the fact that most of the known and at elevated glycoenzymes are either extracellular, or located on the cell surface or within organelles, it has been proposed that glycosylation provides a marker for intracellular transit and secretion.280

3. Cell Wall In Section 111, it was mentioned that cell wall is a complex structure formed by different polysaccharides connected to glycoproteins. Hydroxy-Lproline-rich glycoproteins, such as extensin, have been found in almost all plants surveyed, and in some algae.203328'A network of protein, pectic polymers, and xyloglucan, serving to cross-link the cellulose fibers of the cell wall, has been p r o p o ~ e d . ~However, ~ * * ~ ~ ~covalent links between the different components have not been demonstrated; moreover, some of them can be extracted separately,284and some associations may be artificial.285 Nevertheless, results are consistent with interactions through dipole-dipole (such as hydrogen bonds) or hydrophobic bonds. A fundamental role in cell extension has been proposed for hydroxy-Lproline-rich glycoproteins.286Evidence for this involvement in cell extension (272) (273) (274) (275) (276) (277) (278) (279) (280) (281) (282) (283) (284) (285) (286)

P. M. Dey and J. B. Pridham, Biochem. J., 113 (1969) 49-55. T. W. Okita, R. De Caleya, and L. Rappaport, Plant Physiol., 63 (1979) 195-200. Y. Yasuda, N. Takahashi, and T. Murachi, Biochemistry, 10 (1971) 2624-2630. R. B. Trimble and F. Maley, Biochem. Biophys. Res. Commun., 78 (1977) 935-944. J. H. Pazur, D. L. Simpson, and H. R. Knull, Biochem. Biophys. Res. Commun., 36 (1969) 394-400. J . W. Coffey and C. De Duve, J. B i d . Chem., 243 (1968) 3255-3263. J. H. Pazur, H. R. Knull, and D. L. Simpson, Biochem. Biophys. Res. Commun., 40 (1970) 110-116. W. N. Arnold, Biochim. Biophys. Acta, 178 (1969) 347-353. E. H. Eylar, J. Theor. Biol, 10 (1965) 89-113. D. P. Delmer and D. T. A. Lamport, in Ref. 262, pp. 85-104. D. T. A. Lamport, Annu. Reo. PIanr PhysioL, 21 (1970) 235-270. K. Keegstra, K. W. Talmadge, W. D. Bauer, and P. Albersheim, Plant PhysioL, 51 (1973) 188-196. R. R. Selvendran, Phytochemisrry, 14 (1975) 2175-2180. M. Knee, Phytochemistry, 14 (1975) 2181-2188. D. T. A. Lamport, Adu. Bot. Res., 2 (1965) 151-218.

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as plasticizers or growth-limiting factors is controversial. For example, it is not clear whether the extensin content increases during elongation, or only when it has c e a ~ e d . ~ ~ ’ . ~ ~ ~ 4. Polysaccharide Synthesis Primer requirement for polysaccharide biosynthesis is an old idea that has received abundant experimental support during the past 10 years. In potato, starch synthases catalyze the transfer of D-glucose to glycoproteins, which then accept D-glucose from several donors, to form the (1 + ~ ) - ( Y - D glucan hai in."^*^^^-^^' In the case of phosphorylase, which is a glycoprotein capable of synthesizing amylose-like D-glucan without the addition of a primer, the D-glucan moiety of the enzyme has been postulated as a primer for de nouo synthesis of Formation, by way of lipid intermediates, of a D-glucoprotein that serves as a primer for cellulose biosynthesis in Prototheca zopJi has been discussed in detail in Section II1,l. The possibility that this may be a general mechanism for initiation of polysaccharide synthesis is an attractive idea deserving experimental trial.

5. Storage Glycoproteins Storage proteins in legumes and other species have been characterized as glycoproteins, accumulating in protein bodies that apparently originate from the vacuole. It has been suggested that the carbohydrate moiety may As in be related to the stress-tolerance and water-imbibing the other cases, it may have a function as a “tag” to ensure its proper transport to the right, membrane-limited organelles. 6. Frost Resistance

Frost-resistant (hardy) plants are less sensitive than others to damage by low temperatures that is caused by water loss and intracellular, ice-crystal formation. Production of such highly hydrophilic proteins as glycoproteins would constitute a potential mechanism, through the formation of hydrogen (287) (288) (289) (290) (291) (292) (293) (294) (295)

F. M. Klis and D. T. A. Larnport, Plant Physiol. (Suppl.), 55 (1974) 15. D. Sadava, F. Walker, and M. J. Chrispeels, Deu. Biol., 30 (1973) 42-48. J. S. Tandecarz and C. E. Cardini, Biochim. Biophys. Acta, 543 (1978) 423-429. J. S. Tandecarz, N. Lavintman, and C. E. Cardini, Phytochemistry, 14 (1975) 103-106. J. S. Tandecarz, N. Lavintman, and C. E. Cardini, Biochim. Biophys. Acta, 399 (1975) 345-355. J. F. Friedrick, Plant Sci. Leff.,5 (1975) 131-135. E. Slabnik and R. B. Frydrnan, Biochem. Biophys. Res. Commun., 38 (1970) 709-714. A. Puztai and I. Duncan, Biochim. Biophys. Acfa, 229 (1971) 785-794. D. Racusen and M. Foote, Can. J. Bor., 51 (1973) 495-497.

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RAFAEL PONT LEZICA et al.

bonds with water molecules, to lessen water efflux and to inhibit crystal growth. It has been reported that, in Cornusjorida, variations in protein content would render -30% of the total cell-water osmotically i n a ~ t i v e . ~ ~ ~ , ~ ~ Brown and BixbyZg8reported de novo synthesis of glycoprotein during induction of freezing tolerance in Robinia pseudoacacia. V. CONCLUDING REMARKS

The evidence presented suggests that lipid-linked sugars in bacterial, animal, and plant systems act as sugar donors for transglycosylation reactions across a membrane. On the cytoplasmic side, the glycosyl nucleotides provide the activated sugar to the lipid, which, in turn, carries the activated glycosyl group through the hydrophobic membrane, and polymerization occurs on the external side. This mechanism was selected very early in the evolution of life, and it is used in the biosynthesis of many extracellular, complex glycans in bacteria. It is widely accepted that the glycosylation of some secreted, or membranebound, glycoproteins containing oligosaccharides N-glycosylically linked to L-asparagine proceeds by way of lipid intermediates in animal and plant cells. Why does such a mechanism seem to be restricted to a single pathway in eukaryotic cells? Two explanations are possible: first, the mechanism was not efficient enough, and evolution selected others. Second, eukaryotic organisms during evolution have lost the structures requiring the pathway, making it no longer necessary; this hypothesis seems to fit well for animal cells, but it cannot account for what happens in plant cells, which have cell walls composed of a variety of polysaccharides and glycoproteins whose biosynthesis is at present not well understood. We postulate that the biosynthesis of extracellular polysaccharides and glycoproteins in plants may be mediated by dolichyl derivatives. We should point out that, in addition to the well documented N-glycosylation of L-asparagine-containing glycoproteins, there are other possible pathways in which lipid-activated sugars can be intermediates in plants, namely, ( 1 ) the 0-glycosylation of extracellular glycoproteins; (2) the formation of glycoprotein primers for extracellular polysaccharides; and (3) the synthesis of'such heteropolysaccharides as gums and mucilages. The 0-D-mannosylation of L-serine or L-threonine during the synthesis of D-mannoproteins is mediated by lipid-linked D-mannose in some fungi. Higher-plant cell-walls containing glycoproteins having 0-glycosylically linked D-arabinose, D-galactose, and some D-glucose. Little is known about (296) R. J. Williams, Plant Physiol. (Suppl.), 51 (1973) 25. (297) R. J. Williams, Cryobiology, 11 (1974) 554-555. (298) G . N. Brown and J. G . Bixby, Plant Physiol., 34 (1975) 187-191.

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the glycosylation mechanism of such proteins. Research in this field should be conducted in order to elucidate whether or not lipid intermediates are involved. The synthesis of homopolysaccharides of plant cell-walls is a controversial subject. Knowledge as to the primer requirements for the synthesis of these polysaccharides is scarce, and the experimental evidence is not at present conclusive.29YIt has been suggested that a glycoprotein primer may be a When the universal requirement for the synthesis of polysa~charides.”~ polysaccharide to be synthesized is extracellular, such a primer could be glycosylated in a way similar to that for the glycoproteins secreted in animal cells, namely, by way of lipid-activated sugars. Some experimental evidence is now available indicating that the initiation of P-D-glucans in algae proceeds through lipid intermediates, but, it is not yet known whether such a mechanism occurs in higher plants, or whether the biosynthesis of other extracellular homopolysaccharides proceeds by similar reactions. Finally, details of the synthesis of heteropolysaccharides in plants are as yet completely unknown. The structural similarities among some plant gums and such bacterial exopolysaccharides as xanthan gum suggest that similar mechanisms may be operative in bacteria and in plants. Lipid intermediates could be suggested as potential glycosyl donors in the formation of plant gums and mucilages. It should be emphasized that, although lipid-activated sugars provide the only well known mechanism for transglycosylation reactions occurring across a membrane, other mechanisms cannot be precluded. More, active research is needed in order to elucidate the mechanism of synthesis of several plant glycans. ACKNOWLEDGMENTS We thank the many scientists who have provided us with copies of their work in press. We acknowledge the help of Dr. J. B. Pridham of the Royal Holloway and Bedford New College, University of London, and Drs. H. G. Pontis and R. D. Conde of the Instituto de Investigaciones Biolbgicas, Universidad Nacional de Mar del Plata, for their advice and criticism of the manuscript. R.P.L. and G.R.D. are Career Investigators of the Comisi6n de Investigaciones Cientificas de la Provincia de Buenos Aires (CIC), Argentina. Work in our laboratories is supported by grants from CIC and SUBCYT (Subsecretaria de Ciencia y Tecnologia), Argentina. P.M.D. is indebted to the Royal Society, London, for a short-term grant under an exchange program with CONICET, Argentina, to visit Mar del Plata, where this chapter was planned.

(299) G. A. MacLachlan, in R. M. Brown (Ed.), Cellulose and Other Natural Polymer Systems, Plenum, New York and London, 1982, pp. 327-339.

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ADVANCES IN CARBOHYDRATE CHEMISTRY A N D BIOCHEMISTRY, VOL. 44

GLYCOLIPIDS OF MARINE INVERTEBRATES

BY NICOLAIK. KOCHETKOV A N D GALINAP. SMIRNOVA N. D. Zelinsky Institute for Organic Chemistry, Academy of Sciences of the U.S.S.R., Leninsky Prospect, Moscow B-334, USSR

I. INTRODUCTION Glycolipids constitute a broad class of natural compounds belonging to the glycoconjugates. Depending on the nature of the lipid moiety, glycolipids can be subdivided into two large groups, namely, glycosphingolipids and glycoglycerolipids. Glycosphingolipids contain a long-chain amino alcohol (sphingosine) N-acylated by a fatty acid (R’rO,H), and a glycosyl group of a monosaccharide, or of an oligosaccharide chain, bound by a glycosidic linkage to the primary hydroxyl group of the sphingosine. Their general formula is - 2 follows. GI~COS~I-O-CH,-CH-CHOH-R I NHCO-R

Glycoglycerolipids contain glycerol, acylated by fatty acids, and a carbohydrate group glycosylating one primary hydroxyl group of the glycerol. Marine invertebrates are found to contain mainly the glycosphingolipids; sulfolipids, belonging to the glycoglycerolipids, have been found only in sea urchins. The glycosphingolipids of mammals are known to be cellular-membrane component^,'-^ organized in such a way that their lipid moiety, namely, acylated sphingosine (usually called ceramide), is submerged into the outer leaflet of the plasma-membrane bilayer, and contributes to its structural ( 1 ) B. J. Dod and G. M. Gray, Biochim. Biophys. Actu, 150 (1968) 397-404. (2) D. B. Weinstein, J. B. Marsh, M. C. Glick, and L. Warren, J. Biol. Chem., 245 (1970)

3928-3937. (3) G. Yogeeswaran, R. Sheinik, J. R. Wherrett, and R. U. Murray, J. Biol. Chem., 247 (1972) 5146-5158. (4) D . R. Critchley, J. M. Graham, and 1. Macpherson, FEES Lett., 32 (1973) 37-40. (5) H. D. Klenk and P. W. Choppin, Roc. Nutl. Acad. Sci. U.S.A., 66 (1970) 57-64.

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whereas the hydrophilic, carbohydrate moiety is located on the external surface of the membrane,7.8 and determines the specificity of its interaction with other cells, or biopolymers or both, of the medium, participating in various processes based on the recognition p h e n ~ m e n o n . ~ - ' ~ Glycosphingolipids are cell-surface antigen^,'^*'^-'^ or cell-differentiation markers 12.20-22., they participate in control of cell growth and oncogenic transformation, 10*12-14*23-26 and perform a number of other biological functions. Of greatest biological significance are the so-called gangliosides, which contain in their carbohydrate chain the residues of sialic acids and are thus sialoglycolipids. They constitute one of the major lipid components of nerve-cell membrane^,^^-^' and take part in a large number of biological functions, such as synaptic t r a n s m i ~ s i o nion ,~~ and reception (6) S. Abrahamsson, B. Dahlen, H. Lofgren, I. Pascher, and S. Sundell, in S. Abrahamsson and 1. Pascher (Eds.), Structure of Biological Membranes, Plenum Press, New York, 1977, pp. 1-23. (7) K.-A. Karlsson, in S. Abrahamsson and I. Pascher (Eds.), Structure of Biological Membranes, Plenum Press, New York, 1977, pp. 245-274. (8) T. Yamakawa and Y. Nagai, Trends Biochem. Sci., 3 (1978) 128-131. (9) R. C. Hughes and N. Sharon, Nature, 274 (1978) 637-638. (10) P. H. Fishman and R. 0. Brady, Science, 194 (1976) 906-915. ( 1 1 ) R. C. Hughes, Essays Biochem., 1 1 (1975) 1-36. (12) S. Hakomori, Annu. Reu. Biochem., 50 (1981) 733-764. (13) S. Hakomori, Biochim. Biophys. Acta, 417 (1975) 55-89. (14) S. Hakomori, in J. D. Gregory and R. W. Jeanloz (Eds.), Glycoconjugate Rex, Proc. Int. Symp. Glycoconjugares, 4th, Academic Press, New York, 1979, pp. 965-983. (15) S. Hakomori and W. W. Young, Jr., Scand. J. Immunol., 6 (1978) 97-117. (16) D. M. Marcus and G. A. Schwarting, Adu. Imrnunol., 23 (1976) 203-240. (17) S. Hakomori, E. Nudelman, S . Levery, D. Solter, and B. B. Knowless, Biochem. Biophys. Res. Commun., 100 (1981) 1578-1586. (18) S. Hakomori and A. Kobata, in M. Sela (Ed.), The Antigens, Vol. 2, Academic Press, New York, 1974, pp. 79-140. (19) K. Watanabe, S. Hakomori, R. A. Childs, and T. Feizi, J. Biol. Chem., 254 (1979) 3221-3228. (20) D. R. Critchley and M. G . Vicker, Cell Surface Rev., 3 (1977) 307-370. (21) G. Rebel, J. Robert, and P. Mandel, Ado. Exp. Med. Biol., 125 (1980) 159-166. (22) T. Momoi, K. Sakakibara, K. Nakajima, N. Shinomiya, and Y. Nagai, in T. Yamakawa, T. Osawa, and S . Handa (Eds.), Glycoconjugales, Proc. Int. Symp., 6th, Jpn. Sci. SOC. Press, Tokyo, 1981, pp. 93-94. (23) S. Hakomori, Adu. Cancer Res., 18 (1973) 265-315. (24) G. Yogeeswaran and S. Hakomori, Biochemistry, 14 (1975) 2151-2156. (25) S. Hakomori, W. W. Young, Jr., L. M. Patt, T. Yoshino, and L. Haefpap, Adu. Exp. Med. Biol., 125 (1980) 247-261. (26) S. Shinoda and Y. Eto, in T. Yamakawa, T. Osawa, and S. Handa (Eds.), Glycoconjugates, Proc. Int. Symp., 6th. Jpn. Sci. SOC.Press, Tokyo, 1981, pp. 103-104. (27) H. Wiegandt, Adu. Lipid Res., 9 (1971) 249-289. (28) R. W. Ledeen and R. K. Yu, in L. A. Witting (Ed.), Glycolipid Methodology, American Oil Chemists' Society, Champaign, Illinois, 1976, pp. 187-214.

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of a number of hormone^,'^,^^-^^ interferon, 39*40 some bacterial toxins, "V8.41-46 and The composition of the glycosphingolipids of vertebrates shows species, study of nervoustissue, and individual s p e c i f i ~ i t y . ~A* ~comparative ~-~~

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(52) (53) (54) (55)

1. G. Morgan, J. P. Zanetta, W. C. Breckenridge, G. Vincendon, and G. Combos, Brain Rex, 62 (1973) 405-411. R. W. Ledeen, in R. U. Margolis and R. K. Margolis (Eds.), Complex Carbohydrates of Neruous Tissue, Plenum Press, New York, 1979, pp. 1-23. G. Tettamanti, A. Preti, B. Cestaro, B. Venerando, A. Lombardo, R. Ghidoni, and S. Sonnino, Adu. Exp. Med. Biol., 125 (1980) 263-281. L. Svennerholm, Adu. Exp. Med. Biol., 125 (1980) 533-544. J. P. Behr and J. M. Lehn, FEES Leit., 31 (1973) 297-300. K. Hayashi and A. Katagiri, Biochirn. Biophys. Acra, 337 (1974) 107-1 17. L. D. Kohn, E. Consiglio, M. J. S. De Wolf, E. F. Grollman, F. D. Ledley, G. Lee, and N. P. Morris, Ado. Exp. Med. Biol., 125 (1980) 487-503. G. Lee, S. M. Aloj, R. 0. Brady, and L. D. Kohn, Biochem. Biophys. Res. Commun., 73 (1976) 370-377. M. Deleers, P. Chatelain, A. Poss, and J. M. Ruysshaert, Biochem. Biophys. Res. Commun., 89 (1979) 1102- 1106. F. D. Ledley, B. R. Mullin, G. Lee, S. M. Aloj, P. H. Fishman, L. T. Hunt, M. 0. Dayhoff, and L. D. Kohn, Biochem. Biophys. Res. Commun.,69 (1976) 852-859. T. Aoyagi, A. Okuyama, H. Umezawa, M. Iwamori, Y. Nagai, J. Suzuki, A. Ishii, and S. Kobayashi, Biochem. h i . , 2 (1981) 187-194. V. E. Vengris, B. F. Fernie, and P. M. Pitha, Ado. Exp. Med. Biol., 125 (1980) 479-486. W. E. Van Heyningen, Nature, 249 (1974) 415-417. L. L. Simpson and M. M. Rapport, J. Neurochem., 18 (1971) 1341-1343. R. 0. Brady and P. H. Fishman, Adu. Enzymol., 50 (1979) 303-323. M. Kitamura, M. Iwamori, and Y. Nagai, Biochim. Biophys. Acia, 628 (1980) 328-335. J. Holmgren, H . Elwing, P. Fredman, 0. Strannegard, and L. Svennerholm, Ado. Exp. Med. Biol., 125 (1980) 453-470. L. Svennerholm, P. Fredman, H. Elwing, J. Holmgren, and 0. Strannegard, in C. C. Sweeley (Ed.), Cell Surface Glycolipids, ACS Symp. Ser., Vol. 128, American Chemical Society, Washington, D.C., 1980, pp. 373-390. L. D. Bergelson, A. G. Bukzinskaya, N. V. Prokazova, G. 1. Shaposhnikova, S. L. Kocharov, V. P. Shevchenko, G. V. Kornilaeva, and E. V. Fomina-Ageeva, Eur. J. Biochem., 128 (1982) 467-474. T. Yamakawa, S. Handa, and S. Hamanaka, in R. Schauer, E. Buddecke, M. F. Kramer, J. F. G. Vliegenthart, and H.Wiegandt (Eds.), Glycoconjugaies, Proc. Inr. Symp., Sih, Georg Thieme, Stuttgart, 1979, p. 593. C. C. Sweeley and B. Siddiqui, in M.I. Horowitz and W. Pigman (Eds.), The Glycoconjugares, Vol. I, Academic Press, New York, 1977, pp. 459-540. M. E. Breimer, G. C. Hansson, K.-A. Karlsson, and H. Leffler, J. Biochem. (Tokyo), 90 (1981) 589-609. P. F. Urban, S. Harth, L. Freysz, and H. Dreyfus, Adu. Exp. Med. Biol., 125 (1980) 149-157. S. Ando, N. Chang, and R. K. Yu, Anal. Biochem., 89 (1978) 437-450. G. D. Hunter, V. M. Wiegant, and A. Dunn, J. Neurochem., 37 (1981) 1025-1031. M. Iwamori and Y. Nagai, Biochim. Biophys. Acra, 665 (1981) 214-220. T. Yamakawa, in M. Koike, T. Nagatsu, J. Okuda, and T. Ozawa (Eds.), New Horizons in Biological Chemistry, Japan Scientific Societies Press, Tokyo, 1980, pp. 109-119.

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tissue gangliosides in vertebrates belonging to different taxonomic groups has shown a correlation between the content of them and the level of nervous organization of the animal: the highest concentration of gangliosides has been found in the brains of higher animals, namely, mammals and No gangliosides have been discovered in the nervous tissue of invertebrates, 56*59-63 and they may, therefore, be assumed to have appeared as specific components of nerve-cell membranes at a rather late stage of evolution and, thus, to be characteristic only of vertebrates. In the sixties, however, sialoglycolipids were found in the tissues of some invertebrates, the sea urchins Arbacia p~nctuata,6~Pseudocentrotus d e p r e ~ s u s and , ~ ~ Strongylocentrotus intermedius.66 These compounds were soon found in other species belonging to the phylum Echinodermata, and their systematic study began. The data on the structure, biological functions, and metabolism of gangliosides and other glycolipids in mammals have been summarized in a number of reviews~8.10-14.16.18.20.23,30~67-72a On the other hand, despite the fact that a quite considerable body of information has by now been accumulated, as (56) C. Honneger and T. A. Freyfogel, Helv. Chim. Acra, 46 (1963) 2265-2270. (57) R. Hibbig and H. Rahman, in R. Schauer, E. Buddecke, M. F. Kramer, J. F. G. Vliegenthart, and H. Wiegandt (Eds.), Glycoconjugures, Roc. Inr. Symp., 5rh, Georg Thieme, Stuttgart, 1979, pp. 708-709. (58) H. Rahman, Ado. Exp. Med. Biol., 125 (1980) 505-514. (59) H. Price, M. Mosher, S. Wilson, D. Blatt, and R. Creekmore, Roc. W. Va. Acad. Sci., 46 (1975) 287-292; Chem. Absfr., 84 (1975) 55,541. (60) L. Bolognani, M. Masserini, P. A. Bodini, A. M. Bolognani Fantin, and E. Ottaviani, J. Neurochem., 36 (1981) 821-825. (61) Y. Komai, S. Matsukawa, and M. Satake, J. Biochem. (Tokyo), 70 (1971) 367-369. (62) W. C. McMurray, J. D. McColl, and R. J. Rossiter, in D. Richter (Ed.), Compurarioe Neurochernistry, Roc. Int. Neurochem. Symp., 5th. Pergamon Press, Oxford, 1964, pp. 101- 107. (63) P. Fredman, R. Noren, J.-E. Mansson, and L. Svennerholm, Biochim. Biophys. Acra, 713 (1982) 410-418. (64) L. Warren and R. Hathaway, Biol. Bull. Woods Hole, 119 (1960) 354-355. (65) Y. Isono and Y . Nagai, Jpn. J. Exp. Med., 36 (1966) 461-476. (66) N. K. Kochetkov, 1. G. Zhukova, G. P. Smirnova, and V. E. Vaskovsky, Dokl. Acad. Nuuk SSSR, 177 (1967) 1472-1474. (67) J. Kiss, Ado. Carbohydr. Chem. Biochem., 24 (1969) 381-433. (68) W. Gielen, Chimiu, 25 (1971) 81-86. (69) L. Svennerholm, P.Mandel, H. Dreyfus, and P.F. Urban (Eds.), Srrucrure and Function of Gungliosides, Plenum Press, New York, 1980. (70) B. A. Macher and C. C. Sweeley, Methods Enzymol., 50 (1978) 236-254. (71) C. C. Sweeley (Ed.), Cell Surface Glycolipids, A C S Symp. Ser., Vol. 128, American Chemical Society, Washington, D.C., 1980. (72) R. W. Ledeen and R. K. Yu, Merhods Enzymol., 83 (1982) 139-191. (72a) Y.-T. Li and S.-C. Li, Ado. Carbohydr. Chem. Biochem., 40 (1982) 235-286.

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far as we know no systematic review of the data on the glycolipids of invertebrates has been published. The only exception is some information in Joseph’s reviews on the lipids of sponges, coelenterate^,^^ and marine The present article is an attempt to summarize the information on the glycosphingolipids of marine invertebrates, including sialoglycolipids, on the sulfolipids of sea urchins, and on the glycosphingolipids of fresh-water mollusks; it covers publications that appeared before the end of 1982. 11. OCCURRENCE OF GLYCOSPHINGOLIPIDS,

THEIRISOLATION, A N D PURIFICATION In contrast to the sufficiently comprehensive information at present available on the occurrence of glycosphingolipids in the organs and tissues of vertebrates, such information on invertebrates is far from being complete. This seems to be associated with lesser accessibility of the biological material, as well as with the fact that the most characteristic glycosphingolipids of mammalian brain, namely, cerebrosides, sulfatides, and gangliosides, have not been found in the nervous tissue of some invertebrate^.'^*'^-^^ However, with the rapidly growing interest in studying the oceans of the world, information from that source will undoubtedly increase steadily. Before 1970, only a few reports on the glycolipids of marine invertebrates had appeared in the literature. Thus, at the end of the fifties, Rajagopal and Sohonie found considerable quantities of cerebroside, a glycosphingolipid containing one monosaccharide residue, in the sea a n e m ~ n e , ~and ’ Nakasawa isolated a glycolipid from the oyster.76 In 1963, Soper found sulfated glycolipids in some marine organisms,77and soon afterwards, Isono and Nagai isolated a sulfolipid and a sialoglycolipid from the sea urchin:’ and Kochetkov and coworkers isolated cerebrosides from the sponge and the starfish,78and sialoglycolipids from the sea urchin.66 A substantial contribution to our understanding of the occurrence of glycosphingolipids among marine invertebrates was made by Vaskovsky and coworkers,79 who performed broad screening of marine invertebrates J. D. Joseph, h o g . Lipid Res., 18 (1979) 1-30. J. D. Joseph, b o g . Lipid Res., 21 (1982) 109-153. M. V. Rajagopal and K. Sohonie, Biochem. J., 65 (1957) 34-36. Y. Nakasawa, J. Biochem. (Tokyo),46 (1959) 1579-1585. S. Soper, Comp. Biochem. PhysioL, 10 (1963) 325-334. N . K. Kochetkov, V. E. Vaskovsky, 1. G. Zhukova, G. P. Smirnova, and E. Y. Kostetsky, Dokl. Acad. Nauk SSSR, 173 (1967) 1448-1450. (79) V. E. Vaskovsky, E. Y. Kostetsky, V. 1. Svetashev, 1. G. Zhukova, and G. P. Smirnova, Comp. Biochem. Physiol., 34 (1970) 163-177.

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in order to determine the proportion of glycolipids in lipid extracts, and the composition of monosaccharides by t.1.c. after hydrolysis of the lipids with acid, and to characterize the glycolipids in terms of polarity in t.1.c. in different solvent-systems. More than 50 marine invertebrate species were investigated, belonging to 8 phyla and 15 orders. All of them proved to contain glycolipids, although their number and composition, as well as the monosaccharide composition, differ noticeably according to the phylogenic origin. The highest glycolipid content was found in sponges and echinoderms, and the smallest in coelenterates and arthropods. Appreciable variations in glycolipid content may be observed within one phylum, for example, in mollusks. Echinoderms are the only phylum of marine invertebrates in which sialoglycolipids have been detected. More-detailed information on the occurrence of glycolipids in the representatives of individual, marine-invertebrate phyla will be given in Section V. The procedure of isolating the glycosphingolipids is based on their physicochemical properties. The presence of the lipophilic, lipid moiety results in their solubility in such typical solvents for lipids as chloroform-methanol. On the other hand, the presence of the carbohydrate moiety imparts to them hydrophilic properties that increase with increase in the length of the carbohydrate chain and with the presence of such polar monosaccharides as sialic acid. This very property makes it possible to subdivide the glycolipids further, according to their polarity. In principle, the isolation of glycosphingolipids from marine animals does not differ from their isolation from other sources, a procedure comprehensively elucidated in a number of In brief, the isolation scheme is as follows. First, the lipids are all extracted from the animal tissue, generally by using chloroform-methanol or chloroform-methanol-water. An increase in the polarity of the extracting system makes it possible to extract more fully the polar glycosphingolipids, especially polysialoglycolipids, but may, however, lead to contamination (80) M. Kates, Techniques of Lipidology: Isolaiion, Analysis and Identification of Lipids, Elsevier, New York, 1973. (81) G . Rouser, G. Kritchevsky, and A. Yamarnoto, in G. V. Marinetty (Ed.), Lipid Chromographic Analysis, Vol. 3, Dekker, New York, 1976, pp. 713-776. (82) T. Saito and S. Hakomori, J. Lipid Res., 12 (1971) 257-259. (83) S. Hakomori and B. Siddiqui, Methods Enzyrnol., 32 (1974) 345-367. (84) R. W. Ledeen and R. K. Yu, Res. Merhods Neurochem., 4 (1978) 371-410. (85) A. Nagai and M. Iwamori, Adu. Exp. Med. Biol., 125 (1980) 13-21. (86) L. Svennerholm and P. Fredman, Biochim. Biophys. Acia, 617 (1980) 97-109. (87) H. M. Flowers, Meihods Carbohydr. Chem., 6 (1972) 459-463. (88) L. Svennerholm, Merhods Carbohydr. Chem., 6 (1972) 464-480.

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of the lipids with proteins. Then, in most cases, the lipids are separated according to their polarity, by partitioning between the water-methanol and the chloroform phases (Folch's method," or its modification^^^^^'). This results in sialoglycolipids, oligosylceramides having sufficiently long carbohydrate chains, and part of the sulfated glycosphingolipids in the (upper) aqueous methanol phase, and neutral glycosphingolipids having short carbohydrate chains, sulfated glycosphingolipids, and glycoglycerolipids remaining in the (lower) chloroform phase. The glycolipids of each phase are then separated into classes, using both adsorption chromatography (for example, on silicic U n i ~ i l , and ~ ~ .porous ~ ~ glass97s98) and ion-exchange chromatography on D E A E - c e l l u l ~ s e , ~ ~ DEAE*'~~ S e p h a d e ~ , ~ ~ . "DEAE-Sepharose,823'02 ' Spherosyl-DEAE-dextran, '03-lo5 DEAE-silica gel and DEAE-porous glass,lo6 and Q A E - S e p h a d e ~ .A ~~ technique usual for removal of phospholipids from the glycosphingolipid fraction consists in mild alkaline treatment of the lipid mixture, resulting in splitting of the 0-acyl bonds in phosphoglycerolipids, the amide bonds in glycosphingolipids remaining ~naffected.'~'For the removal of phospholipids, as well as for better separation of polar glycosphingolipids, acetylated glycosphingolipid derivatives are subjected to column chro-

J. Folch, M. Lees, and G. H. Sloane-Stanley, J. Biol. Chem., 226 (1957) 497-509. K. Suzuki, J. Neurochem., 12 (1965) 629-638. P. Hanfland and H. Egli, Vox Sang., 28 (1975) 438-452. D. E. Vance and C. C. Sweeley, J. Lipid Res., 8 (1967) 621-630. M. Sugita and T. Hori, J. Biochem. ( T o k y o ) , 80 (1976) 637-640. L. R. Bjorkman, K.-A. Karlsson, 1. Pascher, and B. E. Samuelsson, Biochim. Biophys. A d a , 270 (1972) 260-265. (95) M. Sugita, J. Biochem. ( T o k y o ) , 82 (1977) 1307-1312. (96) T. Hori, M. Sugita, S. Ando, M. Kuwahara, K. Kumauchi, E. Sugie, and 0. Itasaka, J. Biol. Chem., 256 (1981) 10,979-10,985. (97) S. Ando, M. Isobe, and Y. Nagai, Biochim. Biophys. Acra, 424 (1976) 98-105. (98) T. Momoi, S. Ando, and Y. Nagai, Biochim. Biophys. Acra, 441 (1976) 488-497. (99) G . Rouser, G. Kritchevsky, D. Heller, and E. Lieber, J. Am. Oil Chem. Soc., 40 (1963) 425-454. (100) C. C. Winterbourn, J. Neurochem., 18 (1971) 1153-1155. (101) R. K. Yu and R. W. Ledeen, J. Lipid Res., 13 (1972) 680-686. (102) M. lwamori and Y. Nagai, Biochim. Biophys. Acra, 528 (1978) 257-267. (103) P. Fredman, Adu. Exp. Med. Biol., 125 (1980) 23-31. (104) P. Fredman, 0. Nilsson, J.-L. Tayot, and L. Svennerholm, Biochim. Biophys. Acra, 618 (1980) 42-52. (105) 0. Nilsson and L. Svennerholm, J. Lipid Rex, 23 (1982) 327-334. (106) S. K. Kundu, Merhods Enzymol., 72 (1981) 174-184. (107) H. E. Carter and R. C. Gaver, Biochem. Biophys. Res. Commun., 29 (1967) 886-891.

(89) (90) (91) (92) (93) (94)

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NICOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

matography on Florisil'08 or silicic a ~ i d , ' ~ ~followed -''~ by deacetylation. To obtain the individual glycosphingolipids, the final stage of isolation is, in most cases, preparative thin-layer chromatography on silica gel"' or column chromatography on "Iatrobeads" porous g l a ~ s . ~ ' * ~ * The foregoing methods were developed for isolation of the glycosphingolipids of vertebrates. Although they are also suitable for isolating these compounds from marine invertebrates, and many of them are actually used for this purpose, it is, however, necessary to bear in mind the possible structural peculiarities of this type of compound that can change their physicochemical properties. For instance, the lower hydrophilicity of gangliosides having a short carbohydrate chain, or of oligosylceramides 0acylated by fatty acids, will prevent their quantitative separation on partitioning the total lipids between the aqueous and the organic phases; therefore, all stages of the glycolipid isolation have to be thoroughly controlled. As there is a possibility of alkali-labile groups being present in glycolipids, such chemical treatments as mild, alkaline hydrolysis and acetylation-deacetylation in the course of isolation should be avoided, at least at the first stage of work. Liquid chromatography (I.c.) under elevated pressure has now been introduced into glycosphingolipid separation. Glycolipids are analyzed as perbenzoylated, 112-116 N-acetylated O - b e n z ~ y l a t e d , " ~ and ~ " ~ N- (p-nitrobenzoy1)ated O - a ~ e t y I a t e d ' ' ~ -derivatives, '~~ as well as in their native (108) S. Hakomori and K. Watanabe, in L. A. Witting (Ed.), Glycolipid Methodology,

American Oil Chemists' Society, Champaign, Illinois, 1976, pp. 13-47. (109) J. Angstrom, M. E. Breimer, K.-E. Falk, 1. Griph, G. C. Hansson, and K.-A. Karlsson, J. Biochem. (Tokyo), 90 (1981) 909-921. (110) P. Hanfland, H. Egge, U. Dabrowski, S. Kuhn, D. Roelcke, and J. Dabrowski, Biochemistry, 20 (1981) 5310-5319. ( 1 1 1 ) S. K. Kundu, Methods Enzymol., 72 (1981) 185-204. (112) R. H. McCluer and F. B. Jungalwala, Adu. Exp. Med. B i d , 68 (1976) 533-554. (113) E. G. Bremer, S. K. Gross, and R. H. McCluer, J. Lipid Res., 20 (1979) 1028-1035. (114) R. H. McCluer and J. E. Evans, J. Lipid Res., 17 (1976) 412-418. (115) M. D. Ullman and R. H. McCluer, J. Lipid Res., 18 (1977) 371-378. (116) W. M. T. Lee, M. A. Westrick, and B. A. Macher, Biochim. Biophys. Acra, 712 (1982) 498-504. (117) S. K. Gross and R. H. McCluer, Anal. Biochem., 102 (1980) 429-433. (118) R. H. McCluer and M. D. Ullman, in C. C. Sweeley (Ed.), Cell Surface Glycolipids, ACS Symp. Ser., Vol. 128, American Chemical Society, Washington, D.C., 1980, pp. 1-13. (119) A. Suzuki, S. K. Kundu, and D. M. Marcus, J. Lipid Res., 21 (1980) 473-477. (120) T. Yamazaki, A. Suzuki, S. Handa, and T. Yamakawa, J. Biochem. (Tokyo), 86 (1979) 803-806. (121) A. Suzuki, S. Handa, and T. Yamakawa, J. Biochem. (Tokyo), 82 (1977) 1185-1187. (122) T. Yamakawa, S. Handa, T. Yamazaki, and A. Suzuki, in A. Varmavuori (Ed.), In?. Symp. Pure Appl. Chem., 27rh, Pergamon Press, New York, 1980, pp. 351-358.

GLYCOLIPIDS O F MARINE INVERTEBRATES

395

form. 123,124 This method has not yet been used to isolate glycosphingolipids from marine invertebrates, but its obvious advantages will undoubtedly provide a basis for wide application thereof in the analysis of glycosphingolipids from marine animals. A specific problem in the chemistry of glycosphingolipids, common to the whole chemistry of lipids, is the separation of individual compounds. It is reasonable first of all to dwell on what is meant by the individuality of a glycolipid. A glycosphingolipid is regarded as individual if it has a strictly defined carbohydrate chain structure, but it may include molecules having various fatty acids and sphingosine bases. When determining the glycosphingolipid structures, investigators usually deal with this particular kind of “individual” compound. Apparently, this notion of individuality corresponds to the biological role of glycolipids. Stringent requirements are imposed on the carbohydrate chain structure, as it primarily determines the biological specificity of glycosphingolipids, and even small variations in the composition of sugars, the position of glycosidic bonds, and their configuration change the antigenic or receptor properties of a glycosphingolipid. In contrast, the lipid moiety dictates the positioning of glycolipids in the membrane and their interaction with its other lipid and protein components, and also participates in the regulation of the membrane physicochemical characteristics, in particular, its rigidity and “fl~idity.”~.’ This function seems to be best accomplished by ceramide, containing not merely a certain single fatty acid and one sphingosine base but a set of these, including compounds differing in polarity, degree of saturation, chain length, and branching. A similar situation is observed in the lipids of other classes, where every “individual” lipid also has a corresponding set of molecules having the same structure of the polar moiety and a different composition of the fatty acids. In connection with the foregoing, the isolation of individual glycosphingolipids acquires a somewhat different meaning. As already noted, the final operation in a multi-stage procedure of isolating the individual glycosphingolipids is adsorption chromatography on silica gel or porous glass, wherein glycosphingolipids are separated according to the structure of their carbohydrate chains. The criterion of glycolipid individuality is the behavior in t.1.c. in several solvent-systems. Sometimes, to determine the purity of the isolated compound, the oligosaccharide chain of the glycolipid is split off, and its homogeneity is tested. The individuality of the glycosphingolipid is finally determined in the course of establishing its structure. (123) U. R. Tjaden, J. H. Krol, R. P. Van Hoeven, E. P. M. Oomen-Meulemans, and P. Emmelot, 1. Chromatogr., 136 (1977) 233-243. (124) K. Watanabe and Y. Arao, J. Lipid Rex, 22 (1981) 1020-1024.

396

NICOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

111. COMPOSITION OF GLYCOSPHINGOLIPIDS

A glycosphingolipid molecule of a marine invertebrate can contain a different combination of long-chain alcohols (sphingosines), fatty acids, and monosaccharides. Although this composition is, in its general features, close to that of vertebrate glycosphingolipids, the compounds isolated from marine invertebrates are, however, more diversified and also contain some unusual components, among them monosaccharides having polar groups. 1. Sphingosine Bases

The composition of sphingosine bases of marine invertebrate glycosphingolipids is wider than that of vertebrate glycolipids. In mammals, the principal sphingosine base of glycosphingolipids in most tissues is the Cis: dihydroxy base (Csphingenine), and considerable proportions of its Cz0: homolog were found in brain gangliosides, whereas, in the glycolipids of marine animals, in addition to these compounds, have been found, as the major components, C16:1(Refs. 125 and 126), C19:l (Ref. 127), and Czz:l (Ref. 128) dihydroxy bases, compounds with two double bonds that may have both a linear129-132and a branched chain,I3’ and trihydroxy bases (phytosphingosines) especially characteristic of the glycosphingolipids of echinoderms. In glycosphingolipids of starfish, a considerable proportion of the phytosphingosines (40 to 80%) has a branched s t r u c t ~ r e . ” ~ - ’ ~ ~ 2. Fatty Acids

As in the case of the glycosphingolipids of vertebrates, in the glycolipids of marine invertebrates are encountered both unsubstituted and a-hydroxy (125) T. Matsubara and A. Hayashi, Biochim. Biophys. Acra, 711 (1982) 551-553. (126) M. Sugita, S . Shirai, 0. Itasaka, and T. Hori, J. Biochem. (Tokyo),77 (1975) 125-130. (127) M. Iwamori, S. Okumura, and T. Hori, Shiga Daigaku Kyoku Gakubu Kiyo, Shizenkagaku, 22 (1972) 11-15; Chem. Absrr., 80 (1974) 143,235. (128) F. Matsuura, J. Biochem. (Tokyo), 85 (1979) 433-441. (129) A. Hayashi and T. Matsubara, Biochim. Biophys. Acra, 202 (1970) 228-230. (130) Y. Mishima and A. Hayashi, Yukagaku Zasshi, 27 (1978) 92-97; Chem. Abstr., 88 (1978) 118,058. (131) A. Hayashi and F. Matsuura, Chem. Phys. Lipids, 22 (1978) 9-13. (132) F. Matsuura, Chem. Phys. Lipids, 19 (1977) 223-242. (133) K.-A. Karlsson, H. Leffler, and B. E. Samuelsson, Biochim. Biophys. Acra, 574 (1979) 79-93. (134) M. Sugita, J. Biochem. (Tokyo), 86 (1979) 289-300. (135) M. Sugita, 1. Biochem. (Tokyo), 86 (1979) 765-772. (136) G. P. Smirnova and N. K. Kochetkov, Biochim. Biophys. Acra, 618 (1980) 486-495. (137) N. K. Kochetkov, G . P. Smirnova, and I. S. Glukhoded, Biochim. Biophys. Acra, 712 (1982) 650-658.

GLYCOLIPIDS OF MARINE INVERTEBRATES

397

acids, and both of these types of acid may contain double bonds. In the glycosphingolipids of numerous marine animal species, palmitic (hexadecanoic) and a- hydroxypalmitic (2-hydroxyhexadecanoic) acid preponderate, although, in some cases, the major component of the acids is the C2*: acid'38*'39or higher (C22-C24) a-hydroxy a ~ i d ~ . ~ On ~ * the ' ~ ~ - ' ~ ~ other hand, the glycolipids of some invertebrate species (for example, sea urchins) contain complex mixtures of acids, including unsubstituted and a-hydroxy acids of different chain-lengths (from 13 to 25 carbon atoms) and degrees of u n s a t ~ r a t i o n . ' ~ ~ - ' ~ ' 3. Monosaccharides

The composition of the monosaccharides in the oligosaccharide chains of glycosphingolipids of marine invertebrates differs markedly from that usually observed for the glycolipids of vertebrates. Despite the remarkable variety of structures of vertebrate glycosphingolipids, the variety of monosaccharides in their carbohydrate chains is rather limited. In an overwhelming majority of cases, only D-glucose, D-galactose, and 2-amino-2deoxy-D-glucose and -galactose are encountered; in addition, sialic acids are present in gangliosides, and L-fucose is characteristic for the bloodgroup-active glycolipids. In the glycosphingolipids of marine invertebrates, along with these monosaccharides characteristic of vertebrates, mannose, arabinose, xylose, glucuronic acid, and rhamnose have also been found. The distinguishing feature of a number of glycosphingolipids in aquatic organisms is the presence of methylated monosaccharide derivatives, among them, methylated sialic acids, as well as 0-sulfated sialic acids. 4. Other Components

The glycosphingolipids of marine invertebrates sometimes include groups unusual for this class of compound. Thus, in the glycolipids of gastropods, (2-aminoethyl)phosphonicand [2-(methylamino)ethyl]phosphonic groups

(138) N. K. Kochetkov, 1. G. Zhukova, G. P. Smirnova, and 1. S. Glukhoded, Biochim. Biophys. Acta, 326 (1973) 74-83. (139) M. Hoshi and Y. Nagai, Biochim. Biophys. Acta, 388 (1975) 152-162. (140) G. P. Smirnova, N. V. Chekareva, and N. K. Kochetkov, Bioorg. Khim., 7 (1981) 123- 130. (141) N. K. Kochetkov, G. P. Smirnova, and N. V. Chekareva, Biochim. Biophys. Acta, 424 (1976) 274-283.

398

NICOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

are bound to galactose,128~131*132*142-146 and, in the glycolipid from a freshwater bivalve, a (2-aminoethy1)phosphoric acid group bound to mannoseI4’ has been found.

IV. DETERMINATION OF THE STRUCTURE OF GLYCOSPHINGOLIPIDS Structural studies of glycosphingolipids involves determination of the structure of the oligosaccharide chain and of the lipid moiety. For the oligosaccharide chain, it is necessary to determine the composition, molar ratio, and sequence of the monosaccharides, their pyranose or furanose nature, and the position of glycosidic bonds and their configuration; for the lipid moiety, the composition of the fatty acids and sphingosine bases must be determined. Used for these purposes are the classical, chemical methods, conventionally accepted in the chemistry of carbohydrates and lipids and based on the degradation of compounds, enzymic, and physicochemical methods, primarily mass spectrometry and n.m.r. spectroscopy. 1. Determination of the Sphingosine Base Composition Sphingosine bases are usually isolated from glycosphingolipids by treatand characterized, in ment with methanolic HCI containing some the form of the free bases or their 2,4-dinitrophenyl (DNP)derivatives,149-151 by t.1.c. For quantitative determination of bases, a colorimetric procedure is used, based on color formation of the base with Methyl Orange.”* The composition of the sphingosine bases, as their Me,% derivatives, is determined by g.1.c. and g . l . c . - m . ~ . , ’ ~ ~as , ~well ’ ~ * as ~ ~by ~ using for this purpose (142) A. Hayashi and F. Matsuura, Biochim. Biophys. Acta, 248 (1971) 133-136. (143) A. Hayashi and T. Matsubara, in T. Yamakawa, T. Osawa, and S. Handa (Eds.), Glycoconjugares, Froc. Int. Symp., 6rh, Japan Scientific Societies Press, Tokyo, 1981, pp. 30-31. (143a) A. Hayashi and T. Matsubara, Ado. Exp. Med. B i d , 152 (1982) 103-114. (144) S. Araki, Y. Komai, and M. Satake, J. Biochem. (Tokyo), 87 (1980) 503-510. (145) S. Araki and M. Satake, Neurosci. Lett., 22 (1981) 179-182; Chem. Abstr., 94 (1981) 171,273. (146) S. Araki and M. Satake, in T. Yamakawa, T. Osawa, and S. Handa (Eds.), Glycoconjugates, Proc. Int. Symp., 6th, Japan Scientific Societies Press, Tokyo, 1981, pp. 66-67. (147) 0. Itasaka and T. Hori, J. Biochem. (Tokyo),85 (1979) 1469-1481. (148) R. C. Gaver and C. C. Sweeley, J. Am. Oil Chem. Soc., 42 (1965) 294-298. (149) K.-A. Karlsson, Chem. Phys. Lipids, 5 (1970) 6-43. (150) K.-A. Karlsson and E. Martensson, Biochim. Biophys. Acra, 152 (1968) 230-233. (151) K.-A. Karlsson, B. E. Samuelsson,’and G. 0. Steen, Biochim. Biophys. Ada, 316 (1973) 336-362. (152) C. J. Lauter and E. G. Trams, J. Lipid Rex, 3 (1962) 136-138. (153) H. E. Carter and R. C. Gaver, J. Lipid Res., 8 (1967) 391-395. (154) K.-A. Karlsson, Acra Chem. Scand., 19 (1966) 2425-2427.

GLYCOLIPIDS OF MARINE INVERTEBRATES

399

aldehydes obtained following oxidation of the free bases with p e r i ~ d a t e , ' ~ or ~ . ' ozonolysis ~~ of unsaturated basesi5' Aldehydes are analyzed either as or as the corresponding alcohols'56 and their trimethylsilyl or trifluoroacetyl derivative^'^^ or methyl ethers.136Also, the methyl esters of acids obtained after oxidation of unsaturated, long-chain bases with lead t e t r a a ~ e t a t eor '~~ KMn0, are used. The composition of trihydroxy bases is determined by periodate oxidation of the glycolipid; the aldehydes formed are treated with KBH,, and the alcohols obtained are analyzed by g . l . ~ . ' ~ * ~ ' ~ ' ~ ' ~ ~ 2. Determination of the Fatty Acid Composition

As a rule, qualitative and quantitative determinations of fatty acids are performed by g.1.c. of their methyl esters, obtained by treating the glycolipid with HCl in methanol.'60 To raise the volatility of the methyl esters of a-hydroxy acids, they are methy1atedl6' or trimethylsilylated.'62 3. Determination of the Structure of the Carbohydrate Chain

~ ~acid ' ~ ~ hydrolysis (acetolysis a. Chemical M e t h o d ~ . - M e t h a n o l y s i s ~ ~or is frequently used prior to h y d r o l y ~ i s ) is '~~ used for determination of the monosaccharide composition of glycosphingolipids. Methyl glycosides of amino sugars and sialic acids, formed upon methanolysis are N-acetylated to protect the amino groups, trimethylsilylated, and the products analyzed by g.l.c. 160,163,165 The free sugars, formed upon hydrolysis, are reduced with KBH, to alditols, which are acetylated, and the acetates analyzed by g.1.c. 164~166Both of these methods have been successfully applied to analysis of the monosaccharide composition of glycolipids from marine invertebrates. In quantitation of the monosaccharides, an internal standard ( Dmannitol or myo-inositol) is added in an amount equivalent to the content of sphingosine base in the sample. (155) (156) (157) (158) (159) (160) (161) (162) (163) (164) (165) (166)

C. C. Sweeley and E. A. Moscatelli, J. Lipid Res., 1 (1959) 40-47. N . Z. Stanacev and E. Chargaff, Biochim. Biophys. Acta, 98 (1965) 168-181. J . E. Evans and R. H. McCluer, J. Neurochern., 16 (1969) 1393-1399. R. Wood, J. Gas Chromatogr., 6 (1968) 94-96. M. Sugita, 0. Itasaka, and T. Hori, Chem. Phys. Lipids, 16 (1976) 1-8. A. Makita and T. Yarnakawa, J. Biochem. (Tokyo), 51 (1969) 124-133. N. K. Kochetkov and G. P. Smirnova, Bioorg. Khim., 3 (1977) 1048-1054. P. Capella, C. Galli, and R. Fumagalli, Lipids, 3 (1968) 431-438. C. C. Sweeley and R. V. P. Tao, Methods Carbohydr. Chem., 6 (1972) 8-13. H. Yang and S. Hakomori, J. Bid. Chem., 246 (1971) 1192-1200. C. C. Sweeley and B. Walker, Anal. Chem., 36 (1964) 1461-1466. M. Holm, J.-E. Mansson, M.-T. Vanier, and L. Svennerholrn, Biochim. Biophys. Acta, 280 (1972) 356-364.

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NICOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

It is necessary to mention the determination of sialic acids which, as already noted, occur in the glycolipids of echinoderms. Neither of the previously described methods of analyzing the monosaccharide composition of glycolipids is applicable to the characterization of sialic acids, because methanolysis results in the splitting not only of the glycoside bonds but also of the amide bonds, and subsequent N-acetylation produces N-acetylneuraminic acid regardless of which sialic acid was originally incorporated in the native glycolipid, and, on total hydrolysis with acid, the sialic acids are decomposed. They are, therefore, isolated by mild, acid hydroly~is,'~' and analyzed by t.1.c. on silica gel impregnated with 0.2 A4 NaH2P04, and by g.1.c. as their Me,Si derivatives,I6' or as the Me,Si derivatives of sialic acid methyl esters.'69 The methyl esters of sialic acid methyl ketosides are isolated by mild methanolysis, and analyzed by g.1.c. and g.1.c.-m.s. as their Me,Si derivative^.'^^.'^^ The latter procedure is likewise used for quantitative determination of sialic acids in a glycolipid. Also applied for this purpose are the colorimetric methods based on color formation upon heating sialic acids with resorcinol in an acidic m e d i ~ m , ' ~or' in the reaction with the sodium periodate-thiobarbituric acid reagent.'72 The latter method is more sensitive, but is suitable only for free sialic acids that have no 0-substituents, especially at 0 - 4 and 0-7. The resorcinol method is less sensitive, but more universal, because it can be used to determine not only free but also ketoside-bound sialic acids, and is suitable for determining 0-substituted sialic acids. It is, therefore, more reliable in analyzing the glycolipids of echinoderms, which, as has been shown, may have diverse substituents. To determine the structure of the sialic acids, mass spectrometry is used (see later). An article on sialic acids has been published in this To establish the structure of the oligosaccharide chain here, as in the chemistry of other glycoconjugates, partial hydrolysis with acid, and partial methanolysis, are widely used. This results in mono- and oligo-saccharide fragments, and glycosylceramides having a carbohydrate chain shorter than in the initial glycolipid; these are then separated, and analyzed. Partial, acid hydrolysis primarily results in the splitting of the (most labile) ketoside bonds of sialic acids in sial~glycolipids,'~~ as well as fucosyl bonds in (167) L. Svennerholrn, Acra Chem. Scand., 12 (1958) 547-554. (168) J. Casals-Stenzel, H.-P.Buscher, and R. Schauer, Anal. Biochem., 65 (1975) 507-524. (169) J. P. Kamerling, J. F. G. Vliegenthart, C. Versluis, and R. Schauer, Carbohydr. Res., 41 (1975) 7-17. (170) R. K. Yu and R. W. Ledeen, J. Lipid Res., 11 (1970) 506-516. (171) L. Svennerholrn, Biochim. Biophys. Acta, 24 (1957) 604-611. (172) L. Warren, J. Bid. Chem., 234 (1959) 1971-1975. (172a) R. Schauer, Adu. Carbohydr. Chem. Biochem., 40 (1982) 132-234. (173) R. Kuhn and H. Wiegandt, Chem. Ber., 96 (1963) 866-880.

GLYCOLIPIDS O F MARINE INVERTEBRATES

40 1

fucoglycolipids L64,174*175 and arabinosyl bonds in arabinosialoglycolipids. 134-136 The other glycoside bonds are also partially split, and a series of related oligosylceramides having shortened oligosaccharide chains is formed, from the analysis of which can be drawn conclusions as to the sequence of the monosaccharide units in the glycolipid. This nonspecific splitting of glycoside bonds proceeds to a greater extent in partial methanolysis. Glycosylceramide fragments are formed in better yields, and, in the case of sialoglycolipids, it is possible to obtain sialo-containing, glycolipid fragments.176 From glycolipids having 4-sphingenine as the long-chain base, nondegraded carbohydrate chains are isolated by oxidative cleavage of the double bond of 4-sphingenine by o z o n ~ l y s i sor , ~by ~ ~the action of sodium metaperiodate in the presence of Os04 upon the acetylated g l y ~ o l i p i d ' ~ ~ and subsequent, mild, alkaline hydrolysis. The oligosaccharide block that is split off is then analyzed by the conventional methods of carbohydrate chemistry. To determine the position of glycosidic bonds in glycosphingolipids, periodate oxidation and methylation are widely used. A characteristic feature of the periodate oxidation of glycosphingolipids is the stability, to oxidation under mild condition^,'^^ of the D-glucose residue that is located at the beginning of the carbohydrate chain and substituted at 0-4; this seems to be associated with steric hindrance caused by the close location of the lipid moiety. Methylation of glycosphingolipids, with the use of methylated derivatives in their structural analysis, deserves special mention. The rapid and efficient Hakomori procedurelso is the most applicable at present for permethylation of glycolipids. Standard methods may be applied to determine the type of bonds in the oligosaccharide chain of these derivatives, with glycosidic bonds being split by methanolysis or acid hydrolysis. The partially methylated methyl glycosides formed by methanolysis are analyzed by g.l.c., either as or as their trimethylsily1181or acetyl derivative^.'^^^'^^ The partially methylated sugars formed by hydrolysis are converted into partially methyl(174) (175) (176) (177) (178) (179) (180) (181) (182) (183)

A. Suzuki, I . Ishizuka, and T. Yamakawa, J. Biochem. (Tokyo), 78 (1975) 945-954. A. Slomiany and M. 1. Horowitz, J. Biol. Chem., 248 (1973) 6232-6238. R. Ledeen and K. Salsrnan, Biochemisrry, 4 (1965) 2225-2233. H . Wiegandt and G . Baschang, Z. Naturforsch., Teil B, 20 (1965) 164-166. S. Hakomori, J. Lipid Res., 7 (1966) 789-792. G. A. Johnson and R. H. McCluer, Biochim. Biophys. Acta, 84 (1964) 587-595. S. Hakornori, J. Biochem. (Tokyo), 55 (1964) 205-208. T. Matsubara and A. Hayashi, Biomed. Mass Spectrom., 1 (1974) 62-65. S. K. Kundu, R. W. Ledeen, and P. A. J. Gorin, Carbohydr. Res., 39 (1975) 179-191. S. K. Kundu, R. W. Ledeen, and P. A. J. Gorin, Carbohydr. Res., 39 (1975) 329-334.

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NICOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

ated alditol acetates, and g.1.c. and g.1.c.-m.s. may be used for their analysis. 184-187 To determine the substitution sites in neutral sugars and hexosamines, both the methyl glycosides and alditol acetates have been successfully used, and sialic acids are more often analyzed, in the form of derivatives of the partially methylated, acetylated188or trimethyl~ilylated'~~ methyl glycosides of methyl esters, by g.1.c. and g.1.c-m.s. The methylated methyl ketosides of sialic acids are stable under conditions of methanolysis. In the polysaccharide and the glycoprotein series, permethylated derivatives are almost exclusively used for their solvolytic decomposition and for the analysis of the mono- and oligo-saccharides formed, whereas, in the glycosphingolipid series, the investigation of the permethylated derivatives themselves by physicochemical methods has acquired great importance. They proved to be very convenient both for mass-spectrometric analysis and for n.m.r. spectroscopy (see later). The configuration of glycosidic bonds is established by oxidation with chromium t r i ~ x i d e ' ~and ' by the action of specific enzymes (see later), and n.m.r. spectroscopy has been increasingly used for this purpose. b. Physicochemical Methods.-As in the case of other carbohydratecontaining biopolymers, the use of mass spectrometry and n.m.r. spectroscopy has brought about fundamental changes in the methodology of the structural analysis of glycosphingolipids. (i) Mass Spectrometry. Structural studies of glycosphingolipids constituted one of the first examples of especially successful application of mass spectrometry to the analysis of complex glycoconjugates. Mass spectrometry has also been routinely applied here in the analysis of partially methylated monosaccharides in the form of the acetates of their glycosides, or the acetates of the corresponding alditols. Mass spectrometry has been used successfully to establish the structure of sialic acids, and the type of their bonds in the oligosaccharide chain. For this purpose, the fragmentation of various derivatives containing both the cyclic and the acyclic forms of sialic acid has been comprehensively studied. The methyl esters of sialic acid methyl ketosides, obtained as a (184) W. Stoffel and P. Hantland, Hoppe-Seyler's Z.Physiol. Chem., 354 (1973) 21-31. (185) K. Stellner, H. Saito, and S . Hakomori, Arch. Biochern. Biophys., 155 (1973) 464-472. (186) P.-E. Jansson, L. Kenne, H. Liedgren, B. Lindberg, and J. Lonngren, Chem. Commun. Uniu. Srockholm, 8 (1976) 1-75. (187) B. Lindberg and J. Lonngren, Merhods Enzymol., 50 (1978) 3-33. (188) H. Halbeek, J. Haverkamp, J. P. Kamerling, J. F. Vliegenthart, C. Versluis, and R. Schauer, Carbohydr. Res., 60 (1978) 51-62. (189) C. Bruvier, Y. Leroy, J. Montreuil, B. Fournet, and J. P. Kamerling, 1. Chromarogr., 210 (1981) 487-504. (190) R. A. Laine and 0. Renkonen, J. Lipid Res., 16 (1975) 102-106.

GLYCOLIPIDS OF MARINE INVERTEBRATES

403

result of mild methanolysis of sialoglycolipids, are analyzed in the form of the M e,Si 16%170.191 or acetyl derivative^."^ Although trimethylsilylation takes less time than the preparation of acetylated derivatives, the fragmentation patterns are more complex, and the interpretation of the mass spectra can cause difficulties, especially in the case of unusual sialic acids. Acetyl derivatives are stable, and convenient to work with, and yield readily interpretable spectra. Mass-spectral analysis of acetylated derivatives was used for identification of sialic acids from the sialoglycolipids of some echinoderm species,I4’ and for determination of the structure of N-acetyl-80-methylneuraminic acid isolated from the sialoglycolipid of the starfish Distolasterias n i ~ 0 n . I ’ ~ Simple spectra containing the necessary information on the structure of sialic acids are yielded by derivatives of the acyclic forms, obtained by KBH4 reduction of the keto group of the sialic acid to an alcohol. Subsequent treatment with diazomethane, and acetylation, resulted in the acetates of methyl 5-(acylamino)-3,5-dideoxynononates,in whose mass spectra the most characteristic peaks are those of the molecular ion and of nitrogencontaining fragments formed by cleavage of the C-C bonds located next (see Scheme 1). to the amide C0,Me

I

M’ = 577 (635)

CHOAc

I

CHZ I _ _ _ _ _ _ - CHOAc I__----______ 360 (418) CHNHCOCH,(CH,OAc)

~ _ - - - - - _ _ _ _ _ _ _ _288 _ _(346) _____

CHOAc

I I

CHOAc CHOAc

I

CHZOAC SCHEME1.

This type of derivative was used to analyze sialic acids from the sea urchin S. intermediu~.”~ Especially useful, however, is the use of mass spectrometry for the structural analysis of nondegraded glycosphingolipids. (191) J. P. Kamerling, J. F. G . Vliegenthart, and J. Vink, Carbohydr. Rex, 33 (1974) 297-306. (192) N. K. Kochetkov, 0. S. Chizhov, V. 1. Kadentsev, G . P. Smirnova, and 1. G. Zhukova, Carbohydr. Res., 27 (1973) 5-10. (193) G. P. Smirnova, N . V. Chekareva, 0.S. Chizhov, B. M. Zolotarev, and N. K. Kochetkov, Carbohydr. Res., 59 (1977) 235-239.

NICOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

404

The permethylated derivatives were found to be the most convenient, because of good volatility, and they yield clear-cut and simple fragmentaderivatives are tion-patterns. 194-197 Trimethylsilyl 198-201 and acety1202*203 suitable only for the analysis of glycolipids having short oligosaccharide chains, as higher oligosylceramides yield very complex spectra, or may be subject to partial pyrolysis when the mass spectra are r e ~ 0 r d e d . I ~ ~ Mass spectrometry of permethylated glycosphingolipids directly provides information on their structure. For the oligosaccharide chain, this includes the number, type, and sequence of the monosaccharide units, the presence and site of branching (fragments of series A and fragment B in Scheme 2), and, in some cases, also the positions of the glycosidic bonds; for the lipid moiety, it gives the composition of the fatty acids and sphingosine bases (fragments C, D, and the rearrangement ions formed from the sphingosine base 195.204 1. I

A, I

A, I

A4 I

A,I

1'

1

1

B I

Sug-cO-Sug+O-Sug+O-Sug-tOcCH,-CHtCH-CH+CH-CH=CH-(CH~)" I l l I I I I I I I I I CH,N I OCH3 1 0 1 I - - -1-1- - - I co I sug A2 I I I I R I I D I I- - - - - _ I where Sug = glycosyl group or residue.

-CH3

-,

SCHEME 2.

Along with permethylated derivatives, the permethylated, LiAlH4reduced derivatives of glycolipids are also used for mass-spectrometric analysis. The amide groups of acetamido sugars and the ceramide part are reduced to alkylamino groups, and, in the case of gangliosides, the ester (194) H. Egge, Chem. Phys. Lipids, 21 (1978) 349-360. (195) K.-A. Karlsson, I. Pascher, W. Pimlot, and B. E. Samuelsson, Biomed. Mass Spectrom., 1 (1974) 49-56. (196) K.-A. Karlsson, Adu. Exp. Med. Biol., 125 (1980) 47-61. (197) K.-A. Karlsson, in L. A. Witting (Ed.), Glycoliplid Methodology, American Oil Chemists' Society, Champaign, Illinois, 1977, pp. 97-122. (198) C. C. Sweeley and G. Dawson, Biochem. Biophys. Res. Commun.,37 (1969) 6-14. (199) K.-A. Karlsson, 1. Pascher, B. E. Samuelsson, and G. 0. Steen, Chem Phys. Lipids, 9 (1972) 230-246. (200) G. Dawson and C. C. Sweeley, 1. Lipid Res., 12 (1971) 56-64. (201) K. Samuelsson and B. Sarnuelsson, Eiochem. Biophys. Res. Commun.,37 (1969) 15-21. (202) B. A. Anderson, K.-A. Karlsson, I. Pascher, B. E. Samuelsson, and G. 0. Steen, Chem. Phys. Lipids, 9 (1972) 89-1 11. (203) S. P. Markey and D. A. Wenger, Chem. Phys. Lipids, 12 (1974) 182-200. (204) R. W. Ledeen, S. K. Kundu, H. C. Price, and J. W. Fong, Chem. Phys. Lipids, 13 (1974) 429-446.

GLYCOLIPIDS OF MARINE INVERTEBRATES

405

group of the methylated sialic acid is reduced to a primary hydroxyl group, which is then silylated.'95*'96.205.206 In addition to the aforementioned series A fragments, of great diagnostic significance for determination of the structure of the oligosaccharide chain are the fragments of series E, which include the monosaccharide residues, starting with the monosaccharide bound to the ceramide moiety (see Scheme 3). The same E fragments revealed the composition of the fatty acids. Information on the lipid moiety is also contained in fragment F. The use of both types of derivative is especially A,

I

A,

I

A,

I

I

I F S u g i O - S u g i O - S u g ~O-Sug-O-CH2+CH+CH-CH=CH-(CH,),CH, 1

I

1

1 - _ - - _0 t- I

A2

sug

I I I

I I

I/

I I

I

I I

I

I

I

1-

R

L

I

El

1

I I

- - - - - - - - - - - - I

I I

I

CH,N I OCH, I 1 CH2 I

-______

E2 __

____-_--

I I I

SCHEME3.

effective in investigation of the glycosphingolipids having complex carbohydrate chains. For marine invertebrates, this approach was applied in determination of the structure of an unusual disialoglycolipid isolated from the starfish Patiria pe~tinifera.~"This glycolipid contains N-acetylneuraminic acid and its 8-0-methyl derivative, with both sialic acid residues located inside the oligosaccharide chain. Mass-spectrometric analysis of the permethylated and the permethylated, LiAlH,-reduced disialoglycolipid made it possible to determine the sequence of monosaccharides, and the position of the N-acetyl-8-0-methylneuraminic acid in the chain. Other mass-spectrometric methods, using chemical and field d e ~ o r p t i o n , ~have ~ ~ ,also ~ ' ~been applied to analysis of glycosphin(205) K.-A. Karlsson, Biochemistry, 13 (1974) 3643-3647. (206) M. E. Breimer, G. C. Hansson, K.-A. Karlsson, and H. Leffler, in C. C. Sweeley (Ed.), Cell Surface Glycolipids, ACS Symp. Ser., Vol. 128, American Chemical Society, Washington, D.C., 1980, pp. 79-104. (207) N. K. Kochetkov and G . P. Smirnova, Biochim. Biophys. Acfa, 759 (1983) 192-198. (208) M. Oshima, T. Ariga, and T. Murata, Chem. Phys. Lipids, 19 (1977) 289-299. (209) T. Murata, T. Ariga, M. Oshima, and T. Mijatake, J. Lipid R e r , 19 (1978) 370-374. (210) T. Ariga, T. Murata, M. Oshima, M. Maezawa, and T. Mijatake, J. Lipid Rex, 21 (1980) 879-887. (21 1 ) T. Ariga, R. K. Yu,M. Suzuki, S. Ando, and T. Mijatake, J. Lipid Res., 23 (1982) 437-442. (212) Y. Kushi and S. Handa, J. Biochem. (Tokyo), 91 (1982) 923-931. (213) C. E. Costello, B. W. Wilson, K. Biemann, and V. N. Reinhold, in C. C. Sweeley (Ed.), Cell Surface Glycolipids, ACS Symp. Ser., Vol. 128, American Chemical Society, Washington, D.C., 1980, pp. 35-54.

406

NICOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

golipids, but, up to now, they have not been applied so extensively as electron-impact, mass spectrometry. There is, nevertheless, an example of field-desorption, mass spectrometry that was used for a study of a glycolipid from the sea (ii) N.m.r. Spectroscopy. For 'H-n.m.r. spectroscopy, permethylated derivatives, as well as unsubstituted, intact compounds, have proved to be the most convenient. Signals in the spectrum of permethylated glycolipids characterize not only the configuration of the anomeric center, but also the type of sugar, and, to a certain extent, the carbohydrate sequence in the oligosaccharide chain. 109*215-217 The use of permethylated, LiA1H4-reduced derivatives, along with permethylated derivatives, for the glycosphingolipids containing amino sugars has simplified the spectrum; and, besides, the removal of the amido oxygen atom produces, on the anomeric proton of the hexose bound to 0-3 of the hexosamine,a strong deshielding which is indicative of the type of hexosamine substitution.216The use of permethylated derivatives of glycolipids for 'H-n.m.r. spectroscopy is convenient, because these derivatives are used in the mass-spectrometric analysis of glycolipids, and in obtaining partially methylated sugars. However, the use of intact glycolipids for analysis is no less important, especially when only a small quantity of a substance is available, as this excludes the stage of obtaining the derivative, and allows use of the glycolipid for further investigation after the spectrum has been recorded. This is an Me2SO-d6-containing D 2 0 was suggested as a solvent.21B-221 excellent solvent for practically all glycosphingolipids, and makes it possible to record the spectra of different compounds under the same conditions, which is important for comparison of the spectra of the compounds under investigation and of the models. This method was successfully used to

(214) 1. Kitagawa, Y. Hamamoto, and M. Kobayashi, Chem. Pharm. Bull., 27 (1979) 19341937. (215) K. E. Falk, K.-A. Karlsson, and B. E. Samuelsson, Arch. Biochem. Biophys., 192 (1979) 164- 176. (216) K. E. Falk, K.-A. Karlsson, and B. E. Samuelsson, Arch. Biochem. Biophys., 192 (1979) 177- 190. (217) K. E. Falk, K.-A. Karlsson, and B. E. Samuelsson, Arch. Biochem. Biophys., 192 (1979) 191-202. (218) J. Dabrowski, P. Hanfland, and H. Egge, Biochemisrry, 19 (1980) 5652-5658. (219) J. Dabrowski, H. Egge, P. Hanfland, and S. Kuhn, in C. C. Sweeley (Ed.), Cell Surface Glycolipids, ACS Symp. Ser., Vol. 128, American Chemical Society, Washington, D.C., 1980, pp. 55-64. (220) J. Dabrowski, P. Hanfland, H. Egge, and U. Dabrowski, Arch. Biochem. Biophys., 210 (1981) 405-411. (221) J. Dabrowski, P. Hanfland, and H. Egge, Methods EnzyrnoL, 83 (1982) 69-86.

GLYCOLIPIDS OF MARINE INVERTEBRATES

407

analyze gangliosides,222and complex glycosphingolipids having a chain length of up to 10 monosaccharide units,"0,221where it provided the possibility not only of determining the anomeric configuration but also of confirming the data, obtained by other methods, on the type of bonds between monosaccharides, their sequence, the presence of branching, and the structure of the ceramide moiety. For marine invertebrate glycolipids, 'H-n.m.r. spectroscopy has thus far been mainly used to determine the configuration of glycosidic bonds. 13 C-N.m.r. spectroscopy has also been successfully applied. The method allows determination of the number of sugar residues, their furanose or pyranose form, the position of glycosidic bonds, and the anomeric configuration, as well as permitting assessment of the degree of unsaturation of the lipid moiety. I3C-N.m.r. spectroscopy has been used to analyze neutral g l y ~ o s y l c e r a m i d e s and ~ ~ ~g- a~n~ g~ l i ~ s i d e s ~of~mammals. ~ - ~ ~ ' The increasing number of interpreted spectra for different types of glycosphingolipids, and the development of techniques making it possible to use smaller quantities of substances for recording the spectra, will undoubtedly facilitate a wider application of this method for the structural analysis of glycosphingolipids. For the glycolipids of marine invertebrates, "C-n.m.r. spectroscopy was used to analyze the sulfated sialic acid isolated from the sialoglycolipid of the sea urchin E. cordatum. When the spectrum of this sialic acid was compared with that of N-glycolylneuraminic acid, it was found that they proved to have the same number of signals, some of which coincided. However, the C-8 signal in the spectrum of the sulfated sialic acid is shifted downfield by 8.5 p.p.m., and the C-7 and C-9 signals are shifted upfield by 1.1 and 1.95 p.p.m., respectively, as compared with the corresponding signals (222) S. Gasa, T. Mitsuyama, and A. Makita, in T. Yamakawa, T. Osawa, and S. Handa (Eds.), Glycoconjugates, Roc. Int. Symp., 6rh, Japan Scientific Societies Press, Tokyo, 1981, pp. 202-203. (223) T. A. W. Koerner, Jr., L. W. Cary, S.-C. Li, and Y.-T. Li, J. Biol. Chem., 254 (1979) 2326-2328. (224) C. C. Sweeley, J. R. Moskal, H. Nunez, and F. Matsuura, in A. Varmavuori (Ed.), Int. Congr. Pure Appl. Chem., 27th, Pergamon Press, New York, 1980, pp. 233-244. (225) T. A. W. Koerner, Jr., L. W. Cary, S.-C. Li, and Y.-T. Li, Biochem. J., 195 (1981) 529-533. (226) J. Dabrowski, H. Egge, and P. Hanfland, Chem. Phys. Lipids, 26 (1980) 187-196. (229) H. A. Nunez and C. C. Sweeley, J. Lipid Res., 23 (1982) 863-867. (228) P. L. Harris and E. R. Thornton, J. Am. Chem. Soc., 100 (1978) 6738-6745. (229) L. 0. Sillerud, J. H. Prestegard, R. K. Yu, D. E. Schafter, and W. H. Konigsberg, Biochemistry, 17 (1978) 2619-2628. (230) R. K. Yu, L. 0. Sillerud, and D. E. Schafter, in T. Yamakawa, T. Osawa, and S. Handa (Eds.), Glycoconjugates, Proc. Int. Symp., 6th, Japan Scientific Societies Press, Tokyo, 1981, pp. 79-80. (231) L. 0. Sillerud, R. K. Yu, and D. E. Schafter, Biochemistry, 21 (1982) 1260-1271.

408

NICOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

in the spectrum of N-glycolylneuraminic acid. These data showed that the sialic acid isolated is N-glycolylneuraminic acid having a sulfate group on C-8. Interestingly, there was a marked upfield shift of the C-2 signal in the spectrum of the sulfated sialic acid; this may indicate steric proximity of the sulfate group to the ketosidic center C-2. c. Enzymic Methods.-The monosaccharide sequence and the anomeric configuration of glycosidic bonds in glycosphingolipids are traditionally determined by use of a set of specific exo-glycosidases, from different sources, that catalyze the splitting of monosaccharides from the nonreducing end of the carbohydrate chain. Enzymic hydrolysis of glycosphingolipids often requires the presence of such detergents as, for example, sodium t a u r o c h ~ l a t e ? ~and, ~ sometimes, of activators as well.233-236After the enzymic hydrolysis of glycolipids, not only are the liberated monosaccharides analyzed, but also the glycolipids formed that have a shorter oligosaccharide chain. The use of glycosidases in analyzing mammalian glycosphingolipids has been dealt with in a number of review^.^^'-^^^ This approach has also been applied in studying the glycosphingolipids of marine invertebrates; see, for example, Refs. 126, 240, and 241. For the structural analysis of glycosphingolipids, not only exoglycosidases but also an endo-glycosidase, namely, the endo-P-D-galactosidase from Escherichia freudii, has now come into use. It splits the P-D-galactoside bond in glycolipids having the following monosaccharide sequence. R-P-GlcNAc-( 1 + 3)-P-Gal-(1 + 4)-Glc (or -GlcNAc),

where R is hydrogen or a glycosyl g r o ~ p . It~ is~ of~ interest * ~ ~ ~that the presence of sialic acid in the chain decreased the stability of the internal

(232) S. Hakomori, B. Siddiqui, Y.-T. Li, S.-C. Li, and C. G. Hellerqvist, J. Bid. Chem., 246 (1971) 2271-2277. (233) Y.-T. Li, M. Y. Mazzotta, C. C. Wan, R. Orth, and S.-C. Li, J. Biol. Chem., 248 (1973) 7512-7515. (234) Y.-T. Li and S.-C. Li, Ado. Exp. Med. Biol., 152 (1982) 223-226. (235) M. W. Ho, J. S. O’Brien, N. S. Radin, and J. S. Erickson, Biochem. J., 131 (1973) 173-176. (236) S.-C. Li and Y.-T. Li, J. Biol. Chem. 251 (1976) 1159-1163. Li and S.-C. Li, Methods Enzymol, 28 (1973) 714-720. (237) Y.-T. (238) Y.-T. Li and S.-C. Li, in M. 1. Horowitz and W. Pigman (Eds.), The Glycoconjugates, Vol. I, Academic Press, New York, 1977, pp. 52-67. (239) A. Kobata, Anal. Biochem., 100 (1979) 1-14. (240) T. Matsubara and A. Hayashi, 1. Biochem. (Tokyo), 89 (1981) 645-650. (241) T. Hori, M. Sugita, J. Kanbayashi, and 0. Itasaka, J. Biochem. (Tokyo), 81 (1977) 107-1 14. (242) M. N. Fukuda, K. Watanabe, and S. Hakomori, J. Bid. Chem., 253 (1978) 6814-6819.

GLYCOLIPIDS OF MARINE INVERTEBRATES

409

&Gal-( 1 + 4)-Glc bond, making it possible to obtain sialo-containing oligosaccharides in high yield.242

V. GLYCOLIPIDS O F VARIOUSGROUPS O F MARINEINVERTEBRATES In this, the main section of the present article, are presented data on the glycolipids that are found in the tissues of marine invertebrates. It is to be noted that, although these compounds have been studied quite intensively, different phyla of marine invertebrates have been studied to widely differing extents. Indeed, there are marine invertebrate phyla that have not been studied at all in this respect (for example, Protozoa, Bryoza, and Pogonophora), others that have been studied but little (for example, Spongia, Arthropoda, and Brachiopoda), and some others, in greater detail (Mollusca and Echinodermata). The degree to which the glycolipids have been studied is also different. For some compounds, only the monosaccharide composition has been determined, and they have been characterized in terms of polarity, depending on their t.1.c. mobility (those with low polarity have the mobility of monoglycosylceramides, that is, cerebrosides and their derivatives; those having medium polarity, the mobility of oligosylceramides containing several neutral monosaccharides; and polar compounds having the mobility of sialoglycolipids, and neutral oligosylceramides with longer carbohydrate chains). For some other glycosphingolipids, many structural features have been ascertained, but some questions remain unclarified, and there are glycosphingolipids whose structures have been completely elucidated. The data on glycolipids will now be separately presented for each phylum of marine invertebrates, in the ascending order of their evolutionary level. 1. Spongia

Sponges are the most primitive multicellular animals ( M e t a z o a ) , whose bodies do not yet have true tissues and organs. The phylum Spongia is subdivided into three classes: Calcarea, Hexactinellida, and Demospongia. Data on glycolipids exist only for the last class. As has already been noted, sponges show a high glycolipid content (from 2.8 to 4.3% of monosaccharides in the lipid extracts) and, in this respect, are inferior only to echinoderm^.'^ The t.1.c. patterns of glycolipids from three species of sponge (Haliclona aqueducta, Halichondria panicea, and Myxilla incrustans) differ markedly from each other, and from those for other animal species. The glycolipid composition is the richest in H. aqueducta, where up to 10 bands of glycolipids having various polarities are observed. In H. aqueducta and H. panicea, considerable proportions of

410

NICOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

lipids having the mobility of monohexosylceramides (cerebrosides) are present, and, in H.aqueducta, was also detected a glycolipid, slightly more polar than cerebroside, containing hydroxy fatty acids.79In all sponges, the glycolipids of medium and high polarity are colored by the orcinol reagent with a tinge characteristic of hexoses, whereas similar glycolipids of the majority of other phyla of marine invertebrates produce a great number of spots that take on the pentose coloring. Among the monosaccharides have been found galactose, fucose, glucose, and unidentified sugars.79 In the sponge Polymastia sp., a considerable proportion of sulfatide has been detected by paper c h r ~ m a t o g r a p h y . ~ ~ From the sponge M. incrustans, two cerebrosides and a polar glycolipid were isolated.78 Both cerebrosides contain galactose and sphingosine, but differ in the nature of their fatty acids: the less polar cerebroside contains normal, and the more polar, monohydroxy, acids. The structure of the polar glycolipid was not established, but it was shown to contain sphingosine, fatty acids, galactose, arabinose, fucose, and pho~phorus.~' From the sponge Chondrilla nucula, a glucocerebroside was isolated that had a lipid moiety more polar than that in the cerebrosides from M. incrustans. It contains trihydroxy, long-chain bases (phytosphingosines) having chain lengths of 17, 18, and 19 carbon atoms, respectively, and a-hydroxy acids from c16 to c 2 6 . The structure of the cerebroside was established by acid methanolysis, followed by analysis of the degradation products by g.1.c. and g.1.c.-m.s., as well as by analysis of the cerebroside acetate by n.m.r.

2. Coelenterata Coelenterates are the most ancient, and the least organized, of the true multicellular animals (Eumetazoa). This phylum is characterized (along with arthropods) by the lowest content of glycolipids: 0.6-0.9% of monosaccharides in lipid extract^.'^ The glycolipid composition has been characterized for two species of sea anemone, Metridium senile Jimbriatum and Anthopleura sp., and the jellyfish Aurelia aurita. In every one of the animals investigated has been found one zone of low-polarity glycolipids having the mobility of cerebrosides. In A. aurita and M. senile, no other glycolipids were found, whereas Anthopleura sp. additionally has two other glycolipids of medium polarity. The major monosaccharides in the lipids from the jellyfish A. aurita are glucose and galactose, and in the lipids of sea anemones, only glu~ose.'~ (243) F. J. Schmitz and T. J. McDonald, J. Lipid Res., 15 (1974) 158-164.

GLYCOLIPIDS OF MARINE INVERTEBRATES

41 1

Appreciable proportions of galactocerebrosides were found in the sea anemone Gyrostoma sp., the whole animal and its individual organs being studied; the glycolipid was not, however, purified and studied in detail.75 From the sea anemone M. senile was isolated p-glucopyranosyl-( 1 + 1)-ceramide, the structure of which was determined by mass spectrometry, 'H-n.m.r. spectroscopy, and chemical splitting. The major sphingosine in the cerebroside is a new base containing two double bonds and branching at one double bond, namely, 2-amino-~-erythro1,3-dihydroxy-9-methyl-( E, E)-octadeca-4,8-diene. The major fatty acids are C,,:, and C,,:, a-hydroxy Cerebrosides seem to be the major glycolipids in this phylum of marine invertebrates. 3. Worms

This large group of invertebrates, including four individual phyla, is subdivided into lower and higher worms. Their glycolipids have not been studied to any significant extent. The glycolipid composition has been characterized for three species of the higher worms (Annelida):Chaetopterus variopedatus, Serpula vermicularis, and Urechis unicinetus. The lipid extracts contained 0.9-2.29'0 of monosaccharides. C. variopedatus contains a larger proportion and number of glycolipids than the other two species. These are mainly cerebrosides and glycolipids of medium polarity, and there are very few polar glycolipids. In S. vermicularis and U.unicinetus, glycolipids of medium polarity are preponderant, and there are dnly traces of cerebrosides and polar glycolipids. Among the monosaccharides, along with glucose and galactose, there is a considerable proportion of arabinose, and some xy10se.~~ 4. Mollusca

In the number of species, mollusks constitute one of the most extensive groups of invertebrates, second in this respect only to insects. Many of the mollusks have for a long time been used as food, and this seems to have stimulated the investigation of the chemical composition of these animals, in particular, their lipids (for the lipids of mollusks, see a re vie^'^). With regard to glycolipids, this invertebrate phylum (along with echinoderms) has been studied more comprehensively than other phyla. The glycolipid composition has been characterized for 19 mollusk species belonging to 4 orders.79 The proportion of monosaccharides in lipid extracts varies from 0.7 to 2.9%, and even related species can differ sharply in their glycolipid content: 0.7% of monosaccharides has been found in the lipids of the mussel Crenomytilis grayanus, and 0-2.0% in those of the mussel Modiolus diflcilis, belonging to the same family.79

412

NICOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

a. The Class Lon’cara.-One species, Tonicella sp., has been characterized. It contains two low-polarity and two medium-polarity glycolipids. The main monosaccharides are galactose and xylose; also present are glucose, rhamnose, and arabinose.”

b. The Class Custropoda.-The glycolipids of eight species have been characterized: Collisella sp., Acmaea pallida, Tegula rustica, Littorina brevicula, L. squalida, Nucella heyseana, Rapana thomasiana, and Neptunea c o n s t r i c ~ aIn . ~ ~most of the species studied, the preponderant glycolipids are those of high polarity, whose number can vary from one (L. squalida and L. brevicula) to four (Collisella sp.). A. pallida, however, contains no polar glycolipids, but only those of medium polarity. In many of the species are also present low-polarity glycolipids having the mobility of monoglycosylceramides. The main monosaccharides in most of the species are galactose and glucose. The only exception is A. pallida, wherein the major monosaccharide is arabinose, and the glucose content is somewhat smaller.79 In the lipid extracts of Aplysia punctata, Archidoris britannica, and Coryphella rufubranchialis, sulfatides were detected by paper chromatography .77 From the visceral tissues of the sea snail Turbo cornutus (the subclass Prosobranchia, the family Turbinidae) two types of glycosphingolipid, neutral and acidic, have been isolated. Among the neutral glycosphingolipids, four compounds constitute a series of glycosphingolipids of a new type whose carbohydrate chain contains galactose only. From the data obtained by methylation, periodate oxidation, enzymic hydrolysis, and n.m.r. spectroscopy, the structures of three of them have been established as @-Gal-(1 + 1)-Cer, @-Gal-(1 + 6)-@-Gal-(1 + 1)-Cer, and @-Gal-(1 + 6)@-Gal-(1 + 6)-@-Gal-(1 + 1)-Cer. The fourth glycolipid was not isolated in pure form, but qualitative and quantitative analysis of the monosaccharides, and its enzymic hydrolysis with @-D-galactosidase showed that it is a tetragalactosylceramide of the same s e r i e ~ . The ~ ~ composition ~.~~ of the fatty acids is very similar in these glycolipids. In all of the compounds, palmitic acid is preponderant (from 47 to 70% of the total acids), and only the galactosylceramide contains a considerable proportion (35%) of ahydroxypalmitic acid. The sphingosine base in all of the galactolipids is mainly represented by a dihydroxy base having a chain length of 16 to 22 carbon atoms, with one or two double bonds; the main component is 4-s~hingenine.~~’ (244) T. Matsubara and A. Hayashi, in R. Schauer, E. Buddecke, M. F. Kramer, J. F. G . Vliegenthart, and H. Wiegandt (Eds.), Glycoconjugates, Roc. Inr. Symp., 5th, Georg Thieme, Stuttgart, 1979, pp. 55-56.

GLYCOLIPIDS OF MARINE INVERTEBRATES

413

Previously, a glycolipid having a carbohydrate chain of the same type, but with a-galactosidic bonds, had been isolated from a plant source, namely, rice bran, but this glycolipid is a g l y c ~ g l y c e r o l i p i d . ~ ~ ~ From T. cornutus, another neutral glycosphingolipid was isolated, the carbohydrate chain containing240 galactose, glucose, fucose and 2acetamido-2-deoxy-~-glucosein the ratios of 2 : 1 : 1 : 1. The composition of fatty acids and sphingosine bases in this glycolipid differs somewhat from that in galactosphingolipids: it contains fatty acids having longer chains (Cz0:o,C22:0,and C 2 3 : 0 )the , content of C , , : , sphingenine is twice as high as, and that of c l 8 : l sphingenine, two thirds of, that in galactosphingo lipid^.'^^ The structure of this glycolipid has not as yet been determined. Acid glycolipids isolated from T. cornutus constitute a new class of glycosphingolipids, the O-phosphinicoglycosphingolipids.'31.'32~142~'43~246 The acid glycolipid from visceral tissue contains142 2-(methylamino)ethylphosphonic acid, fatty acids, sphingosine base, and galactose in the ratios of 1 : 1 : 1 : 1. Based on the results of partial hydrolysis with acid, alkaline treatment, and periodate oxidation, with the degradation products being identified mainly by g.1.c.-m.s., the glycolipid was assigned the structure of 1-0-{6-0-[2-(methylamino)ethylphosphinico]-~-galactopyran0syl)ceramide. 132,246 The fatty acids in this glycolipid include normal and 0

I1

H2CO-P-(CH2)2-NHMe I

I

0 - C HI-C H-C H- R'

I I

NH OH

I

OH

COR

a-hydroxy acids in approximately equal amounts. The main components of the acids are hexadecanoic (palmitic; 32.6% ) and 2-hydroxyhexadecanoic (a-hydroxypalmitic; 31.2%) acid. Diene dihydroxy bases (c18:214.7% and Czz:223.5%) are preponderant among the sphingosine bases, and -20% of them are comprised of phytosphing~sines.'~~ From the muscle of T. cornutus were isolated two phosphonoglycosphingolipids, the major of which has the same structure as that of that isolated from visceral tissue; the minor differs from it by the phosphate component, and has the structure of 1-0-(6-0-[(2-aminoethyl)phosphono]-~-ga1acto(245) Y. Fujino and T. Mijazawa, Ezochim. Eiophys. Acra, 572 (1979) 442-451. (246) A. Hayashi, F. Matsuura, and T. Matsubara, Yukagaku Zasshi, 25 (1976) 501-502; Chem. Absrr., 85 (1976) 119,912.

414

NILOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

pyranosy1)~eramide.'~' The major components of the fatty acids are palmitic (52% of the total acids) and a-hydroxypalmitic (14.6%) acid, and the sphingosine bases are ~ o m p r i s e d ' ~of' dihydroxy bases, mainly C22:2,CIS:1, and C18: 2. The total concentration of phosphonocerebrosides in the complex lipids from T. cornutus muscle-tissue is approximately a third that in the visceral tissue. Galactosylceramides that could be the precursors of these compounds were isolated from the muscle and the visceral tissues, and the composition of fatty acids and sphingosine bases in the galactocerebrosides and their phosphonates, has been shown to be very similar, which is indicative of their relationship with regard to bio~ynthesis.'~' From T. cornutus have also been isolated 2-(methylamino)ethylphosphoric acid derivatives of di- and tri-galactosylceramides, wherein the substituted phosphoric acid group is located 143*143aat 0 - 6 of the D-galactosyl terminal residue. Sphingophosphonolipids containing galactose were isolated from another species of gastropoda, Monodonta labio, belonging to the same subclass and order, but a different family ( Trochidae). The major phosphonoglycolipid component here is also 2-(methylamino)ethylphosphonogalactocerebroside, and the minor, the (2-aminoethy1)phosphonogalactocerebroside.'28 Among the fatty acids, the preponderant ones are also hexadecanoic (50% of the total acids) and 2-hydroxyhexadecanoic (12%) acid, and among the sphingosine bases, mono-unsaturated dihydroxy bases having a linear chain (40% of the total bases) and a branched one (34%), as well as compounds having two double bonds (-20% ).I2' Complex phosphonoglycolipids containing 2-(methylamino)ethylphosphonic acid have been found in the gastropod Chlorostoma argurostoma turbinatum.1437143a One of the glycolipids is a ceramide pentasaccharide comprised of glucose, galactose, fucose, 3-O-methylgalactose, and 2-amino2-deoxygalactose, and the other is an octasaccharide containing one glucosyl and one 3- 0-methylgalactosyl residue, and two residues each of fucose, galactose, and 2-amin0-2-deoxygalactose.'~~ The structures of these compounds have not as yet been established. From the sea hare Apfysia kurodai, belonging to the subclass Prosobranchia, two phosphonoglycolipids having complex, carbohydrate chains have also been i ~ o l a t e d . ' ~ ' -One ' ~ ~ of the lipids contains one residue each of sphingosine base, fatty acid, glucose, 2-acetamido-2-deoxygalactose, and 3-O-methylgalactose, and two residues each of galactose and (2aminoethy1)phosphonic acid; that is, it has the structure of a ceramide bis[(2-aminoethyl)phosphono]pentaoside.'44 The other phosphonolipid seems to be a ceramide mono-[ (2-aminoethyl)phosphono]pentaosidecontaining one residue each of glucose, 2-acetamido-2-deoxygalactose,and fucose, and two galactosyl residues in the oligosaccharide chain. These

GLYCOLIPIDS OF MARINE INVERTEBRATES

415

glycolipids were detected in the skin of the mollusk,144and also found as the major components of the glycosphingolipids in nerve fibers and ganglion. 145*146 Cerebrosides, sulfatides, and gangliosides usual for the nervous tissue of vertebrates are practically absent.145 From the abalone Haliotisjaponica has been isolated a neutral pentaglycosylceramide containing one glucosyl residue, one galactosyl residue, one 2-acetamido-2-deoxygalactosylresidue, and two fucosyl residues.125The structure of this compound has been identified, from the data given by partial and total acid hydrolysis, methylation, and 'H-n.m.r. spectroscopy, as a-Fuc-( 1 + 3)-a-GalNAc-( 1 3)-[a-Fuc-( 1 2)]-P-Gal-( 1 + 4)-P-Glc-(1 + I)-Cer. --f

--f

When the lipid composition of the ganglion in four species of gastropod (Helix pomatia, Lymnaea stagnalis, Murex trunculus, and M . brandaris; the last two species inhabit the sea) was investigated by biochemical and histochemical methods, the glycosphingolipids were found to be present mainly in the form of sulfatides, .whereas gangliosides are absent.60

c. The Class 43iualuia.-(i) Marine Bivalves. The glycolipid composition of eight bivalve species, namely, Anadara broughtoni, Modiolus dificilis, Crenomytilus grayanus, Patinopecten yessoensis, Chlamys J: nipponensis, Ostrea gigas, Callista brevisiphonata, and Spisula sachalinensis, has been ~ h a r a c t e r i z e dAlmost .~~ all of them contain all three types of glycolipid (of low, medium, and high polarity), with the exception of M. dificilis, in which only a polar glycolipid was found, and C. brevisiphonata, having no mediumpolarity glycolipids. Low-polarity glycolipids in most of these species have the mobility of cerebrosides containing hydroxy fatty acids, and only S. sachalinensis has two cerebrosides, one of which contains normal, and the other, hydroxy, fatty acids. The major monosaccharides in all are glucose and galactose; in addition to these, considerable proportions of fucose, or arabinose, or both, have been found in many of the species.79 From the pearl oyster Pinctada martensii, a glycosylceramide was isolated in which -80% of the fatty acids are comprised of a-hydroxy acids, and the main, long-chain base is C19:I ~ p h i n g o s i n e . ' ~ ~ From the muscle tissue of the bivalve Barnea dilatata japonica, a galactocerebroside has also been isolated, but here, the major fatty acid component is hexadecanoic acid (92.3%), and the major sphingosine base is C l S r s2p h i n g ~ s i n e . ' ~ ~ From oyster gills, a glycosphingolipid was isolated whose oligosaccharide chain contains247 glucose, galactose, fucose, 3-0-methylfucose, and (247) A. Hayashi, T. Matsubara, and 1976, p. 85.

F. Matsuura, Abstr. Int. Symp. Carbohydr. Chem., Sth,

416

NlCOLAl K. KOCHETKOV AND GALINA P. SMIRNOVA

2-acetamido-2-deoxy-glucose and -galactose in the ratios of 1:3 : 1 : 1 : 1 : 1. Subsequently, it was found that the carbohydrate chain in this glycolipid is branched, the galactose being at 0-3 and 0-2, and the glycolipid contains 2-acetamido-2-deoxy-3-O - m e t h y l g a l a ~ t o s eThe . ~ ~struc~ ture of this complex glycolipid has not yet been established conclusively; it has, however, been that the fucose and 3-0-methylfucose are terminal and attached to galactose residues at 0-2; the 2-acetamido-2-deoxy3-0-methylgalactose also occupies a terminal position, and is attached to a hexose by a (1 + 3) bond.249 From oyster mantle, a complex glycolipid containing glucose, galactose, fucose, 3-0-methylfucose, 2-acetamido-2-deoxyglucose,and an amino sugar phosphate was also isolated.250In the ceramide moiety, among the fatty acids, the preponderant ones are c16:0, C17:0, and Cls:oacids, and the major component of the sphingosine bases is octadeca-4,8-sphingadienine (57% of the total bases), whose structure was identified by g.1.c.-m.s. of the Me,Si derivatives of the N-acetylated sphingosine bases, and of the products of their oxidation with periodate and periodatepermanganate. 29*251 (ii) Fresh-water Bivalves. From the bivalve Corbicula sandai, a complex mixture of glycosphingolipids was isolated that contained neutral and acid glycolipids. 126*252-257 This mixture was separated into individual, ceramide oligoside fractions by column chromatography on Florisil, silicic acid, and QAE-Sephadex, and t.1.c. on silica gel. The ceramide monoglycoside fraction contained glucosylceramide as the major component (77%), and galactosylceramide as the minor one2',; the ceramide disaccharide fraction contained one glycolipid, having the structure126*253-258 /.?-Man-(1+ 4)-p-Glc-(1 + 1)Cer, and the ceramide trisaccharide fraction, a glycolipid whose structure was126,258 p-Man-( 1 + 4)-p-Man-(1 + 4)-p-Glc-( 1+ 1)-Cer. A distinguishing feature of the last two ceramide oligosides is the presence, in the oligosaccharide chain, of mannose, instead of the galactose that is usually included in the ceramide oligosides of mammals and other animals. (248) (249) (250) (251) (252) (253) (254) (255) (256) (257) (258)

T. Matsubara and A. Hayashi, J. Biochem. (Tokyo),74 (1973) 853-856. T. Matsubara and A. Hayashi, J. Biochem. (Tokyo),83 (1978) 1195-1197. A. Hayashi and T. Matsubara, J. Biochem. (Tokyo),65 (1969) 503-511. A. Hayashi and T. Matsubara, Biochim. Biophys. A m , 248 (1971) 306-314. T. Hori, 0. Itasaka, and T. Hashimoto, J. Biochem. (Tokyo), 55 (1964) 1-10. T. Hori, 0. Itsaka, and M. Kamimura, J. Biochem. (Tokyo),64 (1968) 125-128. 0. Itasaka, J. Biochem. (Tokyo),60 (1966) 52-55. 0. Itasaka, J. Biochem. (Tokyo),63 (1968) 347-350. 0. Itasaka, M. Sugita, and T. Hori, Abstr. Inr. Symp. Cnrbohydr. Chem., 8rh, 1976, p. 84. 0. Itasaka, J. Biochem. (Tokyo), 60 (1966) 435-438. 0. Itasaka, M. Sugita, H. Yoshizaka, and T. Hori, J. Biochem. (Tokyo), 80 (1976) 935-936.

GLYCOLIPIDS OF MARINE INVERTEBRATES

417

The three more-polar glycosphingolipids from C. sandai, designated GL-1, GL-2, and GL-3, have similar i.r. spectra, resembling those of mammalian, hexosamine-containing glycosphingolipids. The structure of GL-3 has been established; its carbohydrate chain contains glucose, mannose, xylose, fucose, 4-0-methylgalactose, 2-acetamido-2-deoxyglucose,and mannose phosphate. 147v2s4-256 From the results of hydrolysis of the glycolipid by HCl and H F, methylation, oxidation with C r 0 3 , Smith degradation, and enzymic hydrolysis with alkaline phosphatase, the glycolipid was assigned the structure of an octaglycosylceramide whose carbohydrate chain has, as a branch, a single xylosyl group, and the branching point is constituted of mannose 6-(2-aminoethyl)phosphate (see top structure on p. 418).147 The presence, in the glycolipid, of an aminoethylphosphonomannosyl residue (see bottom structure on p. 418), which is a zwitterion, makes, in the opinion of the authors, a unique ionogenic contribution to the cellular membrane of the mollusk. Apart from the complexity of its monosaccharide composition and the presence of the mannose 6-phosphate derivative, this glycolipid is also unusual, in that fucose is located inside the oligosaccharide chain, which is the first time that this has been found in glycolipids. For GL-2, the composition of sugars has been determined. Glucose, mannose, xylose, and 3- 0-methylfucose were detected,257and the compound was shown to contain a free amino group and phosphorus, ~imilarly’~’ to GL-3. Among the sphingosine bases in the C. sandai sphingolipid mixture, a high content (44% of the total bases) of branched mono-unsaturated dihydroxy bases has been observed.1s9 In the spermatozoa of the fresh-water bivalve Hyriopsis schlegelii, the major components of the glycolipids are monoglycosylceramide and oligosylceramides. 259.260 The monoglycosylceramide has been identified as galactocerebroside, but glucocerebroside was detected in traces.259 By column chromatography on Unisil, silicic acid, and QAE-Sephadex, and preparative t.1.c. on silica gel, four oligosylceramides were isolated, whose structures were elucidated on the basis of the results of acid hydrolysis, methylation, oxidation with 0-0, , enzymic hydrolysis, and n.m.r. spectroscopy. The major component of the oligosylceramides, having the highest mobility in t.l.c., has a linear, tetrasaccharide chain containing one glucosyl, one 2-acetamido-2-deoxyg1ucosy1, and two mannosyl residues.241 (259) S. Higashi and T. Hori, Biochim. Biophys. Acta, 152 (1968) 568-575. (260) T. Hori, 0. Itasaka, and M. Sugita, in R. Schauer, E. Buddecke, M. F. Kramer, J. F.

G. Vliegenthart, and H. Wiegandt (Eds.), Glycoconjugates, Proc. In?. Symp., Jth, Georg Thierne, Stuttgart, 1979, pp. 46-47.

POdCHZ)ZNHZ

I

6 QO-Me-P-Gal-( 1 + 3)-pGalNAc-( 1 + 3)-a-Fuc-(1 + 4)-B-GlcNAc-(1 + 2)-a-Man-(l+ 3)-p-Man-(l+4)Glc-B-(l+ 1)-Cer 2

T

1

a-Xyl

GLYCOLIPIDS OF MARINE INVERTEBRATES

419

P-GlcNAc-( 1 + 2)-a-Man-(1 + 3)-P-Man-(1 + 4)-P-Glc-(1 + I)-Cer

The other three oligosylceramides contain the same tetrasaccharide backbone, to which other mono- or oligo-saccharide residues are attached. The two neutral oligosylceramides were identified as261 P-GlcNAc-( 1 + 2)-a-Man-(1 + 3)-[p-Xyl-( 1 + 2)]-P-Man-( 1 + 4)-P-Glc-( 1 + I)-Cer

and96 3-O-Me-a-Fuc-(l-+2)-3-0-MeXyl p-Xyl-I

1

1

1

4 2 a-Fuc-( 1 + 4)-P-GlcNAc-( 1 + 2)-a-Man-(1 + 3)-P-Man-(1 + 4)-Glc-Cer 3

t

1

3- 0-Me-a-GalNAc

and the third oligosylceramide is of an acidic nature, and contains 4 - 0 methylglucuronic 4-0-Me-p-GlcA 1

1 4

a-Fuc-( 1 + 4)-P-GlcNAc-(1 + 2)-a-Man-(1 3)-P-Man-( + 1 + 4)Glc-Cer 3 2

t

1

3-0-MeGalNAc

t

1

P-XYI

The authors assumed that this glycolipid creates ionogenic centers in cellular membranes, similar to those that appear because of the presence of a sialic acid in sialoglycolipids.262 The lipid composition in all of these oligosylceramides is similar. The major fatty acids are hexadecanoic and octadecanoic acid, and the major sphingosine base is octadeca-4-sphingenine. Besides these major glycolipids, minor glycosphingolipid components were isolated from the spermatozoa of Hyriopsis schlegelii. These are o l i g o s y l ~ e r a m i d e having s ~ ~ ~ a shorter carbohydrate chain, and lacking any amino sugars, namely, the ceramide disaccharides p-Gal-( 1+ 4)-Glc-Cer (261) T. Hori, H. Takeda, M. Sugita, and 0.Itasaka, J. Biochem. (Tokyo),82 (1977) 1281-1285. (262) T. Hori, 0. Itasaka, M. Sugita, S. Ando, M. Iwama, and K. Kumauchi, in T. Yamakawa, T. Osawa, and S. Handa (Eds.), Glycoconjugares, Roc. In?. Symp., 6?h,Japan Scientific Societies Press, Tokyo, 1981, pp. 35-36. (263) T. Hori, 0.Itasaka, M.Sugita, and S. Ando, Ado. Exp. Med. B i d , 152 (1982) 93-101. (264) M.Sugita, T. Yamamoto, S. Masuda, 0. Itasaka, and T. Hori, J. Biochem. (Tokyo), 90 (1981) 1529-1535.

420

NICOLAI K. KOCHETKOV A N D GALINA P. SMIRNOVA

and p-Man-( 1+ 4)-Glc-Cer, the ceramide trisaccharide a-Man-( 1-* 3)-pMan-( 1 + 4)-Glc-Cer, and the ceramide tetrasaccharide a-Man-( 1-* 3)[p-Xyl-(1 + 2)]-p-Man-( 1+ 4)Glc-Cer. With the exception of lactosylceramide, these glycolipids form a series of mannose-containing lipids that can be the precursors in the biosynthesis of the more-complex oligosylceramides already described. This assumption is based not only on the structure of the oligosaccharide core, which is identical in these compounds, but also on the similarity of their lipid moieties. In all of the glycolipids from H. schlegelii spermatozoa, the major fatty acid components are hexadecanoic and octadecanoic acid, and 90% of the sphingosine bases are 4-sphingenine.96,241.261.263.264 The hepatopancreas of H. schlegelii was found to contain an unusual cerebroside, a mannosylceramide, isolated from the monohexosylceramide fraction by t.1.c. on borate-impregnated silica gel.265Its structure was shown to be p-D-mannosylceramide, whose major sphingosine base is 4-sphingenine, and the fatty acids constitute a mixture of normal (77% of the total acids) and a-hydroxy acids, with hexadecanoic acid preponderant (43.7% of the total A mannose-containing cerebroside was found here (for the first time), and this extends the number of cerebrosides known today. An overwhelming majority of the cerebrosides in animals contain glucose or the salt glands of the herring gull were, however, found to contain a x y l ~ s y l c e r a m i d eand , ~ ~ ~from a human adenocarcinoma, a fucosylceramide has been isolated.268 Concluding the discussion of the glycolipids of fresh-water bivalves, it is necessary to note that the glycosphingolipids of both mollusk species, H. schlegelii and C. sandai, have in common some structural features distinguishing them from the glycosphingolipids of other animals. One of them is the presence of mannose in the oligosaccharide chains, instead of the galactose that is usually included in the glycosphingolipids of animal origin, although mannosylglycosphingolipids had previously been isolated from a plant source.269*270 Mannose-containing glycosphingolipids were isolated from another invertebrate species, the larvae of the greenbottle (265) T. Hori, M. Sugita, and H. Shimizu, Biochim. Biophys. Acra, 665 (1981) 170-173. (266) S. Hakomori and I. Ishizuka, in G . D. Fasman (Ed.), Handbook of Biochemistry and Molecular Biology, 3rd edn., CRC Press, Cleveland, 1975, pp. 416-425. (267) K.-A. Karlsson, B. E. Samuelsson, and G. 0. Steen, J. Lipid Res., 13 (1972) 169-176. (268) K. Watanabe, T. Matsubara, and S. Hakomori, J. Biol. Chem., 251 (1976) 2385-2387. (269) R. A. Laine and 0. Renkonen, Biochemistry, 13 (1974) 2837-2843. (270) H. E. Carter, D. R. Strobach, and J. N. Hawthorne, Biochemistry, 8 (1969) 383-388. (271) M. Sugita, M. Nishido, and T. Hori, J. Biochem. (Tokyo), 92 (1982) 327-334. (272) M. Sugita, Y. Iwasaki, and T. Hori, J. Biochem. (Tokyo), 92 (1982) 881-887.

GLYCOLIPIDS OF MARINE INVERTEBRATES

42 1

The second characteristic feature of glycosphingolipids in fresh-water Bivaluia is the position of the fucosyl residue inside the oligosaccharide chains of fucose-containing glycosphingolipids, whereas, in mammalian fucoglycosphingolipids, it is always in a terminal position. Finally, attention is drawn to the presence of 0-methylated derivatives of sugars in the complex glycolipids of these mollusks. 0-Methylation of the carbohydrate chain may make it possible to regulate the physical properties of a lipid by increasing its hydrophobicity, as well as to change its antigenic proper tie^.^^ d. The Class Cephalopoda.-Cephalopods are the most advanced of the mollusks; they have reached a high stage of development, and possess the most-complex nervous system, in comparison with the other invertebrates. However, the content of glycolipids in them is unexpectedly low. In the octopus Octopus sp. and the cuttlefish Rossia pacijica, only 1.4 and 0.8% of monosaccharides, respectively, have been found in their lipid extracts, each of these species contains only one polar glycolipid, and medium- and low-polarity glycolipids are absent.79 Monosaccharides in the glycolipid from the Octopus sp. have been found to include glucose, galactose, and xy10se.’~ In the nerve tissue of the cuttlefish Loligo, no glycolipids at all have been detected.62Sialoglycolipids have been shown to be absent in the

5. Brachiopoda Brachiopods constitute a group of exclusively marine animals. With regard to glycolipids, they have received practically no study. Only one species of this phylum, Coptothyris grayi, has been characterized. It contains an appreciable proportion of glycolipids (2.2% of monosaccharides in the lipid extract)79; their number is, however, not great. Two glycolipids, one of low, and one of medium, polarity have been detected, as well as a trace of a polar glycolipid. Galactose preponderates among the monosaccharides; there is somewhat less glucose and arabinose, and a little xy10se.~~ 6. Arthropoda

Arthropods constitute the most numerous group of animals, many species of which inhabit the sea. The glycolipids of marine arthropods have not been studied to any considerable extent. The glycolipid composition has been characterized for five species of one class: Pandalus latirostris, Pagurus ochotensis, Hemigraspus sanguineus, Erimacrus isenbeckii, and Cymadoce japonica. Among the other marine invertebrate phyla, they contain the (273) L. Svennerholm, in A. Lajtha, (Ed.), Handbook of Neurochernistry, Vol. 3, Plenum Press, New York, 1969, pp. 425-452.

422

NICOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

smallest proportion of glycolipids (0.4-0.8% of monosaccharides in the lipid extracts79). All of the species have been found to contain a small proportion of medium- and high-polarity glycolipids, and one species ( P . latirostris) also contains a glycolipid having the mobility of a cerebroside. The major monosaccharides are glucose, galactose, and a r a b i n ~ s e . ~ ~ 7. Echinodermata Echinoderms constitute a peculiar phylum of invertebrates belonging to the most highly organized animals, the Deuterostomia, including, also, all the phyla of chordates. Among the invertebrates, echinoderms are the richest in glycolipids, and differ from all of the other marine invertebrate phyla in that they contain ~ialoglycolipids.~~ This conforms to the data of Warren, who studied the occurrence of sialic acids in Nature, and found that all of the vertebrates have them, but, of the invertebrates, only the echinoderms; practically all of the Protostomia contain no sialic The lipid extracts from echinoderms have been found to contain 3.3 to 8.5% of monosaccharides. The only exception is the sea cucumber Cucumariajaponica, whose lipid extract contains 1.6% of monosaccharides, although a related species, C. fraudatrix, has been found to contain the highest proportion thereof (8.5%). It should, however, be noted that some carbohydrate-containing compounds in the lipid extracts of echinoderms may be represented not by glycolipids, but by steroid or triterpene glycosides. 275-282 a. The Class Echinoidea.-This class is subdivided into two subclasses: Regularia and Irregularia. The glycolipid content has been characterized for four species of sea urchin, two of which belong to the subclass Regularia (Strongylocentrotus nudus and S. intermedius), and two to the subclass Irregularia (Echinarachnius griseus and Echinocardium ord datum).^^ In all (274) L. Warren, Comp. Biochem. Physiol., 10 (1963) 153-171. (275) T. Yasumoto and Y. Hashimoto, Agric. Biol. Chem., 29 (1965) 804-808; Chem. Absrr., 63 (1965) 18,540. (276) I. Kitagawa and M. Kobayashi, Tetrahedron Lert., (1977) 859-862. (277) F. De S h o n e , A. Dini, E. Finamore, L. Minale, C. Pizza, R. Riccio, and F. Zollo. J. Chem. Soc., Perkin Trans. 1 (1981) 1855-1862. (278) S. Ikegami, K. Okano, and H. Muragaki, Tetrahedron Lerr., (1979) 1769-1772. (279) I. Kitagawa, T. Nishino, T. Matsuno, H. Akutsu, and Y. Kyogoku, Tetrahedron Lett., (1978) 985-988. (280) 1. Kitagawa, T. Nishino, M. Kobayashi, T. Matsuno, H. Akutsu, and Y. Kyogoku, Chem. Pharm. Bull., 29 (1981) 1942-1950. (281) I. Kitagawa, T. Nishino, M. Kobayashi, and Y. Kyogoku, Chem. Pharm. Bull., 29 (1981) 1951-1956. (282) I. Kitagawa, T. Sugawara, and I. Yoshioka, Chem. Pharm. Bull., 24 (1976) 275-284.

GLYCOLIPIDS OF MARINE INVERTEBRATES

423

of the species, the monosaccharide content in the lipid extracts is high (4.9 to 6.8%); the preponderant monosaccharide is glucose; only in E. griseus, was galactose found, along with glucose. The glycolipid composition in all of the species is rather broad: 1-2 cerebroside-like glycolipids, 2-3 mediumpolarity glycolipids, and 3-7 polar glycolipids, most of which contain sialic acid, are present.79 (i) Sialoglycolipidsof Sea Urchins. In 1960, Warren and Hathaway reported isolating, from the eggs of Arbacia punctulata (the subclass Reguluria), a glycolipid containing sialic acid; the latter was identified as N-glycolylneuraminic acid by paper chromatography. The other lipid components were not identified.64 In 1966-1967, Isono and Nagai isolated from two Regularia species, Pseudocentrotus depressus6’ and Hemicentrotus pulcherwhose carbohydrate chains contained only glucose and r i m ~ s , glycolipids ~’~ sialic acid. Independently, Kochetkov and coworkers isolated four glycosphingolipids from S. intermedius gonads; these contained glucose, sialic acid, fatty acids, and a trihydroxy long-chain base (phyto~phingosine).~~~’~~ For the major component of glycolipids, whose carbohydrate chain contains two glucosyl and two sialic acid residues, two alternative structures were proposed on the basis of acid hydrolysis, methanolysis, methylation, and periodate oxidation data. The most probable is the one whose carbohydrate chain is composed of two NeuAc(G1)-(2 + 6)-Glc disaccharide cr-NeuAc(GI)-(2+6)-Glc-(l+ 8)-NeuAc(GI)-(2+ 6)-Glc-(1+ 1)-Cer

In this glycolipid, a new type of sialic acid linkage, a (2 + 6) bond with the glucosyl residue was demonstrated for the first time. The sialoglycoconjugates are known to contain ( 2 + 3 ) and ( 2 + 6 ) bonds of sialic acids with galactose or 2-acetamido-2-deoxygalactose,as well as the (2 + 8) bond with the other sialic acid The glycolipid includes N-acetyl- and N-glycolyl-neuraminic acids in the ratio of 2 : 1. It should be noted that there is a high content of N-acetylneuraminic acid, as, previously, echinoderms were found to contain N-glycolylneuraminic acid almost exc~usively.~’~ The fatty acids include normal and a-hydroxy acids, the latter accounting for -25% of the overall, acid mixture. The major components are the Cz2: (283) Y. Isono, Jpn. J. Exp. Med., 37 (1967) 87-96. (284) N. K. Kochetkov, I. G. Zhukova, and G. P. Smirnova, Dokl. Acad. Nauk SSSR, 180 (1968) 996-999. (285) N. K. Kochetkov, I. G . Zhukova, and G. P. Smirnova, Dokl. Acad. N a u k SSSR, 192 (1970) 344-347. (286) H. Wiegandt, Adu. Exp. Med. Biol., 125 (1980) 3-10. (287) M. 1. Horowitz and W. Pigman (Eds.), The Glycoconjugates, Vol. 1, Academic Press, New York, 1977.

424

NICOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

acid (41% of the mixture of normal acids) and the C,,:, a-hydroxy acid (71% of the mixture of hydroxy acids). Sphingosine bases are represented by only the trihydroxy bases, and the major components of the mixture are C16:oand C1g:o phytosphingosines.138*288 Later, from the eggs of S. interrnedius, two sialoglycolipids were isolated that had the same type of structure as the sialoglycolipid from the gonads, but they contained only N-glycolylneuraminic acid, and, in one of them, this acid was sulfated.289v290 In the spermatozoa of the sea urchin Anthocidaris crassipina, nine sialoglycolipids were found, two of which were isolated and their structures established from the results of acid hydrolysis, methylation, and periodate ~xidation.’~’ The major component of the glycolipid mixture is a disialoglycolipid, NeuAc-(2 + 8)-NeuAc-(2 + 6)-Glc-Cer. The least-polar, minor component has the structure of a monosialoglycolipid, NeuAc-(2 + 6)-GlcCer. In these compounds, glucose is again the only neutral monosaccharide, and the sialic acid is attached to it at 0-6. The major, fatty acid component in these glycolipids is the C22:1acid, and no hydroxy acids were found. The sphingosine bases are represented by dihydroxy bases, among which, the preponderant one is the C18: sphing0~ine.I~’ From the S. nudus gonads, mono- and di-sialoglycolipids were isolated as well.291Their structures are similar to those of the sialoglycolipids from A. crassipina. a-NeuG1-(2+ 6)-P-Glc-(l+ I)-Cer a-NeuGI-(2+ 4)-a-NeuGI-(2+ 6)-P-Glc-(I + 1)-Cer

The difference is that, here, N-glycolylneuraminic acid is present, and its two residues in the disialoglycolipid are joined by a (2 + 4) bond unusual for sialic acids. The sphingosine bases in the sialoglycolipids from S. nudus are phytosphingosines, among which, -70% are accounted for by CIS phytosphingosine. The fatty acids were found to contain saturated, normal, and a-hydroxy acids. The major components of the normal acids in both sialoglycolipids are palmitic and stearic acid, and, among the a-hydroxy acids in the monosialoglycolipid, the preponderant ones are CIS,C,, , C,, (288) I. G. Zhukova, G. P. Smirnova, 1. S. Glukhoded, and N. K. Kochetkov, Dokl. Acnd. Nauk SSSR, 192 (1970) 563-566. (289) N. V. Prokazova, S. L. Kocharov, V. L. Sadovskaya, U. V. Moshenskii, L. D. Bergelson, and N. D. Zvezdina, Bioorg. Khim., 5 (1979) 458-467. (290) N. V. Prokazova, A. T. Mikhailov, S. L. Kocharov, L. A. Malchenko, N. D. Zvezdina, G. Buznikov, and L. D. Bergelson, Eur. J. Biochem., 115 (1981) 671-677. (291) N. K. Kochetkov, G. P. Smirnova, and I. S. Glukhoded, Bioorg. Khim., 4 (1978) 1093- 1099.

GLYCOLIPIDS OF MARINE INVERTEBRATES

425

a-hydroxy acids, whereas, in the disialoglycolipids, they are the C,4, C I S , C22,and CZ3a-hydroxy acids.29' From the gonads of the sea urchin Tripneustes uentricosa (the subclass Reguluria), inhabiting the Atlantic Ocean, three mono- and one di-sialoglycolipid were isolated. The major components are two monosialoglycolipids, one of which contains N-acetyl-, and the other, N-glycolyl-neuraminic acid, namely, a-NeuAc-(2+ 6)-P-Glc-(1 + 1)-Cer and a-NeuG1-(2+ ~~ + 8)-a6)-P-Glc-(1 + 1)-Cer, and a d i s i a l ~ g l y c o l i p i d , ~a-NeuAc-(2 NeuAc-(2+ 6)-P-Glc-(1 + 1)-Cer. The carbohydrate chains in these compounds are seen to be similar to those in sialoglycolipids from the other sea urchin species of the subclass Regularia. In the subclass Irregularia, the sialoglycolipid composition has been studied for two species of sea urchins belonging to different orders: E. cordatum (the order Spatongoida) and Echinurachnius parma (the order Clypeastroida). From the E. cordatum gonads, three sialoglycolipids were isolated, two major and one minor, whose structure has been elucidated by partial and total acid hydrolysis, methanolysis, methylation, periodate oxidation, oxidation with chromium trioxide, and enzymic hydrolysis with neuraminidase. One of the major sialoglycolipids, having the least acidic nature, proved to be a monosialoglycolipid containing one N-glycolylneuraminic acid and one glucosyl residue joined by an a-(2 + 6) bond.I4' N-Glycolylneuraminic acid in the glycolipid has, on 0-4, an alkali-labile substituent that makes it resistant to the action of neuraminidase from Vibrio cholerue. After mild, alkaline treatment of the glycolipid, the sialic acid is completely split offby the neuraminidase. The nature of the substituent has not yet been elucidated. It has been shown, however, that this substituent is not an 0-acetyl or 0-glycolyl group (usual for sialic acids), nor is it a formic, lactic, or pyruvic acid group. For the glycolipid, the following structure has been propo~ed,'~'where X is the unknown acyl group. 4-O-X-a-NeuGI-(2+ 6)-P-Glc-( 1 + 1)-Cer

The minor glycolipid contains two N-glycolylneuraminic acid residues and one glucosyl residue; the residues of the sialic acids are bound to one another by a (2 + 4) bond, as in the case of the disialoglycolipid from S. nudus, and the disialyl group is attached293to glucose at 0-6. a-NeuGI-(2+4)-(r-NeuGI-(2+6)-P-Glc-(l+ 1)-Cer

(292) N. V. Chekareva, Absrr. A//-UnionConj, Chem. Biochem. Carbohydr., 7rh, Pushchino, 1982, pp. 95-96. (293) G . P. Smirnova, N. V. Chekareva, and N. K. Kochetkov, Bioorg. Khim., 4 (1978) 937-942.

426

NICOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

The composition of the ceramide moiety of these glycolipids is quite similar. Among the fatty acids, normal and a-hydroxy acids were detected; the latter accounted for -10% of the mixture of acids. In the monosialoglycolipid, the major components of the normal acids are C14:o and C16:O acids, and of the a-hydroxy acids, the C17:o,and C24:o a-hydroxy acids. The sphingosine bases are phytosphingosines; the preponderant ones are and C19 phytosphingosine~.'~~~~~~ The second major sialoglycolipid from E. cordatum was the most acidic. Its carbohydrate chain contained one sialic acid residue and one glucosyl residue, as well as a sulfate group at C-8 of the sialic This was the first example found in Nature of a sialoglycolipid containing a sulfated sialic acid. The structure proposed for the glycolipid is as follows. a-NeuGI-8-O-SO3H-(2+ 6)-P-Glc-(1

1)-Cer

The sulfate group protects the sialic acid from the action of V. cholerae neuraminidase, whereas, in the desulfated glycolipid, the sialic acid is completely split off on treatment with the neuraminidase. The method that proved to be most convenient for sialoglycolipid desulfation was solvolysis, previously proposed for the desulfation of carbohydrate sulfates.294When the sulfated sialoglycolipid is boiled in 1P-dioxane in the presence of pyridine hydrochloride, the sulfate group is completely removed after 15 min, but the glycosidic bonds remain ~naffected.'~' Other desulfation methods, such as treatment with HCI in methanol,295and boiling in anhydrous 1 , 4 - d i o ~ a n e ,were * ~ ~ unsuccessful. In the first case, along with removal of the sulfate group, the sialic acid ketoside bond was also partially split, and, in the second, no desulfation took place. The sphingosine bases in sulfated sialoglycolipid are phytosphingosines having a chain length of C I 6to CI9;the major component was found to be C18-phytosphingosinewith a linear chain (56% of the total bases); -20% of the mixture is composed of phytosphingosines having an isostructure. The fatty acids consist of a mixture of normal and a-hydroxy acids, in the ratio of 3 : 1, that include a broad range of acids differing in their chain length (from 13 to 25 carbon atoms). The normal acids are mainly saturated; the major component is the C,, acid (32.5%). Among the a-hydroxy acids, -30% ofthe mixture is made up of unsaturated acids; the major components are c24:0, C22:0,and C,,:, a-hydroxy acids.141 Sulfated sialoglycolipids have also been found as the major sialoglycolipid components in the gonads of another sea urchin species, E.parma, belonging (294) N. K. Kochetkov, A. I. Usov, and K. S. Adamyants, Zh. Obshch. Khim., 42 (1972) 1617-1622. (295) 1. Ishizuka, M. Suzuki, and T. Yamakawa, J. Biochem. (Tokyo),73 (1973) 77-87. (296) G . L. Mayers, M. Pousada, and T. H. Haines, Biochemistry, 8 (1969) 2981-2986.

GLYCOLIPIDS OF MARINE INVERTEBRATES

427

to the same subclass of Zrregularia, but to another order (the Clypeastr~idea).*~ ’ sulfated monosialoglycolipids were isolated, whose carTwo bohydrate chains contain, in addition to sialic acid, one glucosyl residue each, and the sulfate group is located on C-8 of the sialic acid residue. The glycolipids differ only in the type of sialic acids: one is the 8-sulfate of N-acetyl-a-neuraminosyl-(2-+ 6)-p-glycosyl-(1 + 1)-ceramide, and the other, the 8-sulfate of N-glycolyl-a-neuraminosyl-(2 + 6)-p-glucosyl-( 1 + l ) - ~ e r a m i d e . ~The ~ ’ lipid moiety composition of these two compounds is similar. Among the fatty acids, the normal ones are preponderant, and a-hydroxy acids account for not more than 10% of the mixture. The sphingosine bases are mainly phytosphingosines, and 10% of the mixture is composed of dihydrosphingosine~.~~’ Thus, in the sea urchins E. cordaturn and E. parrna of the subclass Irregularia, sulfated gangliosides having a sulfate group on the sialic acid residue have been found for the first time; these are the major components of the sialoglycolipid mixture. Later, such compounds were also isolated from sea urchins of the subclass Regularia, from the eggs and embryos of S. i n t e r r n e d i u ~and , ~ ~from ~ ~ ~the ~ ~gonads of T. u e n t r i c ~ s aSulfated . ~ ~ ~ monoand di-sialogangliosides containing sulfated sialic acid have also been found in mammals, in bovine gastric mucosa, where they are minor components of the g l y c o s p h i n g ~ l i p i d s . ~ ~ ~ ~ ~ ~ ~ From the data just presented, it may be seen that the sialoglycolipids of both subclasses of sea urchin have a common type of structure: their carbohydrate chains contain only glucose and sialic acid, the latter being attached to 0 - 6 of the glucose. Carbohydrate chains of such a type have not been found in the gangliosides of the vertebrates. The gangliosides of the vertebrates include, in addition to glucose and sialic acid, galactose and, often, a hexosamine (2-acetamido-2-deoxy-galactose or -glucose), and the sialic acids are usually linked by a (2-+ 3) bond to galactose or, by a (2 + 8) bond, to another sialic acid residue.286The lipid moiety of sea urchin sialoglycolipids includes in most cases a trihydroxy base, except for the sialoglycolipids from A. crassispina, where only dihydroxy bases have been found. In the gangliosides of the majority of normal tissues of mammals, the sphingosine bases are dihydroxy bases. Phytosphingosines have been found in considerable proportions only in the hematosides of small-intestine

-

(297) G. P. Smirnova, N . V. Chekareva, and N. K. Kochetkov, Bioorg. Khim., 6 (1980) 1667- 1673. (298) J . Dabrowski, in preparation. (299) B. L. Slomiany, K. Kojima, 2. Banas-Gruszka, V. L. N. Murty, N. I. Galicki, and A. Slomiany, Eur. J. Biochem., 119 (1981) 647-650. (300) A. Slorniany, K. Kojirna, 2. Banas-Gruszka, and B. L. Slomiany, Biochem. Biophys. Res. Commun., 100 (1981) 778-784.

428

NICOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

epitheli~m'~''' and in the gangliosides of kidney and ~ p l e e n ~ in ' ~some -~~ mammalian species. (ii) Sulfolipids of Sea Urchins. As early as 1965,Isono and Nagai reported isolating, from the lipid extracts of eggs and sperm of P. depressus, a sulfolipid that differed from the sulfatides of animals, as it contained no sphingosine and no galactose, and, in its i.r. spectrum, no sulfate absorption bands were pre~ent.~'*~'' The isolated sulfolipid proved to be a glycoglycerolipid similar to plant 6-sulfoquinovosylglycerides. Its i.r. spectrum showed intense absorption bands characteristic of sulfonic acids (at 1170 and 1035 cm-I), and the absorption spectrum of the product of reaction of the carbohydrate component with anthrone produced the same pattern as in the case of sulfoquinovose (A,,, 595 nm). Soon afterwards, the same sulfolipid was isolated from the eggs and the developing embryos of the ~ ~ ~sulfolipids were detected by t.1.c. in the sea urchin H. p u l c h e r r i r n ~ sand .~~ of the lipid extracts from A. crassipina and Clypeaster j a p o n i ~ aAnalysis fatty acids of the sulfolipids of sea urchins showed that the only ones present are hexadecanoic and tetradecanoic acid,305whereas, in the sulfolipids' of plants, unsaturated acids are prep~nderant.~"This difference in the fatty acid composition is indicative of the fact that the sulfolipids of sea urchins are of endogenous origin, that is, they do not receive them with food from algae that contain sulfolipids. The proportion of sulfolipids in the gonads of sea urchins changes during early development of the embryos.z83 It has also been found that the sulfolipid from P. depressus exerts a stimulating effect on the respiration of the spermatozoa of the sea ~ r c h i n . ~ " From the shell of A. crassipina has been isolated a sulfoquinovosylmonoglyceride, and its structure has been established by chemical splitting (alkaline methanolysis and acid hydrolysis), as well as by such physicochemical methods as 'H- and "C-n.m.r. spectroscopy, g.1.c.-mass spectrometry, and field-desorption mass ~ p e c t r o m e t r y .The ~ ' ~ major component was shown to (96% of the be l-O-hexadecanoyl-3-O-(6-C-sulfo-a-~-quinovosyl)glycerol mixture), and the minor one was its analog containing tetradecanoic acid.

(301) (302) (303) (304) (305) (306) (307)

J.-E. Bouhours and R. M. Glickman, Biochim. Biophys. Acra, 487 (1977) 51-60. K. Puro and A. Keraenen, Biochim Biophys. Actu, 187 (1969) 393-400. A. Hara and T. Taketomi, J. Biochem. (Tokyo), 78 (1975) 527-536. S. Gasa and A. Makita, J. Biochem. (Tokyo), 88 (1980) 1119-1128. Y. Nagai and Y. Isono, Jpn. J. Exp. Med., 35 (1965) 315-318. Y.Nagai and M. Hoshi, Biochim. Biophys. Acra, 388 (1975) 146-151. C. F. Allen, P. Good, H. F. Davis, and S. D. Fowler, Biochem Biophys. Res. Commun.,

(308)

Y. Isono, H. Mohri, and Y.Nagai,

15 (1964) 424-430. Narure, 214 (1967) 1336-1338.

GLYCOLIPIDS OF MARINE INVERTEBRATES

429

0

I/

H,C-S-ONa II I

0

HO

CHOH

I

HICOC(CHI),,CH~

I1

0 where n = 14 (96%) and 12 (4%)

b. The Class Aszeroidea.-Starfish constitute one of the most ancient groups of animals. The starfish existing today are subdivided into three orders, namely, Phanerozonia, Spinulosa, and Forcipulata. The glycolipid composition has been characterized for six starfish species, five belonging to the order Forcipulata (Lysastrosoma anthosticta, Distolasterias nipon, Lethasterias fusca, Aphelasterias japonica, and Asterias arnurensis), and one to the order Spinulosa (Patiria p e ~ t i n i f e r a )Their .~~ lipid extracts contain 3.3 to 4.4% of monosaccharides, and have a similar chromatographic pattern for low- and medium-polarity glycolipids: in each of the species, two cerebroside-like glycolipids and two (or three) glycolipids of medium polarity have been found. T.1.c. of the polar glycolipids showed that, depending on the species, from one to five sialic acid-containing zones are present. The major monosaccharide in all of the species is glucose; in most of them, there are also considerable proportions of galactose and pentoses. (i) Neutral Glycolipids. From P. (Asterina)pectinifera have been isolated glucosylceramides78~95~309 and lactosyl~eramide~~ whose ceramide moieties are distinguished by a higher degree of hydroxylation, and contain phytosphingosines and a considerable proportion of a-hydroxy fatty acids. Among the phytosphingosines, compounds having both linear and branched chains, and containing 16, 17, and 18 carbon atoms, are present. Branched phytosphingosines account for -80% of the total bases in the glucosylceramide, and -70% in the l a c t o s y l ~ e r a m i d e . ~ ~ ~ ~ ~ ~ Considerable proportions of cerebroside have been detected in the starfish Asterias rub en^.^^.^'^ It is a glucosylceramide having a-hydroxy fatty acids (from cl6 to C26) and dihydroxy bases whose major components are C , , and C22bases, with one, or two, double bonds. (309) V. A. Vaver, G. I. Shaposhnikova, and T. N. Simonova, Bioorg. Khim., 2 (1976) 594-600. (310) L. R. Bjorkman, K.-A. Karlsson, and K. Nilsson, Comp. Biochem. Physiol., B, 43 (1972) 409-41 1.

430

NICOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

(ii) Sialoglycolipids. From the lipid extract of the hepatopancreas of D. nipon, a major sialoglycolipid was isolated, and its structure has been established by partial and total hydrolysis with acid, methanolysis, methylation, periodate oxidation, and enzymic splitting with ne~raminidase.~"It has been shown to be a trisialosyllactosylceramide containing N-acetylneuraminic acid, whose three sialic residues are linked to one another by a-(2+ 8) bonds, and the trisialyl chain is attached to 0 - 3 of the galactosyl residue. a-NeuAc-(2+8)-a-NeuAc-(2+ 8)-a-NeuAc-(2+3)-P-Gal-(l+4)-Glc-(l-+ 1)-Cer

A trisialoganglioside of the same type was later also found in the tissues of vertebrates; it was shown to be present in the brain of el as mob ranch^,^'^ and it was isolated from the brain of cod hog kidney,314and erythrocyte membranes of cat.315 In the trisialoglycolipid from D. nipon hepatopancreas, some of the N-acetylneuraminic acid residues are in the form of the 8-0-methyl derivative, whose structure was proved by mass s p e c t r ~ m e t r y . ' ~ ~ The fatty acids of the sialoglycolipid constitute a complex mixture containing normal (from C,, to C3,) and a-hydroxy acids (from cl5 to (&), many of which are unsaturated acids. In the mixture of normal acids, the preponderant onesare the c16:O(29%),C1~:0(14%), C,,:, (12%),andC2,:, (10%) acids, and the major components of the a-hydroxy acids are the C,,:, (30%) and Cz4:l (36%) a-hydroxy acids. The sphingosine base in the glycolipid is phyto~phingosine.~" From the hepatopancreas of Evasterias retijiera, a disialoglycolipid The containing 2-acetamido-2-deoxygalactose has been glycolipid includes two N-acetylneuraminic acid residues joined by a bond that is unusual for sialoglycolipids, a (2 + 9) bond, and the disialyl fragment is attached to 0-3 of the 2-acetamido-2-deoxygalactose. a-NeuAc-(2+ 9)-a-NeuAc-(2 + 3)-P-GalNAc-( 1 + 3)-P-Gal-(l+ 4)-P-Glc-(l+ 1)-Cer

The position of the bond between the sialic acids was determined by g.1.c.-m.s. of the acetates of partially methylated sialic acid derivatives obtained after methanolysis of the methylated sialoglycolipid, and was confirmed by the periodate-oxidation data. The (2 + 9) bond between (311) I. G. Zhukova, T. A. Bogdanovskaya, G. P. Smirnova, N. V. Chekareva, and N. K. Kochetkov, Dokl. Acad. Nauk SSSR, 208 (1973) 981-984. (312) N. F. Avrova, Y.-T. Li, and E. L. Obukhova, J. Neurochem., 32 (1979) 1805-1815. (313) R. K. Yu and S. Ando, Adu. Exp. Med. B i d , 125 (1980) 33-45. (314) K. Murakami-Murofuski, K. Tadano, 1. Koyama, and 1. Ishizuka, J. Biochem. (Tokyo), 90 (1981) 1817-1820. (315) N. Ando and T. Yamakawa, J. Biochem. (Tokyo), 91 (1982) 873-881. (316) G. P. Smirnova and N. K. Kochetkov, Bioorg. Khim., 8 (1982) 102-108.

GLYCOLIPIDS OF MARINE INVERTEBRATES

43 1

the residues of sialic acids in the sialoglycolipid was found here for the first time, although this type of bonding had previously been shown in a sialic acid homopolymer isolated from a micro-organism, Neisseria meningitidi~.~ l7 The sphingosine bases in the sialoglycolipid from E. retifera are phytosphingosines having both linear and branched chains; the latter account for -40% of the mixture of phytosphingosines. The major components are n - C l , , oand i ~ 0 - Cphytosphingosines. ~ ~ : ~ Among the fatty acids, the preponderant ones are a-hydroxy acids (80% of the total acids), and the major components are CI4, C I S ,and C,, a-hydroxy acids. Among the normal acids, palmitic and stearic acids make up >6O% of the m i ~ t u r e . ' ~ ' , ~ ' ~ From the hepatopancreas of A. amurensis was also isolated a disialoglycolipid containing, along with glucose and galactose, 2-acetamido-2-deoxygalactose having the sialic acid residues bound to This glycolipid, however, includes two N-glycolyl-S- O-methylneuraminic acid groups attached to 0 - 3 and 0 - 6 of one 2-acetamido-2-deoxygalactosylresidue. 8- 0-MeNeuGI 2

5-

6 P-GalNAc-(I + 3)-P-Gal-(1 +4)-P-Glc-(1 + I)-Cer 3

t

2 8-0-MeNeuGI

The positions of glycosidic bonds were determined by g.1.c.-m.s. analysis of methylated methyl glycosides and of acetates of partially methylated methyl glycosides obtained after methanolysis of the permethylated sialoglycolipid. Such location of sialic acids in the oligosaccharide chain is not found in the sialoglycoconjugates of vertebrates. Both of the N-glycolyl-8-0methylneuraminic acid groups are stable towards the action of V . cholerae neuraminidase. This stability does not seem to be associated only with the presence of the methyl group on 0-8 of sialic acid, as a bulkier substituent, the acetyl group situated on 0 - 7 or 0 - 9 of sialic acids, has been shown to decrease the degree of liberation of the sialic acid, but not to protect against the enzyme action ~ o m p l e t e l y . ~ ' Most ~ - ~ ~probably, ' this resistance towards (317) A. K. Bhattacharjee, H. J. Jennings, C. P. Kenny, A. Martin, and I. C. P. Smith, J. Bid. Chem., 250 (1975) 1926-1932. (318) G . P. Smirnova, I. S. Glukhoded, and N. K. Kochetkov, Bioorg. Khim., 8 (1982) 971-979. (319) R. Schauer and H. Faillard, Hoppe-Seyler's Z. Physiol. Chem., 349 (1968) 961-968. (320) R. Ghidoni, S. Sonnino, G. Tettamanti, N. Baumann, G. Reuter, and R. Schauer, J. Bid. Chem., 255 (1980) 6990-6995. (321) R. Schauer, Abstr. Int. Symp. Carbohydr. Chem. loth, 1980, 2L2.

432

NICOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

neuraminidase can be explained by the spatial position of the sialic acids in the oligosaccharide chain. Both of the sialic acid groups may be regarded as located at the branching point of the carbohydrate chain. It is known that V. cholerae neuraminidase does not split the a - ( 2 + 3) bond joining sialic acid to a galactosyl residue that contains a substituent on 0-4, as, for example, in173,L76 gangliosides GM1 and GM2,and it was also shown that bacterial neuraminidases cannot liberate N-glycolylneuraminic acid bound to 0-3 of a 2-acetamido-2-deoxygalactosylresidue located inside the oligosaccharide chains obtained from the polysialoglycoproteins of salmon eggs.322 The composition of the lipid moiety in the sialoglycolipid from A. arnurensis is similar to that of the ceramide part of the sialoglycolipid from E. retifera. The content of a-hydroxy acids here is also high (50% of the total acids), and the sphingosine base is phytosphingosine. The major components of the normal acids are hexadecanoic and octadecanoic acid, and those of the a-hydroxy acids are C16, CZ2,and C2., a-hydroxy acids. The major, sphingosine-base components are C17,C18,and CI9phytosphingosines having the is0 s t r u ~ t u r e . ' ~ ~ * ~ ' ~ Thus, the sialoglycolipids from E. retifera and A. arnurensis starfish, which are closely related, contain an amino sugar, absent from the sialoglycolipids of other species of echinoderm, and possess the same trisaccharide chain-structure for the asialo derivative. They differ, however, in the type of sialic acids, and their location in the carbohydrate chain. From the starfish P. (A) pectinifera (the order Spinulosa) have been isolated complex sialoglycolipids of unusual structure, containing (in addition to glucose, galactose, and sialic acid), arabinose, which is not encountered in the gangliosides of vertebrates, and the sialic acid residue is located in the inner part of the oligosaccharide chain and is glycosylated by galactose.93.1

34-136.161.207.323

From the whole starfish were isolated three monosialoglycolipids whose carbohydrate chains contain glucose, galactose, arabinose, and N-glycolylneuraminic acid. From the results of partial hydrolysis with acid, methanolysis, methylation, and C r 0 3 oxidation, the following structures have been proposed for these sialoglycolipids. P-Arap-(l-+6)-P-Galp-(l-+4)-8-0-MeNeuGI-(2-*3)-P-Galp-(l-+4)-P-Glcp-(l+ 1)-Cer (Ref. 135)

(322) S. Inoue, M. Iwasaki, and G . Matsumura, in T. Yamakawa, T. Osawa, and S. Handa (Eds.), Glycoconjugates, Proc. Inr. Symp., 6fh, Japan Scientific Societies Press, Tokyo, 1981, pp. 271-272. (323) N. K. Kochetkov and G.P. Smirnova, Bioorg. Khim., 3 (1977) 1280-1283.

GLYCOLIPIDS OF MARINE INVERTEBRATES

433

P-Arap-(1 +6)-P-Galp-(l+4)-NeuG1-(2 + 3)-P-Galp-(l+4)-P-Glcp-(l+ 1)-Cer (Ref. 135)

Araf;p-(l+6)-P-Galp-(l+4)-[P-Gal-(l+8)]-NeuGI-(2+3)-~-Galp-(l+4)-P-Glcp-(l+ 1)-Cer (Ref. 134)

In the first sialoglycolipid, the N-glycolylneuraminic acid is in the form of its 8-0-methyl derivative. The third, the most polar, sialoglycolipid, preponderant in the sialoglycolipid mixture of A. pectinifera, has another structural peculiarity: the N-glycolylneuraminic acid here is located at the branching point, and is glycosylated at 0-4by arabinosyl-galactose, and at 0 - 8 by a galactosyl residue. Such a position for the sialic acid has not thus far been found in carbohydrate chains of sialoglycoconjugates from other animals. The composition of the lipid moiety in all three of the sialoglycolipids is very similar, and resembles that of the ceramide moiety in neutral glycolipids isolated from the whole A. pectinifera starfish. Only a-hydroxy fatty acids whose major components are C22,C23, and C,, a-hydroxy acids were found there. The sphingosine bases are phytosphingosines having chain lengths of 16, 17, and 18 carbon atoms; the chains are linear, and branched, with the branched phytosphingosines accounting for >70% of the mixture of base^.'^^*'^^ From the hepatopancreas of P. pectinifera were isolated two sialoglycolipids having carbohydrate chains containing glucose, galactose, arabinose, and N-acetylneuraminic acid.I6’ The less-polar glycolipid is a monosialoglycolipid having a branched heptasaccharide chain, with a galactosyl residue as a branching point, and an arabinosyl residue as a single branching unit. Both arabinose residues are present in the furanose form, and the N-acetylneuraminic acid is situated inside the oligosaccharide chain and glycosylated at 0 - 4 by the galactosyl r e ~ i d u e . ” ~ * ~ ~ ’ * ~ ~ ~ Araf-(1+3)-a-Gal-(l+6)-[Araf-(1+3)]-~-Gal-(l+4)-NeuAc-(2+3)p-Gal-( 1 + 4)-p-Glc-( 1 + 1)-Cer

The more-polar sialoglycolipid is a disialoglycolipid having a linear octasaccharide chain. Both N-acetylneuraminic acid residues are situated inside the chain and glycosylated at 0-4by galactosyl residues.207 Araf-(I +3)-a-Gal-(l+4)-8-0-MeNeuAc-(2+3)-Gal-(1+3)-Gal-(1+4)-NeuAc-(2+3). P-Gal-( 1 + 4)-P-Glc-(1 + 1)-Cer

The N-acetylneuraminic acid residue situated closer to the nonreducing end of the chain is present in the form of its 8-0-methyl derivative. The lipid moiety of the sialoglycolipids from the hepatopancreas of P. pectinifera includes a-hydroxy fatty acids, among which, the C22,C23, and C2, acids account for >90% of the mixture, and compounds whose major

434

NICOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

components are c16,C17, and C I 8phytosphingosines having both linear and branched chains, with the latter prep~nderating.'~~*'~'*~~' Therefore, from the starfish P. ( A ) pecfinifera of the order of Spinulosa, unique sialoglycolipids not encountered in other species of animals have been isolated. At present, it is impossible to decide whether this type of structure is characteristic for the sialoglycolipids of starfish belonging to this order; for such a decision to be made, it will be necessary to investigate further the other starfish species of Spinulosa. Although, up to now, the sialoglycolipids of only four starfish species have been studied, and it is too early to reach definite conclusions, it may, however, be noted that the structures of their oligosaccharide chains are more complex than those of those in the sialoglycolipids from sea urchins. In contrast to the sialoglycolipids in sea urchins, those of starfish evidently have no common structural type characteristic of the whole class. It is possible that there is no common structural type inside one order either; this is seen from the example of sialoglycolipids from D. nipon and A. amurensis, belonging to the Forcipulata, and the similarity exists for smaller taxonomic groups, for example, subfamilies, or genera. More-extensive investigations of starfish sialoglycolipids will be necessary in order to clarify this point. c. The Class Holothurioidea.-The glycosphingolipids of this class have been poorly studied. The glycolipid composition was ~ h a r a c t e r i z e dfor ~~ three holothurian species, namely, the trepang Stichopus japonicus, and the sea cucumbers Cucumariajaponica and C.fraudatrix. As already mentioned, even closely related species can differ greatly in their glycolipid content; C.japonica contains 1.6% of monosaccharides in the lipid extracts, whereas C. fraudatrix has 8.5%. All of the species produce a complex, chromatographic pattern for glycolipids. Several glycolipids, of low, medium, and high polarity, are present; only C.japonica contains just one sialoglycolipid. In all of the species, the major monosaccharide is glucose; in S. japonicus and C. fraudatrix, there are also considerable proportions of rhamnose and xylose, and in C . japonica, of arabinose, but galactose, the usual monosaccharide for the majority of glycolipids of the other species, is absent.

8. Tunicata

Tunicates constitute one of the most amazing groups of marine animals, close to chordates, whose larvae stand higher, by a number of important features, than the adult forms. Together with the other chordates and echinoderms, tunicates belong to the Deuterostomia. With regard to glycolipids, this phylum has been poorly studied. The glycolipid composition was ~ h a r a c t e r i z e d for ~ ~ three species, namely,

GLYCOLIPIDS O F MARINE INVERTEBRATES

43 5

Halocynthia roretzi, H. aurantium, and Styela claua, that belong to the class Ascidiacea. The proportion of monosaccharides in the lipid extract from H. roretzi is rather high (3.6'/0), whereas, in the other two species, it is half as much. All of the species contain low-polarity glycolipids having the mobility of cerebrosides and their acylated derivatives, as well as low- and highpolarity glycolipids whose proportions differ in different species; H. aurantium contains more of the glycolipids of medium polarity and traces of polar ones; in contrast, in If. roretzi and S. claua, polar glycolipids are preponderant. In all of the species, the polar lipids do not contain any sialic acids. The major monosaccharide in all of these species is glucose; in addition to that, H. roretzi and S. c l a m were found to contain large proportions of galactose, and H. auruntium, of arabinose; in the hydrolyzates of these glycolipids are also present other orcinol-positive compounds that have not yet been ider~tified.'~ VI. BIOLOGICAL ROLEOF

THE

SIALOGLYCOLIPIDS OF ECHINODERMS

The biological role of the sialoglycolipids of echinoderms remains practically unstudied. There have been only a few communications on this problem, and they concerned the sialoglycolipids of sea urchins. As in the case of vertebrates, the gangliosides seem to be present at the outer leaflet of the membrane, and their carbohydrate chains are located on the menibrane surface. This has been shown for the sialoglycolipids of spermatozoa from four species of sea urchin, three of which belong to the subclass Regularia, and one, to the subclass Irreguluria; this was demonstrated by the use of antisera to the various sialoglycolipids of sea In the same study,325the topographic localization of sialoglycolipids on the cell surface was found to be different in different species of sea urchin. The eggs and developing embryos of S. intermedius were investigated with the help of immunofluorescent labelling,290 and sialoglycolipids were also shown to be located on the surface of the cells. Although the content of sialoglycolipids varies slightly during the early development of embryos,326 the content of more-complex sialoglycolipids increases.283The composition of sialoglycolipids in sea urchins is specific for species and organs, as shown for the eggs and the spermatozoa of four species of sea As in the case of the gangliosides in vertebrates, the sialoglycolipids in sea urchins are cell-surface antigens. A study of antigenic specificity for two disialoglycolipids (from the eggs of S. intermedius) whose structures differ in only one respect, that a sulfate group is present on the sialic acid (324) Y. Nagai and T. Ohsawa, Jpn. J. Exp. Med., 44 (1974) 451-464. (325) T. Ohsawa and Y. Nagai, Biochim. Biophys. A m , 389 (1975) 69-83. (326) M . Hoshi and Y. Nagai, Jpn. J. Exp. Med., 40 (1970) 361-365.

436

NICOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

residue in one of them, showed that they carry an individual, as well as a common, antigenic determinant.290The authors assumed that N-glycolylneuraminic acid plays an important role in the antigenic specificity of both sialoglycolipids, whereas the sulfate group determines the immunochemical difference between them.290 Similarly to the gangliosides of higher animals, the sialoglycolipids in sea urchins seem to take part in growth control. For the S. intermedius embryos, the exposure of sialoglycolipids at the cell surface has been shown to depend on the cell density in the incubation medium; in sparse embryos, they are much more exposed than in the dense ones.29oThis phenomenon seems to be similar to the fact, known for mammalian cells, that the synthesis of glycolipids depends on cell ~ o n t a c t . ' ~ , ' ~ The sialoglycolipids of sea urchins have a protective action against some cytotoxic compounds. Addition of certain sialoglycolipid fractions isolated from the fertilized eggs and embryos of S. intermedius was shown to protect the sea-urchin embryos from cytotoxic analogs of biogenic amines (for example, serotonin) and some detergents.327It has also been shown that the sensitivity of embryos to these cytotoxic preparations depends on the population density of the embryos; dense embryos are less sensitive than sparse ones. The authors assumed that this is caused by active substances, primarily sialoglycolipids, that are released into the incubating medium from the embryonal cells of dense population^.^^' VII. CONCLUSIONS

From the data presented herein, it may be seen that glycosphingolipids are widespread in marine invertebrates, although their proportions in the tissues of various animals can differ sharply. No correlation has been found to exist between the quantitative glycolipid content and the evolutionary level of the animal. Thus, members of the most-primitive phylum, the sponges, contain a relatively large proportion of glycolipids. Coelenterates and arthropods contain an approximately equal proportion of glycolipids (and the smallest among marine invertebrates), although, of the true multicellular animals, the former are the least organized, and the latter constitute the highest group of animals, of the branch of Protostornia. However, the presence of specific glycolipids, the sialoglycolipids, seems to be directly associated with the evolutionary position of the animal. Sialoglycolipids, found in all vertebrates, have also been found in echinoderms, which, together with vertebrates and the other chordates, (327) G . A. Buznikov, N. D. Zvezdina, N. V. Prokazova, B. N. Manukhin, and L. D. Bergelson, Expenenria, 31 (1975) 902-904.

GLYCOLIPIDS OF MARINE INVERTEBRATES

431

belong to the Deuterostomia, the most highly organized group of animals. Unfortunately, no data are as yet available on the occurrence of sialoglycolipids in some other phyla of chordates, such as hemi- and cephalochordates, but their presence may be anticipated on the basis of the fact that sialo-containing compounds have been found in the tissues of these animals.274The appearance of sialoglycolipids seems to be associated, not with the growing complexity of the nervous system of animals, as could be assumed from the data on the content of these compounds in different classes of vertebrates, but with the formation of one of the two principal stems of the evolutionary tree, that of the Deurerosromia.Tunicates are an exception; they do not contain any sialoglycolipids, although, by their phylogenic position, they are above the echinoderms. It is, however, possible that the absence of sialoglycolipids in tunicates is a result of a secondary process in the development of these animals that has led to their degradation. The finding of sialoglycolipids in echinoderms is a chemical confirmation of the phylogenetic relationship between echinoderms and vertebrates, previously established from biological data. The sialoglycolipids of echinoderms have the same fundamental, structural elements as gangliosides of vertebrates, as they contain an oligosaccharide chain and a sphingosine base N-acylated by fatty acids; they differ from the latter, however, in some essential structural features, mainly in their carbohydrate chains. Thus, the carbohydrate components of sialoglycolipids from sea urchins are glucose and sialic acid attached to 0-6 of glucose. In sea urchins was found, for the first time, a new type of sialoglycolipid, sulfated sialoglycolipids having a sulfate group on the sialic acid. From starfish have been isolated sialoglycolipids in which one (or two) sialic acid group is attached to a 2-acetamido-2-deoxygalactosylresidue, as well as arabinose-containing sialoglycolipids wherein the residues of sialic acids are located inside the carbohydrate chain. To date, the sialoglycolipids from starfish are the only source where 0-methylated sialic acids have been found. It is of interest that some unusual sialoglycolipids first isolated from the tissues of echinoderms were later also found in vertebrates. Trisialosyllactosylceramide, which is the major sialoglycolipid in the hepatopancreas of the starfish D. nipon, was subsequently found in and mammal^.^'^"'^ Sialoglycolipids containing sulfated sialic acid, found in the gonads of sea urchins, have now been detected in bovine gastric m u c o ~ a . ~ ~ ~ , ~ ~ Other glycolipids from aquatic invertebrates are also characterized by a great variety of structures. Along with compounds known also to be present in vertebrates (gluco- and galacto-cerebrosides and lactosylceramide), new glycosphingolipids have been detected that differ from the glycosphin-

438

NICOLAI K. KOCHETKOV AND GALINA P. SMIRNOVA

golipids of vertebrates as regards the composition of sugars, their location in the carbohydrate chain, and the presence of noncarbohydrate substituents on the monosaccharides ( 0-methyl, 2-amino- and 2-(methylamino)-ethylphosphonic, and (2-aminoethy1)phosphoric groups). In addition to the glycosphingolipids found in several invertebrate phyla (for example, cerebrosides), glycolipids that are characteristic of individual groups of animals have been found. Thus, 0-phosphonoglycosphingolipids were found in gastropods; mannose-containing glycosphingolipids, in freshwater bivalves; sialoglycolipids, in echinoderms; and sialoglycolipids containing glucose and sialic acid or its sulfated derivative, as well as sulfoquinovosylglycerides, only in sea urchins. Further study of the glycolipids from marine invertebrates, along with investigation of other classes of compounds, may well help in creating the foundation for a chemical taxonomy of these animals. The great variety of structures found in the glycosphingolipids of marine invertebrates can provide scientists with rich material for investigations aimed at ascertaining the relationship between the properties and functions of this class of compounds and their structure.

AUTHOR INDEX Numbers in parentheses are footnote reference numbers and indicate that an author’s work is referred to although his name is not cited in the text.

A Abe. A., 185 Abe, J., 225, 275, 322 Abe, K., 258 Abe, M., 228,267 Aberg, L., 207 Abrahamsson, S., 388, 395(6) Adair, W. L., Jr., 288, 351, 352 Adamyants, K. S., 426 Adrian, G. S . , 352 Afanasev, V. A., 25, 35(103),99, 140(65) Agawal, S. K. D., 24 Akai, H., 254 Akiyama, Y.,181, 255 Akopyan, S., Kh., 36 Akutsu, H., 422 Alam, S. S . , 345, 349(29),353(29) Albano, R. M., 252 Albersheini, P., 152, 182(27),183, 185, 186, 229,358,380, 382 Alblas, B. P., 87 Alexander, S., 239 Allen, A. K., 234, 372(204),373 Allen, C. F., 428 Allen, C. M., 352 Aloj, S. M., 389 Aloni, Y.,325, 326 Altwell, W. A,, 253 Amadao, R . , 166, 167(104), 185 Aniano, J., 237 Amano, K., 288, 328(l24) Amano, Y., 159 Amemura, A., 225, 226, 228, 275, 276, 300, 322 Aminoff, D., 324, 325(357) Anderson, H. A., 24 Anderson, J. S., 282, 286, 290, 328, 329(384), 331(91),342 Anderson, M . A., 262. 273. 274(479) Anderson, N. S . , 189 Anderson, R. L., 381 Anderson, W. J . , 298

Anderson-Prouty, A. J., 380 Andersson, B. A., 404 Ando, N., 430, 437(315) Ando, S., 389,393,394(97, 98), 405, 419, 420(96, 263), 421(96), 430, 437(313) Andrew, M. S.,342 Andrewartha, K. A., 163 Andrews, P., 375 Andrianov, V. M., 40, 41(152),42(152), 43(152),49(153),50(153),52 Angstrom, J., 394, 406(109),428(109) Ankel, E., 366 Ankel, H., 244, 288, 298, 366 Anno, K., 199 Anttinen, H., 244 Antoon, M. K., 7, 59(4), 60(6) Anwar, R. A., 305 Aoji, M., 257 Aon, M . A,, 256 Aoyagi, T., 389 Arai, K., 186, 187(144, 147, 149) Arai, Y.,256 Arakawa, H., 288 Araki, C., 186, 187(144, 146, 147) Araki, S., 398, 414(144, 145, 146), 415(144, 145, 146) Araki, T., 165 Araki, Y., 198, 285, 288, 328(124),355, 356(108) Arao, Y., 395 Archer, S.A,, 183, 185(132) Ard, J. S., 23, 76, 80(186) Ariga, T., 405 Armand, G., 202 Armstrong, E. L., 284 Arnold, W. N . , 382 Asakawa, M . , 237 Asano, N . , 154 Asankozhoev, S. A., 11, 17(39), 19(39),21(39), 25(39) Ascarelli, G., 84 Ashfnrd, D., 234

439

440

AUTHOR INDEX

Ashton, F. E., 298 Ashwell, G . , 246, 286, 292(95), 295(164), 295(95),379 Aspinall, G . O., 160, 165, 182, 247(126), 275, 358,375,377(219), 381 Atalla, R. H., 52, 82(157, 158),83(195, 196), 84(199) Atha, D. H., 213 Atkins, E. D. T., 61, 62(172), 63(174) Atkinson, P. H. 236,238 Audrieth, L. F., 92 Augustin, J., 91, 97 Austin, P. R., 374 Austrian, R., 281, 282(14), 287(14), 288, 289(14), 326 Avigad, G . , 234 Avram, M., 10 Avrova, N. F., 430, 437(312) Axelos, M., 353(91), 354,356(91), 359(91), 366(91) Axelrod, B., 149 Ayers, A. R., 380 Azuma, I., 247,300 Azuma, K., 148

B Baardseth, E., 191 Baba, T., 256 Babczinski, P., 362, 363(162), 370, 372(165, 190, 191),382(191) Bach, G . , 204 Back, D. M., 20 Backinowsky, L. V., 295 Backstrom, G., 213, 215 Bacon, J. S. D., 269,275(468) Baddiley, J . , 285, 299, 300, 327, 329 Baenziger, J., 232 Bailey, R. W., 359(148), 360 Baillie, J.. 247 Baker, C. W., 171 Baker, J. R., 202 Balazs, E.A., 204 Ball, D. H., 124,125 Ballou, C. E.,248, 304,305,362,365 Banas-Gruszka, Z., 427, 437(299,300) Banoub, J. H., 295 Bardalaye, P. C., 366 Barker, S. A., 8, lO(7-ll), 18(7-11), 19(7-ll), 20(8-lo), 21, 41(7, 8), 53

Barnes, H. A,, 166 Bamoud, F., 161 Barr, R. M., 346(40), 347,353(95), 354, 366 Barreto-Bergter. E., 267 Barrett, T. W., 81 Bartnicki-Garcia, S., 268, 358, 374 Barton, D. H. R., 123, 126(106) Basch, J. W., 13, 40(46) Baschang, G., 401 Basile, L. J., 8, 57(22) Bathgate, G. N., 253 Bauer, S., 336, 365 Bauer, W. D., 152, 182, 183, 229,379, 380, 382 Baumann, N., 431 Bause, E., 234,359(144), 360, 363 Baxter, B., 251 Bdolah, A., 289 Bearpark, T. M . , 204 Becker, E. J,, 122 Beetz, C. P., Jr., 84 Beevers, L., 383(86), 354,356(86), 363(86), 364(86), 369, 372(185, 187) Behr, J. P., 388(33),389 Behrens, N. H., 342, 343, 344, 345(22), 363(10,364(10) Beilharz, H., 218, 219(304), 220(304), 221(304), 222(304) Belcher, J., 214 Beldman, G., 149 Bell, R. J., 57 Bellamy, L. J., 10 Belocopitow, E., 352 Benedict, C. D., 283 Benitez, T., 266 Bennett, L. G . , 288 Bentley, F. F., 28 Benziman, M.. 325,326 Bergelson, L. D., 389,424, 427(289, 290), 435(290),436(290) Bergmann, C. W., 204 Berman, H. M., 14, 15(54) Bernheimer, H. P., 281, 282(14), 287(14), 288, 289(14), 326 Bernstein, H. J., 9, 29, 87(33) Bernstein, R. L., 281, 305 Bettinger, C. E., 330 Bevill, R. D., 291 Beyaert, G . O., 292, 302(167) Beyer, T. A., 202,244(223), 246(223) Beytia, E. D., 350

AUTHOR INDEX Bhagwat, A . , 380 Bhattacharjee, A. K., 324,431 Bhattacharjee, S. S., 151 Bhavanandan, V. P., 240 Bhoyroo, V. D., 232 Bhuvaneswari, T. V., 380 Biely, P., 160, 162, 163(62) Biemann, K. 405 Bilisics, L., 162 Billeter, 0.. 94 Binkley, S. B., 324 Bionchik, M. A., 40 Birnbaum, G. I., 9, 29, 87(33) Birth, G. S . , 23 Bishop, C. T., 159,179,288 Bixby, J. G., 384 Bjorkman, L. R., 393,429(94) Bjorndal, H . , 158, 163(51),269, 275(470), 276(470),304 Blacklow, S., 283 Blackwell, J.. 9, 26, 33(29,30), 34, 40(28, 29, 30, 143), 41(143), 42(143), 43(143), 44(143),45(143), 46(29, 143), 47(29), 50(29), 51(29,30), 54,55(30),61, 62(172), 75(28, 143), 80(28, 143, 163).82(30, 164), 88(30,164) Blackwood, R. K . , 92 Blake, C. C. F.,196 Blanken, W. M., 244,246 Blatt, D., 390, 391(59) Blumsom, N., 300 Bobrovnik, L. D., 24 Bodini, P. A., 390, 391(60),415(60) Boer, P. 348(55),349, 353(55,88), 354, 356(88) Boerio, F. J., 33,34, 41 Bogacka, J., 100, 106(77, 78), 109(77),110(78), 112(77,78), 122(78) Bogdanovskaya, T. A , , 430 BognBr, R., 99, 100,122 Bohlool, B. B., 379 Bohm, S., 30, 84(136) Boigegrain, R. A., 124,132, 144(l26) Bolognani, L, 390, 391(60),415(60) Bolognani Fantin, A. M . , 390,391(60), 415(60) Bolton, U. H., 124(140), 125,138(140), 143(140) Bondietti, E., 24 Bonner, 0. D., 86,87(213) Bonnet, F.,216

441

Borisova, V. B., 36 Borkowski, B., 99, 106(77, 78). 109(77), 110(78),112(77, 78), 122(78) Borovsky, D., 256 Bose, J. L., 21, 25(83) Bosso, C . , 161 Bouhours, J.-E., 428 Bouquelet, S., 238 Bourne, E. J., 8, lO(7-9, ll), 18(7-9, II), 19(7-9, ll),20(8, 9), 21, 41(7, 8). 53 Bouveng, H . O., 179 Bowles, D. J., 353(83), 354 Boyd, J., 192 Boyer, C. D., 256 Bradley, C. A., 39 Brady, R. 0.. 388, 389(10),390(10) Branefors-Helander. P., 302 Brant, D. A., 167 Bray, D., 310, 315(278) Brazhnik, L. J., 87 Breckenridge, W. C., 388(29), 389 Breimer, M. E., 389, 394, 405, 406(109), 428(109) Brekle, A , , 209, 216(272) Bremer, E. G . , 394 Bresadola, S . , 92 Brett, C. T., 343, 344, 345(20, 23). 348(23, 3% 349(23), 350(30), 3530% 356(30), 358, 359(137, 139), 360, 361, 367(137), 377(30) Bretthauer, R. K., 353(92), 354, 366(92) Brigden, C. J.. 230 Briggs, D., 23 Brill, W. J., 379, 380(244, 245) Brillinger, G . U., 291 Brillouet, J . - M . , 163 Brine, C. J., 374 Bringmann, G., 123, 126(106) Brisson, J. R.,245 Brittain, E. F. H.,L2,36(40) Brockman, R., 23 Brooks, D., 285 Brooks, W. V. B., 13, 34(45), 40(45) Browder, S. K., 369, 372(187) Brown, D. H . , 374 Brown, G. M., 14, 15(51, 59) Brown, G. N., 384 Brown, J. G . , 291 Brown, R. D., 151, 180(20) Brubaker, R. R.,297, 298(201), 301

442

AUTHORINDEX

Brumfitt, W., 197 Brunkhorst, W., 113, 114(88) Bruns, D., 381 Bruvier, C., 402 Bucke, C., 191, 297, 325(205) Buddecke, E . , 200, 203, 204(217) Budovskii, E. J., 124, 126(119),135(119), 144(119) Bugge, B., 354 Bukzinskaya, A. G., 389 Bundle, D . R., 9, 29, 87(33),288 Bunow, M. R., 84 Burgos, J., 351 Burneau, A., 86 Burnet, F., 379 Burns, D. M., 234 Burton, B. A,, 167 Burton, W. A,, 352 Busch, C., 212 Buscher, H.-P., 400 Buslov, D. K., 87 Butler, N. A., 256 Butters, T. D . , 356 Butterworth, P. H. W., 346(43),347, 351 Buznikov, G. A., 424, 427(290),435(290), 436(290)

C Cabassi, F., 30 Cabezas, J. A , , 149 Cabib, E., 374 Cael, J. J.. 9, 33(29, 30). 34, 40(29, 30, 143), 41(143),42(143),43(143),44(143),45(143), 46(29, 143),47(29), 50(29),51(29, 30), 55(30), 61, 62(172), 75(143), 80(143), 82(30, 164), 88(30, 164) Cairncross, I. M., 160 Caldow, G. L., 93, 139(14) Calvo, P., 149 Camarasa, M. J., 95 Cantrell, M. A., 380 Capella, P., 399 Carceller, M . , 360, 383(153) Cardini, C. E., 360, 383(153) Carey, P. R., 84 Carlos, D. J., 288 Carlson, D . M., 237 Carlstedt, I., 217

Carminatti, H., 344, 352 Carolan, G., 257 Carpenter, R. C., 275 Carter, H. E., 393, 398, 420 Carver, J. P., 245 Cary, L. W., 407 Casals-Stenzel, I., 400 Castle, J. E., 374 Castro, B., 124, 129, 130(124),143(l23), 145(123) Casu, B., 28,30, 55, 213 Catley, B. J., 257 Catley, R. W., 359(147),360, 367(149) Ceccarini, C., 238 Ceccon, A., 94 Cech, D., 99, 105(62),112(62),140(62) Cert Ventula, A., 99, 105(62),112(62),140(62) Cestaro, B., 388(31),389 Chadwick, C. M., 348(58),349 Chalk, R . C., 30 Chang, N . , 389 Chapleur, Y., 124, 129(123, 124), 130(l24), 143(123), 145(123) Charbonniere, R . , 253 Chargaff, E . , 399 Charon, D., 296 Chatelain, P . , 389 Chatterjee, A. K . , 281 Chatterjee, A. N., 330 Cheetham, N. W. H., 168,259 Chekareva, N. V., 397, 399(141), 403(140), 425(140),426(140, 141, 293), 427, 430 Chen, S.-C., 274, 275(482) Chen, W. W., 234 ChBnB, L., 325 Cheng, C. C., 91 Cherniak, R., 297 Chiba, S., 149 Chien, J. L., 208, 241(264) Child, J. J., 267 Childs, R. A., 388 Chinchetru, M . A., 149 Chipman, D. M., 195, 196 Chittenden, G . J. T., 299 Chiu, T.-H., 284, 330 Chizhov, 0. S . , 403, 430(192) Choay, J., 213 Chojnacki, T., 281, 284, 346(43), 347 Choppin, G. R . , 85 Choppin, P. W., 387

443

AUTHOR INDEX Chopra, R. K., 217 Chrispeels, M.J., 383 Christensen, J. E., 124, 132, 135(130), 143(129,130) Christison, J., 189 Christner, J. E., 216 Chu, S. C. C., 14. 15(55,58), 40(55) Chu, F. K, 239 Cifonelli, J. A., 55, 323 Clark, A. F., 349 Clark, A. H., 168, 169(113),170(113),176(113), 178(113) Clarke, A. E., 381 Clarke, J., 372(197), 373 Claus, D., 301 Clements, P. R., 214 Clermont-Beaugiraud, S . , 165 Coffey, J. W., 382 Cohen, R. E., 248,304,326 Coleman, M. M., 9, 12(23),13(23), 32(23), 34(23),35(23), 37(23), 38(23) Coleman, W. G., 301 Colthup, N. B., 10 Colucci, A. V., 286, 331(92) Colvin, J. R., 325, 358, 359(132),360 Combes, D., 88 Compte, J., 353(96), 354, 366(96) Comtat, J., 161 Conrad, H. E., 199, 200, 315, 320 Consiglio, E., 389 Conway, E., 190 Cook, A. F., 124, 126, 143(115) Cooke, D., 167, 170(106) Cooley, J. W., 7 Cooper, D., 325 Corfield, A. P., 233 Costello, C. E., 405 Coster, L., 205, 206, 207, 208(251), 217 CBte, G. L., 259 Couchman, J. R., 216 Coupewhite, F., 297, 325(204) Courtois, J. E., 166, 167(103) Couso, R. 0..305, 322(265), 323, 376, 377(229-231) Cowtnan, M. K., 204 Cox, W. G., 93 Creekmore, R., 390, 391(59) Creeth, J. M., 372(204),373 Creitz, E. C., 8, 18(12),19(l2) Cripps, R. E., 157

Critchley, R. D., 387, 388, 390(20) Cross, P. C., 9, 32(27) Currie, A. J., 194 Curtine, J. A,, 256 Curtis, C. A. M., 285, 327(90) Cynkin, M. A., 285, 311(83),315(83),317(83)

D Dabrowski, J., 394, 406, 407(110, 221), 427 Dabrowski, U., 394, 406, 407(110) Dahl, J. B., 380 Dahlen, B., 388, 395(6) Daleo, G. R., 345, 348(28, 53). 350(27, 28), 351(28, 53), 353(82, 98), 354, 355(82), 356(82),359(82, 98). 361(82),362(156), 367(98) Dalessandro, G., 359(149, 150), 360 Dall, G. G . , 23 Daly, L. H., 10 Damewood, P. A , , 256 Damie, S. P., 205, 207(251) Daniel, A., 292 Daniewski, W. D., 346(43), 347 Danilov, L. L., 284, 314, 317, 335, 336(292, 439). 337(296, 439, 448), 338(439) Dankert, M., 285, 305, 310, 313(81), 315(278), 322(265),323(77),325(77),342, 344, 345(23),348(23),349(23), 353(81), 354, 355, 361(31),376, 377(229-231) Daoust, V., 288, 298 Darke, A . , 166 Darvill, A. G . , 152, 182(27),183, 229, 358 Darvill, J. 229 Dashevskii, V. G . , 40, 41(152), 42(152), 43(152),49(153), 50(153),52 Datema, R., 353(100), 354, 367 Daves, G. D., 91 Davidson, E. A , , 240 Davidson, I. W., 193 Davies. H. M . , 368, 369,372(183) Davis, H. B., 18, 30(76) Davis, H . F., 428 Dawson, G . , 404 Dayhoff, M. O., 389 Dazzo, F. B., 379, 380(244, 245, 247) De, K. K., 99, 102(49), 141(49),142(49) Dea, I. C. M., 164, 164(75),166(75),167, 168, 169(113),170(106, 113), 176(113),178(113)

444

AUTHORINDEX

De Caleya, R . , 382 Decius, J. C., 9, 32(27) Deck, J. C., 29 Decker, G. L., 352 Decker, R. F. H., 326 Dedonder, R. A., 326 DeDuve, C., 382 Defaye, J., 124(141),125, 138, 145(141) deFlores, E. A., 187 Dekker, R. F. H., 147, 159(4),160, 247(4) Delaney, S. R., 200 de las Heras, F. G . , 95 Deleers, M., 389 Dell, A., 218, 219(302), 229, 230 Delmer, D. P., 152, 182(27),326, 345, 348(33), 350(33), 351(33), 353(87),354, 358, 360, 368, 369, 372(87, 181, 183), 382, 385(154) deMatus, M. C., 366 Dennis, W. E., 124, 132, 144(127) denUijl, C. H., 185 DerKosch, J., 27 Derrien, M . , 331 Desai, N. N., 234, 372(204),373 De Simone, F., 422 D’Esposito, L., 7, 59(4), 60(6) devries, J. A., 183, 184(133), 185(133) De Wolf, M. J. S., 389 Dey, P. M. 164, 165(76),382 Dickerson, J. P. 124, 132, 144(128) Dickinson, H. G . , 381 Diena, B. B., 298 Dietrich, C. P., 200, 205, 210(219a), 211, 214(219a) Dietzler, D. N. 293 Dietrich, C. P., 286, 331(92) DiFabio, J. L., 218 DiCiloranio, A , , 300 DiCiloramo, M., 300 Dimick, 8. E., 82, 83(195) Dinh, N. D., 80, 81 Dini, A , , 422 Distler, J., 291, 293(162), 326 Dmitriev, 289, 290, 292, 295, 297, 301 Dmochowski, A., 199 Dod. B. J., 387 Doi, A , , 269 Doi, K., 269 Doke, N., 380 Diirfel, H.. 191 Dorfman, A., 202, 203, 205, 323, 324

Dorland, L., 283 Dorman, D. E., 21 Dorn, H., 99, 105,140(56-59) Doss, S. H., 20, 25(79) Douglas, L. J., 285 Drews, G., 290 Dreyfus, H., 389, 390 Drobnica, L., 91,97 Druzhinina, T. N., 313, 314, 315(286), 316(286. 289), 317(286, 289), 318, 335, 336(322, 439), 337(322, 439, 448), 338(322, 439). 339(322) Duckworth, M . , 186, 187(145),188(145), 190(152),329 Duncan, I., 383 Dunham, D. G . , 216 Dunn, A., 389 Dunphy, P. J., 345, 346(32),348(32) Duran, A., 374 Dutton, G. G. S., 218, 219(302), 223(301, 305b). 224(305b) Dzierzyliska, J . , 99, 106(64),112(64),140(64)

E Ebel, J., 380 Eberhard, T., 204 Ebisu, S., 263 Eda, S., 153, 181 Edge, A. S. B., 206 Egge, H., 394, 404, 406, 407(110, 221) Eggerton, F. V., 46 Egli, H., 393 Eidels, L., 300, 301 Elbein, A. D., 147, 179,235, 281, 284, 291, 295(161), 329(67),343, 348(57),349, 350(16),353(57, 80, 85), 354, 355, 356(16, 85, 112, 113, 115), 357(113, 115). 358, 359(133, 141),360, 361(57, 80). 367(16),377(16) Elder, J. H., 239 Eliseeva, G. I., 284, 336 Elkin, Yu. N., 298 Elliot, H., 291 Elsasser-Beile, U., 218, 219(303), 220(303), 223(303) Eking, J. J., 234 Elwing, H., 389 Emdur, L. L., 330 Emi, S., 184

AUTHORINDEX Emmelot, P., 395 Endo, M., 200 Endo, Y.,240 Englar, J. R., 186 English, P. D., 183 Erickson, J . S . , 408 Ericson, M. C., 345, 348(33),350(33), 351(33),353(87),354, 358, 368, 372(87, 181) Eriksson, K.-E., 158, 163(51) Esaki, H., 267 Eskamani, A,, 22, 23(85) Eto, Y . , 388 Evans, B. B., 346(45),347 Evans, J. E., 394, 399 Evans, M. E., 30 Evans, R. M., 253 Eveleigh, D. E., 248 Eustigneeva, R. P., 317 Eylar, E. H., 382

F Fabian, H., 30, 84(136) Fackre, D. S., 217 Faillard, H., 431 Falk, K.-E., 394, 406(109),428(109) Falk, M . , 86 Faltynek, C. R., 199 Fan, D.-F., 290,298 Fan, D. P., 332,333 Farkas, V., 365,374 Farmer, V. C., 269, 275(468) Fartaczek, F., 348(59),349, 353(59),356(59) Fava, A , , 92, 94 Feeney, J., 345, 346(32),348(32) Feingold, D. S . , 215, 289, 290, 298 Feizi, T., 208, 241(264b),388 Fernandez-Resa, P . , 95 Fernie, B. F., 389 Ferrari, T. E., 381 Ferraro, J. R., 8, 57(22) Ferreira, T. M. P. C., 200, 210(219a), 214(219a) Ferrier, W. G., 14, 15(53) Ferrier, R. J . , 93, 95, 127, 142(20,21), 144(20. 21) Fialeyre, M.,29 Fielding, A. H., 183, 185(132) Filer, D., 282, 287(44)

445

Filippov, M. P., 23 Finamore, E., 422 Fincher, G . B., 274,275(484a) Fischer, E., 92, 94, 95(30),114(30),123, 141(30),142(7),143(7),144(7, 30) Fischer, F. G., 191 Fischer, H. D., 379 Fishman, P. H., 388, 389(10),390(10) Fitzgerald, G . L., 212 Fleet, G . H., 269, 270(471, 472), 272, 275(478),276(478) Flemming, H. C., 319 Fleury, G., 34, 75(145) Floss, H. G . , 291 Flowers, H. M., 147, 392 Fobes, W. S . , 299 Folch, J., 344, 393 Fomina-Ageeva, E. V., 389 Fong, J. W., 404 Foote, M . , 383 Ford, L. 0..196 Forsee, W. T., 214, 235, 348(57),349, 353(57, 80, 85), 354, 355, 356(85, 112), 358, 359(133),361(57, 80) Fournet, B., 402 Fowler, S. D., 428 Francotte, C., 84 Fransson, L . - k , 198, 204, 205, 206, 207, 210, 215,217 Franz, G . , 358. 359(134, 146, 360), 364(134) Franzen, J. S . , 289 Fraser, B. A,, 325 Fraser, A. R . , 24 Fraser-Reid, B . , 31 Frazier, W., 379 Fredman, P., 389, 390, 391(63),392, 393 Freeman, L. E., 185 French, D., 253, 256 Frerman, F., 285,320(84) Frey, P. A , , 282 Freyfogel, T. A , , 390, 391(56) Freysz, L., 389 Friebolin, H., 297 Friedenson, B., 372(196), 373, 382(196) Friedrick, J. F., 383 Fries, D. C., 14, 15(57) Friese, R., 23 Fromme, J., 290, 298, 304, 315 Frush, H. L., 8, 18(l2),19(12) Frydman, R. B., 383

446

AUTHORINDEX

Fuchs-Cleveland, E., 331 Fuentes Mota, J . , 99, 105(62),112(62), 140(62) Fujibayashi, S., 192 Fujino, Y.,413 Fukaya, N., 258,260(429) Fukuda, M. N., 241, 408, 409(242) Fukui, T., 269 Fukumoto, J., 162 Fukushima, J., 257 Fukuyama, J., 269 Fulton, W. S., 62, 63(174) Fumagalli, R., 399 Furlong, C. E., 281 Furuhashi, T.,199 Furuhata, I., 99, 105(60),106(60), 112(60), 140(60) Furuichi, N., 380 G Gabriel, O., 280, 286, 288, 291, 294, 296(189, 190) Gade, W., 380 Gdord, J. T., 350 Gahan, L. C., 320 Galat, A., 63, 83(176) Galbraith, L., 298 Galicki, N. I., 427, 437(299) Galli, C., 399 Gallo, G. G., 28 Gander, J. E., 278,299 Garancis, J. C., 366 Garas, N. A., 380 Garcia, R. C., 285, 322, 323(77), 325(77), 345,355, 361(31),376 Garcia-Lopez, M. T., 95 Gardas, A., 208,241(264) Gardner, H. L., 332 Gardner, K. H., 9, 33(29),40(29), 46(29), 47(29),50(29),51(29) Garegg, P. J.. 14, 15(56), 158, 163(51),323 Gasa, S., 407,428 Gaugler, R. W., 294, 296(190) Gaunt, M. A., 288 Gaver, R. C., 393,398 Geiger, B., 204 Geis, A., 332 Gemeiner, P.,97 12,36(40) George, W. 0.. Gero, S. D., 124,126, 135(ll8),144(ll8) Ghai, S. K.,228, 300 Ghalambor, M. A., 283,320

Ghidoni, R . , 388(31), 389, 431 Ghuysen, J.-M., 195, 196(182),197(182, 189) Giangiacomo, R., 23 Gibbs, C. I., 124,134, 143(133) Gielen, W., 390 Gietl, C., 381 Gilbert, J. M., 285, 291, 311(83),315(83), 317(83), 331 Giles, H. A., 346(46),347 Gill, D., 84 Gill, R. E., 375 Gillette, P. C., 60 Gilson, T. R., 9, lO(24) Gilvard, C . , 331 Ginsburg, V., 281, 282, 291, 295(40, 159) Glaser, J. H., 200 Glaser, L. 281, 286, 287, 291, 294, 325, 358, 359(131), 374, 379 Glasgow, L. R., 233, 378 Gleeson, P. A., 244, 381 Glick, M. C., 387 Glickman, R. M., 428 Gloor, U., 346(47),347 Glukhoded, I. S., 284, 396,397, 399(138), 423(138), 424(138), 425(291), 430(137), 431(137),432(137) Gmernicka-Haftek, C . , 99(71), 100, 106(63, 64,66, 67, 69, 71, 77, 78, 79), 107(67,69, 71), 108(71),109(77, 79), 110(78),112(63, 64,66, 67, 69, 71, 77-79), 122(78), 140(63, 64, 66, 67,69) Gogilashvili, L. M., 313, 314, 315(286), 316(286),317(286), 318, 335, 336(322, 439), 337(322, 439, 448), 338(322, 439), 339(322) Gold, M. H., 353(97), 354, 362(97),365(97) Goldeniann, G., 319 Goldman, D. S., 284 Goldman, R.-C., 315 Goldschmid, H. R., 159,160(55) Goldstein, I. J., 233, 258, 259(428), 260(428), 372(200),373 Gombos, G., 388(29), 389 Gonzalez, J. J.. 166, 168(99) Gonzalez Noriega, A., 379 Gonzalez-Porque, P., 292, 295(170, 171) Good, P., 428 Gooday, G . W., 374 Goodman, I., 97 Goodman, L., 124 (142, 143). 125, 132, 135(130, I S ) , 139(142), 143 (129, 130, 135, 140),144(135)

AUTHORINDEX Gordon, A. H., 269 Gorin, P. A. J.. 191, 248, 267, 401 Gorshkova, R. P., 298, 299 Got, R., 353(96), 354, 366(96) Gotschlich, E. C., 325 Gough, D. P., 350 Goulden, J. D. S., 18, 22, 23 Goustin, A. S., 329 Graham, J . M., 387 Graham, T. L., 380 Grange, D. K., 352 Grant, A. C., 212 Grasdalen, H., 168, 191, 193, 195(171) Grasmuk, H., 283 Grass, F., 27 Gray, G. M., 387 Grebner, E. E., 214 Green, J. R., 348(60), 349, 353(60), 354(60), 372(207), 373 Greenberg, E., 282 Gregory, J. D., 202, 205, 207(251) G r e i h g , H., 204, 208, 209, 216(269) Grellert, E., 304 Gremli, H . , 163, 185(71) Grewal, K. K., 254 G r i h v , L. A., 35, 36(146) Grifiths, P. R., 8, 57(21) Grinna, L. S., 234 Griph, I., 394, 406(109),428(109) Grisebach, H., 294, 299 Groleau, D., 190 Grollman, E. F . , 389 Gross, B., 75, 124, 129 (123, 124), 130(124), 143(123, 125). 144(125, 126), 145(123, 125) Gross, S. K., 394 Guilbot, A , , 253 Gumien, D., 100, 106(79),109(79),112(79) Gundlach, M. W., 199 Gunetileke, K. G., 305 Guthrie, R. D., 93, 95, 124,126, 128, 134, 135(118,131, 132), 142(35, 131).144(22, 23, 118) Guy, R. G., 91 Gyorgydeak, Z., 99, 100(46),122(46)

H Haas, C. M., 13, 34(45), 40(45) Habets-Willems, C., 353(88),354, 356(88) 148, 199(8) Habuchi, 0.. Hachisuka, Y . , 267

447

Haddock, J. W., 299 Hadjiioannou, S., 204 Haefpap, L., 388 Hahn, H. J . , 353(97), 354, 362(97), 365(97) Haider, K . , 24 Haines, T. H., 426 Hakimi, J., 236 Hakomori, S., 241, 388, 390 (11-14, 18, 23). 392, 393(82), 394, 399, 401(164), 402, 408, 409(242), 420, 436(12, 13) Halbeek, H., 402 Hall, C. W., 214, 281 Hall, D. O., 87 Hall, L. D., 171 Hall, R. S., 255 Hal1i.n. A., 215 Harnada, A,, 98 Harnada, N . , 268 Hamamoto, Y., 406 Hamanaka, S., 389 Hammerling, G., 301 Hamnies, W. P., 332 Hancock, I. C., 284, 285 Handa, S., 389,394, 405 Handa, T . , 84 Hanessian, S., 91, 291 Hanfland, P., 208, 241(264b), 393, 394, 402, 406, 407(110, 221) Hannon, M. J., 33 Hannus, K . , 346(44), 347 Hanover, J. A,, 362 Hansen, U., 284,285(73) Hansson, G. C., 389, 394, 405, 406(109), 428(109) Hara, A,, 428 Hara, C., 267 Harada, T., 225, 226, 228,253, 254, 275, 300,322 Hardegger, E., 124, 126, 135(121),144(l21) Hardingham, T. E., 198, 216(211) Hare, M. D., 258, 259(423), 260(432), 262(431),264(432) Haring, K. M., 113, 114(87) Harmon, R. E., 99, 102, 141(49), 142(49) Harris, P. L., 407 Hart, D. A,, 185 Hart, J . W., 381 Harth, S., 389 Hartmann, K. A,, 84 Hascall, V. C., 200, 208, 209, 216(271) Hase, S . , 328 Hashinioto, T., 416

448

AUTHOR INDEX

Hashimoto, Y . , 147, 247, 422 Hasilik, A., 370, 372(192),377(192),379 Haskell, T. H., 290 Haskin, M. A , , 286, 331(91),342 Haskins, R. H., 267 Hassell, J. R., 209, 216(271) Hassid, W. Z., 147, 191, 326, 341, 358, 359(130, 148), 360 Hatakeyama, H., 25 Hathaway, R., 390, 423(64) Haug, A., 191, 193, 194(179),297 Haugen, T., 281 Haverkamp, J., 283, 402 Havlicek, J., 161 Havsmark, B., 206, 210 Hawthorne, J . N . , 420 Hay, A. E., 291 Hayashi, A., 396, 398(131),401, 408, 412(240), 413(131, 142, 143, 240), 414(131, 143, 143a), 415(125, 130). 416(129, 247) Hayashi, K., 388(34), 389 Hayashi, T., 154,155, 156(34),172(34) Hayward, J., 200 Hazama, S., 288, 328(124) Heath, E. C., 283, 285, 291, 295(161), 320(84) Hedges, A , , 198 Heimbach, C. J., 82, 83(196) Heinegard, D., 216 Heise, G. L., 99, 102 Heldin, C.-H., 212 Helferich, B., 94, 95(30), 114(30). 123, 141(30),144(30) Heller, D., 393 Heller, J. S., 179, 359(143, 145), 360 Hellerquist, C.-G., 305, 408 Helsper, J. P. F. G., 359(140),360, 367(140) Helting, T., 202, 211, 214(281) Hemmer, P. C., 166, 168(99) Hemming, F. W., 284, 342, 345, 346(34, 36, 37, 39-42, 48), 347, 348(55),349(29), 350, 351,353(29, 55, 95). 354,355,366 Hendra, P. J., 9, lO(24) Hepburn, A., 24 Herscovics, A , , 234, 354 Herzberg, G . , 10 Hettkamp, H., 234 Heydanek, M. G., 332 Heyde, M.E., 84 Heyns, K., 96, 142(36),290 Hiamatsu, M.,228 Hibbig, R., 390

Hickman, J., 286, 292(95), 293(164),295(95) Higa, H., 233 Higashi, S., 228, 417 Higashi, Y., 286 Higuchi, M., 257 Higuchi, T., 313, 331(93).342 Hildesheim, J . , 124(141),125, 138, 145(141) Hill, J., 124, 125(113, 114). 126(113, 122). 127(113),128(113,122), 144(122) Hill, R. L., 202, 244(223),246(223), 378 Himatsu, M., 322 Himmelbach, D. S., 183 Hineno, M., 22, 28, 40, 41(154),42(154), 43(154) Hinrnan, M . B., 359(142, 151),360 Hirabayashi, Y., 237 Hiraiwa, S., 257 Hirano, S . , 198, 208, 216(267, 268) Hirase, S., 186, 187(147) Hironii, K., 149 Hirota, Y., 333 Hirsch, T. M., 333 Hirschfeld, T. B., 59 Hint, E. L., 165, 375 Hisada, K., 258, 260(429) Hisamatsu, M . , 225, 300 Hisatsune, K., 196 Hitomi, J., 237 Hiura, N., 271 Hizukuri, S . , 252, 253, 255 Ho, M. W., 408 Hoffman, J., 166, 168(98, loo), 171(100),297, 298(201),301 Hoffman, P., 204, 208 Hofman, I. L., 289 Hofniann, A. W., 122 Hofstad, T., 298, 301 Hogness, D. S., 287 Hohlweg, R . , 96, 142(36) Hohnson, J , A , , 23 Hollenberg, J. L., 87 Holm, M., 399 Holme, T., 323 Holmgren, J., 389 Hong, K. C., 188,190 Honneger, C., 390,391(56) Honig, H., 29 Honma, T., 96, 113(37),129, 130(37),131(37), 141(37),143(37, 140). 144(37),145(37) Honykaas, P., 229 Hooghwinkel, G. J. M., 244

AUTHOR INDEX Hook, M., 207,212,215, 216 Hopp, H. E., 345, 348(53), 349, 350(27), 351(53),353(82, 89). 354, 355(82), 356(82, 89, 121). 359(82), 361(82), 362(156),370, 371(89),372(89, 121) Hopwood, J. J . , 214 Horecker, B. L., 231, 313, 315(290),342 Hori, H. , 355, 356(113, 115). 357(113, 115) Hori, T., 393, 396, 398, 399, 408(126), 415(127), 416(126), 417(147, 159, 241), 419, 420(96, 241, 261, 263, 264). 421(96), 432(93) Hornig, D. F., 86 Hornling, N. J . , 85 Horowitz, M. I., 198, 297, 401, 423 Horst, M. N., 375 Horton, D., 30, 7.5 Hoseney, R. C., 253 Hoshi, M., 397, 424(139),428, 435(306) Hough, L., 124(140),l25(114),126(113,122), 127(113),128(113),134, 138(140). 143(113, 122, 133). 144(122),375 Hovingh, P., 210, 211(274), 212, 214(275) Howard D. J., 378 Hoyle, F., 31 Hrabak, E. M . , 379, 380(247) Huang, C. C., 232 Hubbard, S. C., 234, 235, 362, 363(161), 364(161) Huber, D. J . , 274, 275 Hughes, R. C., 327, 356, 388, 390(11) Hui, P. A , , 166 Hull, D. M. G., 124, 135(137),145(137) Hunt, L. A., 232, 238(327) Hunt, L. T., 389 Hunter, G. D., 389 Hurlbert, R. E., 298 Hussey, H., 285 Huvenne, J. P., 34, 75(145) I Ichihara, N., 290, 327 Ichimi, Y . , 258, 260(429) Idoyag-Vargas,V., 352 Ielpi, L., 305, 322,(265), 323, 376, 397(229231) Iffland, D. C., 92 Igarashi, K., 96, 113(37),129, 130(37),131(37), 141(37),143(37),144(37),145(37) Ignatova, L. A , , 99, 102(51-53)

449

Igrashi, T., 296 Ikegami, S . , 422 Iki, K., 155, 156(37),275(400) Iliceto, A , , 92, 94 Imae, Y., 287 Imai, K., 264 Itnber, M. J., 233 Imoto, T., 196 Inoue, S . , 432 Inoue, Y., 232, 233(323), 237(323), 238(323) Iochihara, N., 288, 290(115) Irvine, R. W., 95, 142(35) Irwin, W. E., 353(92), 354, 366(92) Isaac, D. H., 61, 62(172) Isakov, V. V., 298, 299 Ishell, H. S., 8, 18(12, 13), 19(12, 13),21(13), 25(15),65(15) Ishaque, A,, 281 Ishihara, H., 216, 239 Ishihara, M . , 179 Ishii, A., 389 Ishii, S.,182, 183 Ishimoto, N., 288, 290(115), 324, 327 Ishino, F., 333 Ishizu, A,, 181 Ishizuka, I., 401, 420, 426, 430, 437(314) Isler, 0.. 346(47),347 Isobe, M . , 393, 394(97) Isono, Y., 390, 391(65),423(65), 428(65, 283), 435(283) Itasaka, O . , 393, 396, 398, 399, 408(126), 416(126),417(147, 159, 241, 254-256), 419, 420(96, 241, 261, 261, 264), 421(96) Ito, E., 198, 285, 288, 290(115), 328(124),355, 356(108) Ito, J., 239 Ito, M . , 208 Ito, S., 155, 156(40),232, 237(324),275(406) Ito, T., 301 Itoh, T., 256 Ivatt, R. J . , 234 Iwaki, K . , 99, 105(60),106(60),112(60), 140(60) Iwama, M . , 419 Iwamori, M., 389, 392, 393, 396, 415(127) Iwasaki, M . , 432 Iwasaki, T., 159, 179 Iwasaki, Y.,420

450

AUTHORINDEX

Iwashita, S., 232, 233(323), 237(323), 238(323) Izaki, K., 331

J Jack, M. A , , 380 Jackson, S . E . , 86 Jacobsson, I . , 215 Jacqmain, D., 84 Jakobsen, R. J., 13, 40(46) James, D. W., 147, 355, 356015). 357(115) James S. R., 91 Janczura, E . , 281 Jane, J.-L., 252 Jankowski, W., 281,284 Jann, B., 230, 292, 295, 302067). 307(276), 308,318 Jann, K., 230, 285, 286, 292, 295, 302(167), 307(276), 308, 318, 319(80) Jansson, P.-E., 156, 218, 219(302), 226, 230, 288,321, 322, 376, 402 Jansze, M., 217, 219(300a) Jantzen, E., 150, 266(19) Jasse, B. 27 Jastalska, D., 99,106(64), 112(64),140(64) Jayne, J.. 88 Jeanloz, R. W., 197, 198, 314,354 Jeffrey, G . A., 13, 14, 15(55. 58), 16(62), 40(55), 88 Jeffries, T. W., 269 Jennings, H. J . , 288, 293, 324, 431 Jensen, J. W., 215 Jermyn, M. A., 380 Jer6nimo, S. M. B., 200, 210(219a),214(219a) Jeuniaux, C., 198 Jochims, J. C., 97, 141(39) Johary, P. C., 24 John, C. E . , 290 John, M . , 148 Johnson, G. A., 401 Johnson, J. G., 150, 266(19), 267(442), 323 Johnson, L. N., 196 Johnson, M. T., 299 Johnson, S. D., 329 Johnson, T. B., 113, I14(87) Johnston, L. S., 332 Jones, D., 269, 275(468) Jones, G. H., 248 I

Jones, J. K. N., 25,375 Jones, R. N., 63, 87(179) Jones, R. S., 191 Joseleau, J.-P., 161, 168, 176(112) Joseph, J . D., 391, 411(74) Julian, R. L., 61, 62(173),63(173) Jung, P., 346(38), 347, 348(38) Jungalwala, F. B., 395 Just, E. K., 75

K Kadentsev, V. I., 403, 430(192) Kadowaki, S., 237 Katlka, K. J., 23 Kahlenberg, A., 97,141(38) Kainuma, K., 148,253,254,255 Kaji, A., 163, 185, 186(72),247(72) Kajiura, T., 84 Kakuto, M., 296 Kalckar, H. M., 288 Kalin, J. R., 352 Kalinchuk, N. A., 314, 317 Kamberger, W., 380 Kamerling, J. P., 217, 219(300a), 400, 402, 403(169) Kamimura, M . ,416 Kamogawa, A., 281,315(15) Kanaya, K., 149 Kanbayashi, J., 408, 417(241), 420(241) Kanda, T., 151, 159 Kandler, O., 297 Kanegasaki, S., 313, 314,319 Kaneko, T., 252, 274 Kanno, M . , 257 Kanter, J. A,, 87 Kaplan, N . , 230 Kapoor, R., 205 Kappel, W. K., 281 Karicsonyi, s., 162 KardoSovB, A . , 24 Karlsson, K.-A., 388, 389, 393, 394, 396, 395(7), 398, 404, 405(195, 196), 406(109), 411(133), 420, 428(109), 429(94) Karson, E. M., 365 Karunaratne, D. N., 218. 223(305b), 224(305b) Kasahara, Y., 117, 140(97) Kashiwabara, Y., 192, 194

AUTHOR lNDEX Kashiyania, E., 148 Kasyanchuk, N . V . , 292 Katagiri, A., 388(34), 389 Katchalski, E.. 367, 373, 373(199) Kates, M., 392 Kato, G., 380 Kate, K., 153, 179, 180(121),181, 263, 267 Kato, S., 203 Kato. Y., 149, 154(17a),155, lSG(37, 40), 162, 273, 274, 275(40c, 48411) Katohda, S., 275 Katon, J . E., 28 Katona, L., 372(206), 373 Katsuhara, M., 124(139),125, 137(139), 145(139) Katsuki, S., 275 Katz, T., 164 Katz, W., 331 Katzenellenbogen, E . , 293, 297 Kaufinan, B., 291, 293(162), 326 Kauss, H., 345, 348(59),349, 353(59), 356(59), 358, 359(127), 381 Kawaguchi, K.. 281 Kawai, € I . , 287 Kawai, Y.,199 Kawamura, T., 288, 290(115) Kawase, M., 372(198),373 Kazantsev, Y I I . E . , 99, 102(51) Keegstra, K . , 152, 182, 183, 382 Keenan, R. W., 352 Keller, F. A . , 374 Keller, J. M . , 232, 305 Keller, R. K., 208, 209, 216(269), 352 Kelley, W. S., 285, 311(81) Kenne, L., 156. 286, 283, 302, 303, 307(98), 321, 322, 376, 402 Kenny, C. P., 324, 431 Kent, 1. L., 315, 316(308),317 Kent, P. W . , 374 Keraenen, A . , 428 Kerr, J. D., 34.5, 346(32),348(32) Kessler, G . , 165 Khan, H.,124(140),125, 138(140),143(140) Khomenko, N . A , , 295 Khorlin, A . Ya.,11. 17(39),19(39),21(39, 71), 25(39), 29(71),95, 104(34),113, 116, 140(92-94, 95). 141(34), 142(34, 95) Kiessling, G . , 290 Kiho, T., 267

451

Kikuchi, M . , 210. 212, 213 Kikuchi. T., 184 Kikumoto, S., 256 Kilker, H. D., 234 Killean, R . C. G . , 14, lS(53) Kilesso, V. A , , 313, 314, 315(286),316(286, 289), 317(286, 289). 318 Kilpoiien, R. G . , 84 Kim, S. H., 14, 15(54) Kiln, Y. S., 211, 212 Kimata, K., 216, 281, 282(17) Kimura. A., 281, 282(22), 287 Kiniura, N . , 288 Kindel, P. K., 185, 353(98), 354,359(98), 367(98) Kindler, S. H . , 282, 287(44) Kinoshita, T., 117, 140(96,97), 141(96), 142(86) Kirkiiian. B. R., 258 Kirkwood, S., 150, 266(19), 267(442), 288 Kiselevu, E. V., 314 Kishore, H.,8, 22(20) Kiss, J.. 390 Kitagawa, I . , 406,422 Kitainikado, M . , 165, 208, 241(264) Kitamura, M . , 389 Kivirikko, K. I., 244 Kiyokawa, M . , l24(139). 125, 137(139), 145(139) Kjell6ri. L., 212 Kjosbakken, J . , 325. 358, 359(132) Klein, U . , 148, 212 Klemer, A , , 113, 114(89),124, 135(134) Klenk, H. D., 387 Klis, F. M . , 383 Knee, M., 183, 185(132),382 Knirel, Y u . A., 289, 290, 292, 297, 301 Knowles, B. B., 388 Knox, K . W., 277 Knox. R. B., 381 Knudson, W., 199 KnuII, H. R., 382 Kobata, A . , 232, 233(323), 236, 237(323, 324), 238(323), 240, 388, 390(18),408 Kobeyashi, h.I., 258, 260(430), 261, 262. 264, 406, 422 Kol)ay;ishi, R . , 269, 275 Kobayashi. S., 252, 254 Kol)ayashi, S., 389 Kobayashi, T., 148, 160, 162, 257

452

AUTHOR INDEX

Kobayashi, Y ,, 269 Kobyakov, V. V.,86 Koch, P., 94 Kocharov, S. L., 389, 424, 427(289, 290), 435(290),436(290) Kocharova, N. A., 290 Kochetkov, N. K., 124,126, 135(119, UO), 144(119,UO), 280, 284, 286(12),289, 290, 292, 295, 297, 301, 314, 317, 319, 335, 336(292, 439), 337(296,439, 448), 338(439),390, 391(66),396, 397(136), 399(136, 138, 141), 401(136), 403(140), 405, 410(78),423(66. 138), 424(138), 425(140, 291), 426(140,141, 293). 427, 429(78),430(137, 192),431(137, 316), 432(136, 137, 161, 207), 433 (136, 161, 207, 323), 434(136, 161, 207) Kochling, H . , 95, 114(32),142(32) Kocsis, B.,300, 301 Kodama, C., 213 Koenig, J. H . , 7 Koenig, J. L., 7, 9, 12(23),13(23),18, 26, 27, 32(23),33(29, 30). 34(23),35(23),37(23), 38(23),40(28, 29, 30, 143),41(143), 42(143), 43(143), 44(143), 45(143), 46(29, 143),47(29),50(29),51(29, 30), 54(75), 55(30),59(4), 60(6),61, 62(172),63, 65, 66(182),67, 69(183), 75(28, 143, 182, 184). 76(182),80(28, 143, 163).82(30, 164). 85(182),88(30, 164) Koerner, T. A. W., Jr., 407 Kofler, M.,346(47), 347 Kogan, G . A., 11, 17(39),19(39),21(39, 71). 25(39),29(71) Kohn, L. D., 389 Kohn R., 23 Koide, N., 232, 233(323),237(323), 238(323, 347) Koike, Y.,288 Kojima, K., 427, 437(299, 300) Kolattukudy, P. E., 348(56),349,353(56), 362(56), 365(56), 371(56),372(56) Komai, Y.,390, 391(61),398, 414(144), 415(144) Kornandrova, N. A., 299 Kornar, V. P., 21 Kondo, W., 301 Konig, H., 297 Konig, J., 99, 105(62),1l2(62),140(62) Konigsberg, W. H., 407 Koningstein, J. A., 9

Kontrohr, T., 297,300,301 Kooiman, P., 151,154(24) Kopmann, H . J . , 319 Koput, J., 63,83(176) Korbecki, M., 99(70), 100, 106(67,69, 70), 107(67,69,70), 108(70),112(67,69, 70), 140(67,69) Korchagina, N. I., 298, 299 Kormos, D. E., 60 Kornblum, N., 92 Kornfeld, R., 231,232, 342 Kornfeld, R. H . , 282, 295(40) Kornfeld, S.,231, 232,234,235,281, 294, 342,364 Kornilaeva, G. V., 389 Korzybski, T.,281 Kostetsky, E. Y.,391, 409(79), 410(78, 79), 411(79),4U(79), 415(79),421(79),422(79), 423(79),429(78, 79), 434(79),435(79) Kotani, S.,263 Kotelnikova, L. P., 24 KovAE, P., 112, 113(85) Koyama, I., 430, 437(314) Kozar, T., 16 Kraevskaya, M. A. 336 Krassig, H . , 27 KrAtkY, Z., 160 Kratzl, K., 27 Krauss, H . , 353(83),354 Kristian, P., 91 Kritchesky G . , 344 Kritchevsky G . , 392,393 Krol, J. H . , 395 Kruczek, M. E., 352 Kuba6kov6, M., 162 Kubala, J., 24 Kubodera, T., 149, 154(17a) Ku6, J., 380 Kuhr, 336 Kudashova, 0. V., 337 Kudo, E., 110, ll2(81),120(81), 140(81) Kuhn, L. P., 17 Kuhn, R . , 400,432(173) Kuhn, S.,394,406,407(110) Kulczycki, A., 232 Kulow, C., 345, 348(33),350(33),353(33) Kulshin, V. A., 95, 104(34),141(34),142(34) Kumagai, H . , 237 Kumauchi, K., 393, 419, 420(96),421(96) Kundig, F. D., 324,325(357) Kundu, S. K., 393,394,401, 404

s.,

AUTHOR INDEX Kuo, T. T., 301 Kupriyanov, V. V., 317 Kurashashi, K., 281, 282, 284,287, 315(15), 316(62) Kusakabe, I., 160, 179, 181 Kusama, S., 179 Kushi, Y.,405 Kusov, Yu. Yu., 124, l26(119, l20),135(119, 120). 144(119, EO),314, 317, 336 Kuwahara, M., 393, 420(96), 421(96) Kyogoku, Y.,422

L Labavitch, J. M., 155,185 L’abb6, G., 99, 101, 102(47) Laborda, F., 183,185(132) Lacher, K. P., 329 Lada, E., 99(72),100, 106(72),108(72),W(72) Lafuma, F., 29 Lahav, M., 284 Laine, R., A., 402,420 Lamblin G., 197 Lamotte, G . , 123,l26(106) Lamport, D. T. A., 372(202, 203,205, 206). 373,382(203),383 Lang, W. C., 355, 371(105), 372(105, 194), 382(194) Langemann, A., 346(47), 347 Lapp, D., 281 Larm, 0.. 296,326(199) Larsen, B., 191, 193, 194(179),195(171),297 Latimer, P. H., 346(46), 347 Lau, A., 30, 84(136) Lau, J. M., 113 Laurent, T. C., 202 Lauter, C. J . , 398 Lavintman, N., 360,383(153) Lawson. C. J., 193, 251 Leach, S., 380 Ledeen, R. W., 388(30), 389.390(30), 392, 393, 400, 401,403(170), 404, 432(176) LeDizet, P., 166, 167(103) Ledley, F. D., 389 Lee, E. Y.C., 256 Lee, G . , 389 Lee, J . , 75, 81(185), 82 Lee, L., 281,282(22),297 Lee, L. J., 282, 287

453

Lee, P. P., 332 Lee, S. L., 282 Lee, S. R., 165 Lee, W. M. T., 394 Lee, Y. C., 75, 76, 81(185, 188) Leek, D. M., 218 Lees, M., 344,393 Leffler, H., 389, 396, 405,411(133) Legler, C., 234 Lehle, L., 248, 348(59), 349, 353(59), 353(83, 93, 99).354, 355, 356(59,93, 109, 111, 116, 118, l20),359(144),360, 362, 3&3(111, 118, 162),364(111),372(109) Lehmann, M., 281 Lehmann, V., 301 Lehn, J. M., 388(33),389 Leive, L., 315 Leloir, L. F., 234, 285, 305, 321(85, 266), 341, 342, 344, 345(22), 348(30),350(30), 35(30),355, 356(13, 14, 30, 114, 119), 357(114),367(13),376, 377(13,30) Lembi, C. A , , 358, 359(138),366(138) Lemieux, R . U., 13 Lengsfeld, W., 94, 95(29), ll4(29), 142(29) Lennarz, W. J., 234,284, 329(69),342, 352, 355(103),362, 363(11), 364(11), 367(11) LeNoble, W. J . , 92 Leontein, K., 230 Leppard, G. G., 325 Leroy, Y., 402 Lesley, S . M . , 157, 158(45) Letoublon, R. C. P., 353(96), 354, 366(96) Levery, S., 388 Levin, I., W., 84 Levinthal, M., 316 Levvy, G . A , , 149 Levy, G . N., 328,329(384) Levy, H. A , , 14,15(51, 59) Lew, H. C., 288,29O(ll7) Lewis, B. A., 271 Leyh-Bouille, M.,196, 197(189) Lhermitte, M., 197 Li, E., 234,364 Li, S.C., 208, 231, 237, 241(264), 390, 407, 408 Li, Y.T., 208, 231, 237, 241, 264, 390, 407, 408, 430, 437(3l2) Liang, C. Y.,26, 27, 46(ll3) Lidaks, M . , 99,140(65) Liddle, W. K., 76,81(188) Lieber, E., 93,393

454

AUTHOR I N D E X

Liedgren, H . , 402 Liener, L. E., 372(196),373, 382(196) Lifely, M. R., 327 Limouzi, J., 86 Lin, T. Y., 191 Lindahl, U., 207, 211,212, 213, 214(281), 215(292), 297 Lindberg, B., 14, 15(56),156, 158, 163(51), 166, 168(98),179, 218, 219(302),226, 269, 275(470),276(470),286, 288, 293, 296, 297, 298(201),301, 302, 303, 304, 307(98),321, 322, 323, 326(199),376, 402 Lindgren, B. O., 346(35),347 Lindquist, L. C., 291 Lindquist, U., 218, 219(302),288 Lindqvist, B., 293 Linhardt, R. J., 211, 212 Linker, A., 191, 203, 204, 210, 211(274),212, 214(275) Lipmann, F., 311 Lippincott, B. B., 381 Lippincott, J. A,, 381 Lis, H., 367, 373(199),373 Little, L. H., I0 Liu, T. Y., 305, 324(271),325 Liunngren, J., 321 Lofgren, H., 388, 395(6) Lomax, J. A., 321 Lombardi, F. P., 296 Lombardo, A , , 388(31),389 Long, W. F., 189, 190(160) Longas, M. O., 204 Lonngren, J., 230, 293, 301, 304, 321, 326, 402 Lord, J. M . , 353(90),354, 369(90),371 Lord, R. C., 84 Lorenz, D. H., 122 Lormeau, J. C., 213 Losick, R . , 313, 315(288) Low, M. J. D., 61 Lucas, J. J., 354, 355(103) Luchsinger, W. W., 274, 275(482) Luderitz, 0..300, 301 Lugowsky, C . , 293,297 Lukyanov, S . I., 36 Lunney, J., 379 Luscombe, M., 216 Luu, C., 16, 73(67),86(67),88(67) Luu, D. V., 16, 35, 73(67), 75(147), 76(147), 77(187), 79(187), 86(67),88(67) L’vov, V. L., 301

Lygre, H., 298 Lynn, W. S., 232

M McArthiir, H. A. I., 285 McCabe, M. M . , 262 McCallum, M . F . , 297, 325(204) McCleary, 13. V., 147, 149(5),159(5).165, 166(88),167(16, 87, 88, 104), 168(16,88), 169(16, 113). 170(16.88, 89, 106, 113), 176(16,112, 113), 178(16, 113), 179(16, 87) 180(16, 87, 89). 182(16),262 McColl, J. D., 390, 391(62), 421(62) McCloskey, M. A . , 324, 325(360) McCluer, R. H., 394, 399, 401 McConnel, M., 315 Mcl)onald, T. J., 410 McDougal, F. J., 257 McDowell, W., 359(147),360, 367(147) McCuire, E. J . , 324 Maclachlan, G . A., 155, 358, 359(135, 136), 385 McLean, M. W., 189, 190(160) McNeil, M., 152, 182(27), 183, 229, 358 Macpherson, I., 387 McMurray, W. C., 390, 391(62),421(62) Macaskie, L. E., 254 Macharadze, R. C., 95, 104(34),113, 116(9294), 140(92-94, 95), 141(34),142(34,95) Macher, B. A . , 390, 394, 420(70) Machin, P. A., 196 Mackie, D. M., 210 Mackie, K. L., 218 Macmillan, J. D., 269 Madden, J. K., 251 Maekawa, A,, 179 Maezawa, M., 405 Mage, J. B., 23 Maglothin, A., 183 Mair, C. A , , 196 Maitra, U . S., 288, 298 Majima, M., 200 Makeli, P. H., 288, 290(117),305, 315, 316, 317 Makita, A., 399, 407, 428 Malchenko, L. A., 424, 427(290),435(290), 436(290) Maley, F., 232, 237, 238(352), 382 Malmqvist, M., 187

AUTHOR INDEX

Malmstriirn, A., 206, 207, 215, 217 Maltser. S. D., 314, 335, 336(292-439), 337(439,448). 338(439) Mancuso, D. J., 284 Mandel, P., 388, 390 Mandels, M., 269, 275, 276 Mankowski, T., 284, 346(43),347 Manley, R. S . J., 325 Manners, D. J., 163, 252, 253, 254, 269, 270(471, 472), 271, 272, 275(470, 478). 276(470, 478) Mansson, 1.-E.,390, 391(63),399 Mantsch, H . H . , 9, 29, 87(33) Manukhin, B. N., 436 Marchessault, R . H., 26, 27, 46(113) Marcus, D. M . , 388, 390(16),394 Marechal, Y . , 63 Markey, S. P., 404, 405(203) MarkoviE, O., 182 Markovitz, A , , 291, 292(160),296(160, 163), 323 Marriot, K. M., 369 Marsh, J. B., 387 Marshall, J. J., 147, 252(1),253(1),258(1), 266(1),269, 273(1) Martensson, E., 398 Martin, A , , 324,431 Martin, H. G . , 350, 351(65) Martin, J. P., 24 Martin, 0. R., 31 Martvoii, A , , 112, 113(85) Maruyama, Y., 380 Marx-Figini, M., 362, 366 Mascaro, L., 291 Mashilova, G. M., 290, 297 Masserini, M., 390, 391(60),415(60) Masson, A. J., 269, 275(470), 276(470) Masuda, S., 419, 420(264) Matheson, N. K., 147, 149(5),159(5),165, 167(16,87). 168(16),169(16),170(16),171, 176(16,116). 178(16),179(16),180(16,87), 182(16),186, 247(141),253 Mathews, M. B., 216 Mathias, A., 93 Mathlouthi, M., 16, 35, 63, 65(68),66(182), 73(67),75(147, 182), 76(147, 182), 77(187), 79(187, 189), 85(182),86(67),88(67,68) Mathys G., 99, 101(47),102(47) Matsubara, T . , 396, 398, 401, 408, 412(240), 413(143, 240), 414(143, 143a), 415(125), 416(129, 247), 420

455

Matsuchita, J., 154 Matsuda, K . , 149, 154(17a),155(17),156(34, 37, 40), 162, 172(34),255, 258, 260(430), 261, 262, 264, 268, 271, 275(40c),281 Matsuhashi, M., 286, 291, 292, 293, 295(168), 331(91),333, 342 Matsuhashi, S., 291, 292, 293, 295(168, 169) Matsukawa, S., 390, 391(61) Matsumoto, A , , 255 Matsurnura, G . , 432 Matsuno, T., 422 Matsuo, M., 162, 164 Matsushima, Y., 328 Matsushita, J., 149, 154(17a) Matsuura, F., 396, 398(128, 131, 132).407, 413(131, 132, 142), 414(128, 131), 415, 416(247) Mattescu, G., 10 Maxwell, J . , 81 Mayer, H. E., 232, 290,298, 299, 300, 304 Mayer, R. M., 281 Mayers, 6. L., 426 Mazzotta, M . Y., 408 Meadow, P. M., 331 Medeiros, M. G. L., 200, 210(219a),214(219a) Meffroy-Biget,A M., 16, 73(67),86(67), 88(67) Meier, H., 164, 179, 358, 359(l29) Meinders, I., 87 Meinelt, B., 99, 105(62),112(62),140(62) Meleiros, M. G. L., 200 Mellor, R. B., 353(90),354, 369(90),371 Melo, A . , 281, 291, 294 Melton, L. D., 156, 322 Mendez-Castrillon. P. P., 95 Mendiara, S., 360, 383(153) Mense, R. M., 353(86),354, 356(86),363(86), 364(86) Mentaberry, A . , 352 Merchant, Z. M., 211, 212 Mercier, C., 253 Merriam, H. F., 113 Merrifield, E. H., 218, 223, (301) Mersmann, G., 124, 135(134),209, 216(272) Mescher, M. F., 284, 285(73) Mesquida, A., 30 Meyer, K.,203, 204, 208, 216(267, 268) Michael, J. M., 234 Micheel, F., 94, 95(29, 31).113, 114, 142(29, 32)

456

AUTHOR INDEX

Michel, G., 84 Michelacci, Y. M., 200, 205, 206 Michell, A. J., 27, 28(118) Michelson, A. M., 291, 294(147) Mijatake, T., 405 Mijzawa, T., 413 Mikawa, Y.,13, 40(46) Mikhailov, A. T., 424, 427(290), 435(290), 436(290) Milanovich, F. P . , 82 Milas, M., 157 Miller, D. H., 372(203, 205). 373, 382(203) Miller, J. T., Jr., 28 Miller, N . , 372(205),373 Milliken, 6 . A , , 253 Mills, G . T., 281, 282(14),287(14),288, 289(14),326,327(374) Min, K. H . , 194 Minakova, A. L., 258, 259(422),262 Minale, L., 422 Mindt, L., 156, 322 Minner, F., 301 Mirelman, D., 197 Misaki, A . , 154, 155, 156(38),159, 233, 247, 254, 258, 259(428),260(428),263, 265, 266, 267(442),296, 300, 372(200),373 Mishima, Y., 396, 415(130) Misra, D. S., 24 Mitchell, J. P. 163 Mitsui, K., 333 Mitsuishi, Y., 258, 260(430), 261 Mitsuyama, T., 407 Miyazaki, T., 248, 288 Mizoguchi, J., 333 Mizouchi, T . , 237 Mizuno, T., 164 Mizuno, T., 164 Mohri, H., 428 Momoi, T., 388, 393, 394(98) Monsan, P., 88 Montgomery, R., 232 Montreuil, J., 238, 402 Moore, R. H., 18, 53 Morell, A. G . , 378 Morgan, I. G . , 388(29),389 Mori, H., 269 Mori, M., 153, 181 Morikawa, N., 287 Morre, D. J., 358, 359(138),366(138) Morrice, L. M., 189, 190(160) Morris, E. R., 166, 251

Morris, N. P., 389 Morrison, A., 164, 165(75),166(75) Mort, A. J . , 229, 380 Morton, R. A . , 346(37, 48), 347, 351 Moscarello, M. A , , 246 Moscatelli, E. A., 399 Moshenskii, U . V., 424, 427(289) Mosher, M., 390, 391(59) Moskal, J. R., 407 Motherwell, R. S. H . , 123, 126(106) Motherwell, W. B., 123,126(106) Mott, C. J . B., 18, 30(76) Moulin, J., C., 163 Mourio, P. A. S., 252 Moyer, J. D., 8, 18(12),19(12) Muir, H., 198 Mukaiyama, T., 122 Miiller, A . , 92, 93(8),94, 123,142(8),144(8) Miiller, E., 208, 209 216(269) Miiller, L., 285, 311(83),315(83),317(83) Muller, V., 214 Miiller, W. M., 20, 25(79) Mullin, B. R., 389 Mumm, O., 94 Munakata, A., 256 Mutioz, E., 197 Murachi, T., 232, 372(195),373, 382(195) Muragaki, H., 422 Murakami, K., 179 Murakami, Y . , 237 Murakanii-Murofuski,K., 430, 437(314) Muramatsu, T., 232, 233(323),237(323), 238(323, 347) Murata, T., 405 Murazumi, N., 285 Murphy, D., 124, 134, 135(132) Murphy, W. F., 9, 29, 87(33) Murray, R. U . , 387 Murthy, A. S. N., 9 Murty, V. L. N., 427, 437(299) Muzzarelli, R. A. A , , 198 Myers, R. W., 76, 81(188) Myllyla, R., 244

N Nader, H. 8..200, 210(219a),214(219a) Nagahashi, J., 369. 372(185) Nagai, A , , 392 Nagai, Y.,388, 389(8),390(8),391(65),393.

457

AUTHORINDEX 394(97, 98), 397, 423(65), 424(139), 428(65),435(306) Nagasaki, S., 266, 269, 275 Nagasawa, K., 205 Naito, T., 97, 142(42) Nakagawa, H., 208,241(264) Nakae, T., 281,316 Nakagawa, J., 333 Nakajima, K., 388 Nakajima, T., 248, 268, 271, 362, 365 Nakakuki, T., 148 Nakamura, K., 211 Nakamura, M., 380 Nakamura, T., 200 Nakanishi, Y., 203 Nakano, J., 181 Nakasaki, C., 25 Nakasawa, Y., 391 Nakatani, T., 198 Nakayama, K., 198,355,356(108) Nakazawa, F., 301 Nakazawa, K . , 209, 216(271) Nambu, H., 122 Nanjo, F., 148, 271(11b),272(11b) Narashimhan, S., 244,246 Nasir-ud-Din, 197 Nathenson, S. G., 238 Natsume, T., 258, 260(427) Neal, I). J., 300 Neely, W. B., 21 Nelsestuen, G. L., 288 Nelson, T. E., 150, 266(19), 267(442),271 Nesbitt, L. R., 267 Neuberger, A., 234,372(204), 373 Neufeld, E. F., 206, 214, 379 Neuhaus, F. C.! 331, 332 Neukom, H., 163, 166, 167(104),185(71) Nevins, D. J., 148, 162, 272(11c), 273, 274, 275(484b) Newsome, D. A., 209, 216(271) Niemann, H., 218, 219(304), 220(304, 305), 221(304), 222(304),224(305),297 Niemann, R . , 200, 203, 204(217) Nikaido, H., 147, 225, 281, 284, 288, 290(117),292(28),299, 313, 316(61) Nikaido, K., 281, 284, 292(28), 316(61) Nilsson, B., 209, 216(271) Nilsson, K., 429 Nilsson, O . ,393 Ninimich, W., 301, 304 Nimura, N., 91, 93, 99, 105(54,55), 112(54,

55), 113, 114(90, 91), 115(91),116(90),

117(17, 55), ll9(55), l20(99), 140(54, 55, 90, 91, 96, 97, 99), 141(90,96), 142(96) Nishibe, H., 239, 240 Nishido, M., 420 Nishimura, D., 325 Nishino, T., 422 Nishio, H., 265 Nisizawa, K., 147,151, 159, 179,180(117),192, 194 Nogami, A., 254 Nordin, J. H., 265, 366 Noren, R . , 390,391(63) Norris, K. H., 23 North, A. C. T., 196 Northcote, D. H., 348(58, 60), 349, 353(60), 354(60),358, 359(137, 149,150), 360, 361, 367(137), 372(207),373 Norval, M., 285, 321(79) Notario, V., 266 Nowakowska, Z., 100,106(74,77, 78), 109(74,77). 110(78),ll2(74, 77, 78). 122(78) Nudelman, E., 388 Nunez, H. A., 407 Nunn, J. R., 218 Nurminen, N., 301 Nurthen, E., 168,176(ll2)

0 Obata, N., 119, 120(99),140(99) O’Brien, J. S., 408 O’Brien, P. J., 281 Obukhova, E. L., 430,437(312) Ockendon, D. J., 381 Ockman, M., 31 OConnor, R. T., 46 Odzuck, W., 358,359(127) Ogamo, A,, 205 Ogasawara, N . , 268, 269 Ogata, K., 281 Ogata-Arakawa, M . , 232, 233(323), 237(323), 238(323) Ogren, s.,212 Ogura, H., 91, 93, 95, 99, 105(33,54, 55, 60, 55, 61, 62), 106(60),110(82),lll, M(54, 60-63,80-83), 113, ll4(91), ll5(91), ll6(90), ll7(55), 117(17), 119(55,98), 120(61,81, 99), 121(61, 84), 139(33),

458

AUTHOR INDEX

140(54, 55, 60-62, 80-85, 90, 91, 96, 98, 99, loo), 141(33, 90,96). 142(33,96, 100) Oguri, S., 239 Ohara, S . , 258, 260(429) Ohashi, H., 292 Ohgushi, S., 160 Ohno, N., 288 Ohokubo, K., 120,140(100),142(100) Ohsawa, T.,435 Ohst, E . , 208, 209, 216(269) Ohtani, K., 154 Ohya, T., 258 Oike, Y., 216 Okada, G., 151 Okaji, S., 258, 260(429) Okamoto, M., 122 Okano, K., 422 Okazaki, R.,291, 294(147) Okazaki, T., 291, 294(147) Okita, T. W., 382 Okumura, S., 396, 415(127) Okuyama, A,, 389 Okuyama, T.,199 Oldberg, A., 212 O’Neill, M., A., 155, 156(39),234 Onn, T.,323 Ono, T.,256 Oomen-Meulemans, E. P. M., 395 Orchard, P. I., 124, 135(137),145(137) Oreste, P., 213 Oriez, F. X., 124, 132, 143(125),144(l25), 145(L25) Orth, R.,408 Ortiz-Mellet, C., 99, 105(62), ll!2(62), 140(62) Osawa, T., 99, lOO(44) Osborn, M. J., 231, 285, 300, 301, 313(82, 83),315(83), 316(307, 308), 317(83),342 Oshima, M .,405 Ostmann, P., 94, 95(30),114(30),141(30), 144(30) Ototani, N., 205, 210, 211, 212, 213 Otsu, K . , 203 Ottaviani, E.,390, 391(60), 415(60) Overend, W. G., 124,126, 143(115) Ovodov, Yu, S., 298,299 Ousepbjan, A. M., 86 Owen, L. N.,124,126, 135, 145(137) Owen, P., 329

Ozaki, H., 281 Ozutsurni, M., 282

P Padmanabhan, M., 8, 22(20) Page, R. L.. 290,328 Painter, P. C., 9, 12(23),13(23),32(23), 34(23), 35(23), 37(23), 38(23) Painter, T. J., 166, 168(98, 99), 193, 251 Paiva, 1. F., 200, 210(219a), 214(219a) Paiva, V. M . P., 200, 210(219a), 214(219a) Palamarczyk, G., 235,355 Palmer, T. N . , 254 Palva, E. T., 315 Panayotatos, N . , 358, 359(128),366 Pankrushina, A. N., 317 Panos, C . , 330 Panov, V. P., 86 Pape, H., 292, 295(170) Pbquet, M. H . , 246 Park, J. T., 197, 290 Parker, F. S., 8, 9, 10(32), 16(17, 32), 20(17), 25, 35(102),36(32),56(17),66(17),67(17) Parodi, A. J., 342, 344, 345(22),348(54),349, 353(54),355, 356(13, 54, 110,117). 359(110, 117),362, 363(110, 117, 160), 364(160),365(160),367(13), 377(13) Parolis, H . , 218 Parrish, F. W., 30, 124,125, 269,275, 276 Pascher, I., 388, 393, 395(6),404, 405(195), 429(94) Patil, J. R., 21, 25(83) Patt, L. M., 388 Patterson, J. C., 269, 275(470),276(470) Paukon, J. C., 202, 233, 246(223), 378 Pazur. J. H., 281, 282, 294(23), 382 Pearson, C. H., 217 Pearson, F. G., 27 PBaud-Lenoel, C., 353(91), 354, 356(91), 359(91),366(91) Pennock, J. F., 345, 346(32, 41,48), 347, 348(32) Pensar, G., 346(44),347 Perchard, C., 86 Perchard, J. P., 86 Percheron, F., 165 Percival, E. G. V., 165 Perila, O:, 179

459

AUTHOR INDEX Perkins, H. R, 277, 278(4), 290, 331 Perlin, A. S.,30, 55, 151, 159, I60(55), 210, 273, 274(480) Pernet, A. G., 91 Perry, M. B., 288, 298 Pertoft, H., 212 Peterson, H., 95, 114(32),142(32) Peterson, K . , 293, 302 Peticolas, W. L., 84 Petit, J. F., 197, 331 Petitou, M., 213 Petrenko, V. A,, 336 Petriella, C., 355, 356(114, IN), 357(114) PhafT, H. J., 269, 272, 275(461, 478), 276(478) Phelan, A. W., 239 Phelps, C. F., 205,216 Philips, T. S., 124(143), 125, 139(143) Phillips, D. C., 196 Phillips, D. R., 163 Pigman, W., 423 Pillat, M., 319 Pilnik, W., 183, 185 Pirnlot, W., 404, 405(195) Pindar, D. F., 191, 297, 325(205) Pinkard, J. M., 8, 10(11), 18(11),19(11) Pitha, J., 63, 87(179) Pitha, P. M., 389 Pitzner, L. J . , 52, 82(158) Pizza, C., 422 Pizzo, S. V., 233, 378 Plantner, J. J . . 237 Plapp, R., 331, 332 Pless, D. D., 329, 332 Plumrner, T. H., 232, 237, 238(352),239 Poblacion, C. A,, 206 Polavarapu, P. L., 20 Pont Lezica, R., 343, 344, 345(23), 348(23, 28, 53), 349, 350(27, 28). 351(28, 53), 353(81, 82, 84, 89, 100). 354, 355(82, 84), 356(15, 82, 89, 121), 359(82), 361(82), 362(156),367(15), 370, 371(89),372(89, 121) Pope, D. G., 372(201),373 Popelis, J., 99, 140(65) Popova, A. N.,313, 316(289),317(289), 318 Popowicz, J., 63, 83 Porter, E. A , , 123, 126(106) Porter, J. W., 350

Porter, R. K., 93 Poss, A,, 389 Pousada, M., 426 Powell, D. A , , 293, 326, 329 Poxton, I. R., 319, 321(333) Pradera, Adrian, A , , 99, 105(62),1l2(62), 140(62) Prehrn, P. 318 Preiss, J.. 147, 256, 281, 282, 290 Preobrazhenzkaya, M. E., 258, 259(422), 262 Pressey, R., 183 Prestegard, J. H., 407 Preti, A., 388(31),389 Price, H., 390, 391(59) Price, H. C., 166,404 Pridham, J. B., 382 Prieels, J. P., 378 Prihar, H. S., 215, 290 Prima, A. M., 21 Pringle, G. A., 217 Prokazova, N. -V., 389, 424, 427(289, 290), 435(290),436(290) Pueppke, S. G . , 380 Pugashetti, B. A.. 290 Pulkownik, A,, 258, 259(426), 260(426) Puls, J. 162 Puro, K. 428 Puztai, A., 383 Quivoron, C., 29

R Racusen, D., 383 Radin, N. S., 408 Radzieiewska-Lebrecht, J., 299 R&, R. A. 292 Rdgg, P. L.,124,126 Rahman, H., 390 Rajagopal, M. V., 391, 411(75) Rakhrnatullaev, J . , 99, 140(65) Ramachandran, J . , 93 Rarnjeesingh, M . , 97, 141(37) Rao, C. N. R., 9 Rao, D. N. R., 93 Rao, S. T.,14, 15(57) Rao, V. S. R., 167 Rappaport, L., 382 Rapport, M. M., 389

460

AUTHORINDEX

Rashbrook, R. B., 165 Ray, P. H.,283 Ray, P.M., 155 Raymond, Y.,358,359(136) Rearick, J. I., 202,232,244(223),246(223) Rebel, G.,388 Recondo, E.,285,323(77),325(77),345, 361(31) Redmond, J. W., 293 Reed, L.A.,111, 124,135(135),143(135), 144(135) Rees, D.A.,14,16(49),156,166, 168, 169(113),170(113),176(113),178(113),187, 189,190,251,322 Reese, E. T., 159,165,179(84),269,275, 276 Reeves, R. E., 13 Reggiani, M., 28 Reid, J. S. G., 164,358,359(129) Reinhold, V. N.,405 Reinking, A.,353(88),354,356(88) Renkonen, O.,402,420 Renovitch, G.,85 Renson, M., 93 Reske, K., 318 Reusch, V. M.,330 Reuter, G.,431 Reuvers, F.,348(55),349,353(55,88), 354, 3w88) RexovCBenkovi, L., 182 Reynolds, C.-C.,200 Riccio, R., 422 Rice, K.-G., 211,212 Richards, A. W., 274,275(482) Richards, G. N.,147,159(4),160, 247(4) Richards, J. B., 346(39),347 Richardson, A. C.,124,l25(ll3),l26(113, 122),127(113),128(113),143(113,122), 144(122) Richter, M., 94 Rieger-Hug, D., 217,218 Riesenfeld, J. 213,215 Rimai, L.,84 Rimon, A., 213 Rinaudo, M.,157 Riolo, R. L.,200,208 Risbod, P.A., l24(143),l25,139(143) Rivas, L.A.,353(100),354 Robbins, J. B., 305,324(271),325 Robbins, P.W., 234,235,281,285,305,310, 313(81), 315(278,288). 342,362,363(161), 364(161)

Roberts, F. M., 285 Roberts, I. N.,381 Roberts, J . D.,21,388 Roberts, L.M., 371 Roberts, W. S. L., 331 Robertson, S., 351 Robin, J. P . , 253 Robinson, H.C.,202,203 Robyt, J. F.,252,259,310 Rod&, L.,198,202,205,214,215(208),297, 306 Rodriguez, I. R., 256 Roelcke, D.,394,407(110) Roelofsen, G.,87 Roerig, S., 375(206),373 Rogers, H. I., 277,278(4) Rohr, T.E.,324,325(361),328,329(384) Rohrniann, K., 203 Rohrschneider, J. M., 377 Romanowska, A., 297 Romanowska, E.,293,297 Rombouts, F.M., 149,183,184(133), 185(133),269,272,275(461,478). 276(478) Rome, L. N.,379 Romero, P.A.,285,345,348(53),349, 350(27),351(53),353(81,82,84,89).354, 355(82,84), 356(82,89,121).359(82), 361(82),362(156),370,371(89),372(89, 121) Romero Martinez, P. 344, 345(23),348(23), 349(23) Roppel, J., 290 Rosell, K. G.,381 Roseman, S., 291,293(162),306,324, 325(357),326 Rosen, O.,208 Rosen, S. M., 231,282 Rosenberg, E.,230,282,287(44) Rosenberg, R. D.,213 Rosenfeld, E. L.,262 Rosenfelder, G.,300 Rosenthal, A., 265 Rosik, J . , 24 Rosseto, 0.. 94 Rossiter, R. J., 390,391(62),421(62) Rothfield, L.,313,342 Rouser, G.,344,392,393 Rowland, R. L.,346(46), 347 Rozhnova, S. Sh., 313,314,315(286),316(286, 289),317(286,289), 318 Rubenstein, P.A.,281, 292,305

AUTHOR INDEX Rudbn, U., 305,321 Ruegg, R., 346(47),347 Ruiz-Herrera, J., 374 Rundell, K., 317 Rupley, J. A., 196 Ruschmann, E., 301 Russell, J. D., 24 Ruysshaert, J . M., 389 Ryan, J. M., 315 Ryazanov, M. A,, 87

S

Sabnis, D. D., 381 Sach, J. 352 Sadava, D., 383 Sadler, J. E., 202, 244(223),246(223) Sadovskaya, V. L., 424,427(289) Saheki, T.,186 sahu, s. c.,232 Saini, H. S., 186, 247(141) Saito, H., 148, 199(8), 205, 206(247). 402 Saito, K.,266 Saito, S., 282 Saito, T., 192,392, 393(82) Saitoh, F., 237 Sakaguchi, M., 91, 110, 1l2(80),14q80) Sakai, H., 119, 120(99),140(99) Sakai, M . , 91 Sakakibara, K.,388 Sakano, Y., 148,257 Sakurai, Y., 179 Salares, V. R., 84 Salmarsh-Andrew, M .,313 Salo, W. L., 289,290 Salsman, K., 401, 432(176) Salton, M. T. R., 329 Sampietro, A. R., 187 Sampson, P., 203 Samuelson, O., 161 Samuelsson, B. E.,393,396, 398, 404,405(195),406,4ll(133), 420, 429(94) Samuelson, K., 301,404 Sandermann, H., 326 Sanderson, G. R.,156,322 Sandford, P. A., 289,320,322(131) San Felix, A., 95 Sano, M., 97, 142(42) Saralkar, C . , 330

461

Sargeant, J. G., 252 Sarma, V. R., 196 Sarvas, M., 299 Sasak, W., 284, 346(43),347 Sasaki, S. F., 194 Sasaki, T., 284, 316(62) Sasaki, Y., 288 Satake, M.,390, 391(61),398, 414(144, 145, 146), 415(144, 145,146) Sato, M., 185, 301 Sato, O., 99, 105(61, 62), ll0(82), lll, ll2(61, 62, 82-83), l20(61), Ul(61, 84). 140(61, 62,82-85) Sato, T., 275 Satoh, A., 271 Saunier, B., 234 Savage, A. V., 218 Sawai, T., 257, 258, 259(428), 260(427, 428, 429) Sawicka, T., 281 Scaletti, J. V., 266, 267(442) Schachter, H., 244, 246,306 Schafter, D. E., 407 Scharf, H. D., 208, 209, 216(269) SchatschneIder,J. H., 13, 40(42, 43) Schauer, R., 233,283,301,400,402, 403(169),431 Scheinberg, I. H., 378 Schenkel-Brunner, H., 246 Scher, M., 284,329(69) Scher, M. G., 342,352 Schilperoort, R., 229 Schlecht, S., 315 Schlesinger, P. H., 379 Schmid, T. M., 199 Schmidt, E. L., 379,380 Schmidt, J.. 148 Schmidt, M., 199 Schmidt, M. F. G., 3777 Schmit, A. S., 329 Schmitz, F. J., 410 Schneider, B., 19 Schneider, H., 162 Schramek, S., 299 Schrevel, J., 252 Schultz, J. C., 284,329(67), 329 Schulz, I., 355,356(Ii6) Schutt, M., 99,105(57,58), 14q57, 58) Schutzbach, J. S., 235,244,366 Schwarting, G. A., 388,390(16) Schwartz, N. B., 202,306,324

462

AUTHOR INDEX

Schwan, J. C. P., 124,135,137(136) Schwan, R. T., 353,(99, la), 354,367,377 Schweiter, U., 346(47), 347 Scott, P. G., 217 Scovenna, G., 55 Scudder, P.208,241(264b) Searle-Van Leeuwen, M. F., 149 Sebjakin, Yu. L., 317 Seeliger, A. 97, 141(39) Segal, L. E., 46 Segura Ramos, F., 99, 105(62),ll2(62), 140(62) Sellers, L., 291 Selvendran, R. R., 155,156(39),234, 382 Seno, N., 199, 208 Sequeira, L., 380 Seto, N., 247, 300 Seuvre, A.-M., 65, 66(182),75(182), 76(182), 85(192) Sevier, E. D., 369 Seyama, Y., 288 Seymour, F. R., 61, 62(173),63(173) Shabadash, A.N., 35,36(146) Shannon, L. M., 369, 372(197),373 Shaposhnikova,G. I., 389,429 Sharma, C. B., 362,363(162) Sharon, N., 147,195,197,293, 367, 388 Shashkov, A. S., 284,290,292,297, 298, 301 Shaw, D. H., 295 She, C. Y.,80, 81 Sheehan, J. K., 61,62(172) Sheinik, R., 387 Sheldrick, B., 14,15(52) Shen, L, 281 Sheppard, N., 27 Sheremet, 0. K . , 289 Sheu, K.-F., 282 Shevchenko, V. P., 389 Shiau, G . T., 99, 102(49),141(49),142(49) Shibaev, V. N., 124, l26(119, l20),135(119, l20),144(119,EO), 280, 284, 286(l2),313, 314, 315(286),316(286, 289), 317(286, 289), 318, 319, 335, 336(292,322, 439, 440,441), 337(296, 322, 439, 448), 338(322, 439,441), 339(322),343, 356(14a) Shibasaki, K., 372(198),373 Shibata, Y., 165,179(84),271,275(477) Shibuya, N., 155,156(38),159,247 Shida, M., 268,271 Shigemitsu,N., 237

Shimahara, H., 179, 180(ll7) Shirnanouchi,T., 39, 40(150) Shimizu, H., 420 Shimizu, K., 179, 257 Shimizu, M., 203 Shimomura, T., 149 Shinoda, S., 388 Shinomiya, N., 388 Shinomura, T., 216 Shiota, M., 271 Shiozawa, R . , 155, 156(40) Shirai, S., 396, 408(l26),416(l26) Shkolenko, G. A., 23 Shoemaker, S. P., 151,180(20) Shore, G., 358, 359(135,136) Shuey, E. W., 281,294(23) Shulman, M. L., 17, 21(71), 29(71), 95, 104(34),141(34),142(34) Shuster, C. W., 317 Shutalev, A. D., 99,102(52, 53) Shwann, G.-G., 14,15(56) Siddiqui, B., 389,392,408 Siddiqui, I. R.,289 Sidebotham, R. L., 258 Siesler, H., 27 Sietsma, J. H., 267, 268(446) Siewert, G., 331 Silbert, J. E., 199, 203 Sillerud, L. O., 407 Silva, M. E., 210, 211 Silverburg, I., 210, 217 Sirnonova, T. N., 429 Simpson, D. L., 382 Simpson, E. K.G., 256 Simpson, L.-L., 389 Sinay, P. 213 Singh, M., 285,311(83),315(83),317(83) Sinha, R. K. 331 Sivchik, V. V., 17, 19, 43(77), 50(70),52, 53(159) Sjoberg, 11, 206 Skjik-Braek, G . , 193 Slabnik, E., 383 Sleeter, R. T., 24, 24(101) Slettengren, K . , 230 Sloane-Stanley, G. H., 344, 393 Slomiany, A., 401, 427,437(299,300) Slomiany, B. L., 427, 437(299, 300) Sly, w. s., 379 Small, D. M., 165, 167(87),180(87) Srnidsrd, 0.. 193, 194(179)

AUTHOR INDEX Smiley, R. A,, 92 Smiljanski, S., 24 Smirnova, G . P., 390, 391(66),396, 397(136), 399(136, 138, 141). 401(136),403(140), 405, 409(79), 410(78, 79), 411(79), 412(79),415(79),421(79), 422(79), 423(66, 79, 138), 424(138),425(140, 291), 426(140, 141, 293), 427, 429(78, 79), 430(137,192), 431(137, 316), 432(136, 137, 161, 207), 433(136, 161, 207, 323). 434(79, 136, 161, 207), 435(79) Smith, C. G., 166 Smith, E. E., 256,262 Smith, E. E. B., 281, 282(14),287(14,), 288, 289(14), 326, 327(374) Smith E. J., 290 Smith, F.,266, 267(442), Smith, F.A , , 8, 18(12),19(12) Smith, I. C. P., 288, 324, 431 Smith, M., 282 Smith, M. M., 353(91),354,356(91),359(91), 366(91) Smith, R., 202 Snaith, S. M., 149 Snipes, C. E., 291 Snippe, H., 217,219(300a) Snyder, R. G., 13, 40(42, 43, 44) Sohonie, K. 391,411(75) Soliday, C. L. 348(56).349, 353(56),362(56), 365(56),371(56),372(56) Soll, D., 331 Solov’eva, L. A., 36 Solter, D., 388 Somogyi, L.,99, 100(45,46). l22(46) Sonnino, S., 388(31),389, 431 Soper, S . , 391, 410(77),412(77) Sousa, J. A,, 30 Southwick,J., 63 Sowden, L. C., 325 Spedding, F. H., 8, 73(19) Spedding, H., 7, 18(1),19(1),56(1), 67(1) Spencer, J. F. T., 191,248 Spik, G . , 238 Spiro, R. G., 206, 232 Spormaker, T.,283 Spurlock, L. A., 93 Srisuthep, R., 23 Stacey, M., 8, 10(7),18(7),19(7),40(7),53 Stahl, P . , 379 Stamm, R. F., 8, 73(19) Stanacev, N. Z., 399

463

Stangk, J., 124,125, 143(109, 110) Staneloni, R. J., 234, 276, 285, 305, 321(85, 266). 342, 344, 355,356(14, 114, 119), 357,(114),376 Stanislavsky, E. S., 290, 297 Stankovic, S., 24 Stark, J. R., 252, 253,271 Stark, N. J., 328, 329(384) Staudte, R. G., 275 Stead, A. D., 381 Steen, G. O., 398, 404, 420 Stein, J. 2..166 Stein, T., 208, 209, 216(269) Stellner, K., 402 Stephen, A. M.,164, 182(78),218 Stephens, A. W., 213 Stephens, R., 8, lO(8-lo), 18(8-lo), 19(8-lo), 20(8-lo), 40(8) Stevenson, J., 347 Stewart, J . E., 8, 18(12, 13), 19(12,13), 21(13) Stickgold, P. A., 332 Stirling, J. L., 204 Stirm, S., 217, 218, 219(303, 304). 220(303, 304, 305), 221(304),222(304), 223(303), 224(305), 297 Stocker, B. A. D., 301,317 Stockert, R. J., 378 Stoffel, W., 402 Stone, B. A., 163, 273,274(479),275(484a), 381 Stone, K.-J., 346(41, 42) Stoolmiller, A. C., 202, 205, 324 Strannegard, G., 389 Straus, A. H., 210 Strecker, G., 238 Strel’tsova, I. F., 25, 35(103) Strobach, D. R., 420 Strominger, J. L., 196, 197(189),203, 281, 282,284,285(73),286,291,292,293, 294(147),295(168, 169,170,171),305, 324, 331(91,92, 93, 94),332, 342, 352 Struve, W. G., 331,332 Stuckey, M., 333 Stuhlsatz, H. W., 204, 208, 209, 216(269) Sturgeons, R. J., 160,271 Suckane, M., 257 Sugahara, K., 324 Sugawara, T., 422 Sugie, E., 393, 420(96),421(96) Sugimoto, H., 184 Sugimoto, K., 254

464

AUTHORINDEX

Sugimura, A., 282 Sugita, M., 393, 396, 397(95, 134, 135). 399, 401(134, 135), 408(l26),416(126),417(159, 241). 419, 420(96, 241, 261, 263, 264), 421(96),429(95), 432(93, 134, 135), 433(134,135) Sugiyama, K., 239 Sugiyama, N., 179, 180(117) Suhadolnik, R. J., 91 Sukeno, T., 232 Sukhova, N. M., 99, 140(65) Sumizu, K., 162 Sundaralingam, M., 13, 14,15(57),16 Sundararajan, P. R., I67 Sundell, S., 388, 395(6) Susi, H., 23, 76, 80(186) Sutherland, I. W., 157, 158, 193, 218, 219(302), 277, 278(1),285, 305, 319, 321(79, 333). 323, 376 Suzuki, A., 394,401 Suzuki, F . , 275 Suzuki, H., 179,180(117),274, 333 Suzuki, J., 389 Suzuki, K., 393 Suzuki M., 405,426 Suzuki, S., 148, 199(8),203, 205, 206(247), 216, 254, 273, 274(480),281, 282(17) Suzuki, T., 148, 179, 271(11b),272(11b) Svennerholm, L., 388(32),389, 390, 391(63), 392, 393(88),399, 400, 421 Svenson, S. B., 230 Svensson, S., 166, 168(100),171(100),259, 260(432),264(432), 301, 321 Svetashev, V. I., 391, 409(79), 410(79), 411(79),4l2(79),415(79),421(79),422(79), 423(79),429(79),434(79), 435(79) Swan, B., 158, 163(51) Swanson, A. L., 358, 359(130) Sweeley, C. C., 285, 286,329(69),331(93), 389, 390, 393,398, 399,404, 407, 420(70) Swissa, M., 325 Symons, M. C. R., 86 Szczerek, J., 99,102 Szilagyi, L., 99, 100(46),l22(46)

T Tabas, I., 234,235,364 Taljora, E., 343 Tachibana, Y., 238

Tadano, K., 430, 437(314) Tagawa, K., 163, 186(72),247(72) Tago, M., 257 Tai, T., 232, 233(323), 237(323, 324), 238(323) Tajmir-Riahi, H. A,, 65 Tajmr, L., 124, 125, 143(109, 110) Takagaki, K . , 200 Takagi, M .,284 Takagi, S . , 15, 16(62),261, 262 Takahashi, H., 91, 93, 95, 99, 105(33,55, 6062), 106(60),110(82),111, 112(54,55, 6062, 80-83), 113, 114(90,91), 115(91), 116(90),117(17,55). 119(55, 98). 120(61, 81, 99), 121(61, 84). 139(33),140(54, 55, 60-62, 80-85, 90, 91, 98, 99, IOO), 141(33,90). 142(33, 100) Takahashi, K., 149, 155(17) Takahashi, N., 216, 232, 239, 240, 372(195), 373, 382(195) Takahashi, R., 179, 181 Takai, M . , 325 Takao, S . , 288 Takasaki, S., 240 Takayama, K., 284,329 Takeda, H., 419, 420(261) Takeda, K., 91, 93, 113, 114(90),117(17), 119(98),140(90,98), 141(90) Takeda, S . 155, 156(40) Takeda, Y., 252,255 Takegawa, K. 237 Takemoto, H., 296 Takenishi, S., 160, 162,163(57) Takeshita, M., 316 Taketomi, T., 428 Takeuchi, E., 239 Takigawa, A., 179, lSO(l21) Taku, A., 305,332,333 Talmadge, K. W., 152,182, 183, 382 Tamaki, S., 333 Tamari, K., 268 Tamura, M .,257 Tamura, T., 332 Tanabe, T., 246 Tanaka, H . , 268,269 Tanaka, J., 284 Tanaka, M.,185 Tanaka, S . , 162 Tandecarz, J. S., 256, 360, 383(153) Tani, Y., 281 Tanida, S . , 281 Tanner, W., 346(38),347, 348(38, 59), 349, 353(59,93, 94),354, 355, 356(59,

465

AUTHORINDEX 93, 109, lll,116),362, 363(111, 162), 364(lll),369,370, 372(109, 165,191, 192). 377(192) Tao, R. V. P., 399 Tarantino, A. L., 237, 238, 239 Taravel, F. R., 168, 176(1l2) Tarelli, E., 327 Tarentino, A. L., 232, 238(352) Tashpnlatov, A. A,, 99, 140(65) Taylor, C., 259 Taylor, I. F., 269, 275(468) Taylor, K. G . , 29 Taylor, R., 16 Tayot, J.-L., 393 Tejima, S.,216, 239 Telser, A,, 202, 203 Tesche, N., 285, 324(76),325(76) Tettamanti, G., 388(31),389, 431 Thanh, V. H., 372(198),373 Thibault, J.-F., 184 Thom, D., 251 Thomas, G. J., Jr., 84 Thomas, J., 380 Thompson, H. W., 93, 139(14) Thorne, K. J., 330 Thorne, K. J. I., 350, 351(65) Thornton, E. R., 407 Thorpe, S. J., 331 Thunberg, L., 213 Thurman, P. F., 327 Thurow, H., 218,220(305),224(305) Tiller, P. R., 230 Timell, T. E., 158, 159(49),161 Tinelli, R . , 197 Tipper, D. J.. 196, 197(189),277, 278(2, 3) Tipson, R. S., 8, 11(16),16,(16, 17),17(16), 18(13),19(13, 16), 20(16, 17). 21(13), 25(15),56(16, 17). 65(15),66(17),67(16, 17) Tjian, R., 196 Tjaden, U. R., 395 Tkacz, J. S., 234 Tobin, M.-C., 9 Tochikura, T., 237, 281, 282(22),287 Toda, N., 208 Tohyama, T., 258, 260(427) Tokuyama, K., l24(139),125, 137, 145(138, 139) Tolmasky, M. E., 276, 285, 305, 321(85, 266), 355, 356(114,119),357(114), 376 Toman, R., 162 Tominaga, Y.,198

Tomioka, S . , 333 Tomiyama, K., 380 Tomshich, S. V., 298 Tonellato, U., 94 Tonn, S. J., 278 Toppet, S., 99, 101(47),102(47) Torgov, V. I., 314, 319, 335, 336(439), 337(439),338(439) Torii, M., 258, 259(428),206(428) Tornheim, J., 204 Torri, G., 213 Toth, G., 198 Touster, O., 234, 235 Trams, E. G., 398 Traxler, C. I., 329 Trejo, A. G . , 299 Trimble, R. B., 382 Troitskiy, M. F., 124,126(120),135(120),284 Tronchet, J. M. J., 31 Trott, G. F., 29 Troy, F. A., 278, 285, 320(84),324(76), 325(76,358,360,361) Truchet, G . L., 379 Tsuboi, M., 30 Tsuchihashi, H., 248 Tsuji, M., 203 Tsujino, I., 192 Tsujisaka, Y., 160, 162, 163(57),198, 268 Tsumuraya, Y., 247,265,296 Tsutsui, Y., 185 Tu, A. T., 8, 75, 76, 80, 81(185, 188),82 Tukey, J. W., 7 Tul’chinsky,V. M., 11, 17(39),19(39),21(39, 71), 25(39), 29(71) Tulsiani, D. R. P., 235 Tung, K. K., 265,305 Tuppy, H., 246 Turco, S. J., 362, 363(161),364(161) Turner, J. C., 25 Turvey, J. R., 187, 189, 190(152),191, 192(173),193(173) TvaroSka, I., 16 Tylenda, C. A , , 296

U Uchida, T., 185, 284, 316(62) Uemura, K., 208, 241(264b) Uemura, S., 258 Ueno, Y., 179, 180(l21),267 Ugalde, R. A., 234, 285, 321(85), 355, 356(ll4), 357(ll4), 376

466

AUTHORINDEX

Ukai, S., 267 Ukita, T., 98 Ullman, M. D., 394 Ullrey, D., 288 Ulrich, H.-P., 148 Umbreit, J. N.,286, 331(94),332 Umekawa, M., 194 Umeki, K., 255 Umemoto, J., 240 Urnemura, J., 9, 29, 87(33) Umezawa, H., 389 Unger, F. M., 301 Unger, P., 302 Unkovskii, B. V., 99, 102(51-53) Urban, P. F., 389,390 Urbano, M. R., 379,380(247) Urbanski, T., 99, 102 Urey, H. C., 39 Ushioda, Y.,268 Usov, A. I., 284,426 Usui, T., l48,271(11b), 272(11b) Utkina, N. S., 314, 335, 336(439), 337(296, 439),338(439)

V Vadas, L., 290 Vagabou, V. M., 365 Valent, B., 380 Valentiny, M., ll2, 113(85) Vance, D. E., 393 VanDam, J. E. G., 217,219(300a) Vandegans, J., 84 VandeKamp, F. P., 94, 95(31), 114(31) Van den Eijnden, D. H., 244,246 VanderWoude, W., 358, 359(138), 366 van Halbeek, H., 217,219(300a) van Heijenoort, J . , 331, 333 van Heijenoort, Y.,331,333 Van Heyningen, W. E., 389 Van Hoeven, R. P., 395 Vanier, M. T., 399 Van Lenten, L., 286 Vann, W. F., 305,324(271) van Veen, R., 229 Vasko, P. D., 9, 26, 40(2E), 54, 75(28), 80(28, 163) V~kovsky,V. E., 390,391(66), 409(79), 4 W 8 , 79). 4U(79), 412(79), 415(79), 421(79), 422(79), 42466, 79), 429(78, 79),

434(79), 435(79) Vattuone, M. A., 187 Vattuone de Sampietro, M. A., 187 Vaver, V. A., 429 Vegh, L., 124,126,135(121),144(l21) Vella, G., 244 Venerando, B., 388(31),389 Vengris, V. E., 389 Vergoten, G., 34, 75(145) Vergunova, G . I., 284 Versluis, C., 400, 402, 403(169) Verstraeten, L. M. J., 18,20(72), 25(72) Veruzzo, D., 352 Vethaviyasar, N., 93, 95, 127, 142(20, 21), 144(20,21) Vicker, M. G., 388, 390(20) Vigevani, A,, 28 Vignon, M.R., 218 Vijay, I. K., 285, 324(76), 325(76, 358) Villa, T. G., 266 Villaneuva, J . R., 266 Villemez, C. L., 179, 185,349,358, 359(128, 130, 142, 143, 145,151). 360,366 Vincendon, G., 388(29), 389 Vink, J., 403 Vinogradov, E. V., 290,297,301 Vithanage, H. I. M. V., 381 Vlaovic, M., 305 Vliegenthart, J. F. G., 283, 400, 402, 403(169),217, 219(300a) Vodnansky, J . , 19 Volk, W. A., 293 Volkova, L. V., 317 Volkovich, G . , 353(85), 354, 356(85) von Figura, K., 148,212 Voragen, A. G . J., 149, 183, 184(133), 185(133) VrSanskB, M., 160, 163(62)

W Wadstrom, T., 196 Waechter, C. J., 342,352,354,355(103), 363(ll), 364(ll), 367(ll) Wagner, E. L. 92 Wahl, H. P., 294 Waibel, R., 166,167(104) Wakabayashi, K., 151 Wako, K., 254 Walczak, E., 99, 106(67), 107(67),ll2(67), 140(67)

AUTHORINDEX Walden, P., 92 Walker, B . , 399 Walker, D. E., 149 Walker, F . , 383 Walker, G. J., 258, 259(423, 426), 260(426, 432), 262(431), 264(432) Walkinshaw, M. D., 166 Wall, T. T., 86 Wallace, D. H . , 381 Wallace, R., 298 Walrafen, G. E., 21, 73(82), 85, 86 Wan, C . C., 408 Wang, M. C., 268 Wang, S.-F., 291 Ward, J. B., 277, 278(4), 285, 327(90), 331 Ward, L., 291 Wardlaw, A. C., 197 Warren, C. D., 314, 354 Warren, L., 387, 390, 400, 422, 423(64, 274), 437(274) Warmer, L., 283 Warth, A. D., 305 Wasteson, A., 206,212 Watanabe, K., 388, 394, 395, 408, 409(242), 420 Watanabe, T., 149, 155(17),255 Watkins, W. M . , 246 Weber, M., 252 Webley, D. M., 269 Weckesser, J., 290, 298,300, 304 Wedgwood, J. F.. 352 Weidmann, H., 29 Weigel, H., 53 Weigl, J., 251 Weiner, I. M., 313, 315, 316(307),342 Weinhouse, H., 325 Weinstein, D. B., 387 Weinstein, L., 186 Weisgerber, C., 285, 319(80) Weissmann, B., 203,204 Welbourn, A. P.,295 Welburn, A. R., 346(34, 36, 37, 41). 347 Welfle, H. 99, 105(59) Wells, C. H. J., 12,36(40) Welsh, E. J., 166 Wenger, D. A , , 404,405(203) Weppner, W. A., 332 Wessels, J. G. H., 267, 268(446) Weston, A,, 331 Westphal, 0..286 Westrick, M. A., 394

467

Wetzel, R., 30, 84(136) Wheat, R. M., 292 Wheeler, H. L., 113 Whelan, W. J., 253, 256, 360 Wherrett, J. R., 387 Whiffen, D. H., 8, lO(7-9, 11). 18(7-9, 11). 19(7-9, ll),20(8, 9), 21, 40(7, 8), 53 Whistler, R. L., 166, 171 Whitaker, D. R., 159 White, J. W., 23 Whitehouse, M. W., 374 Whitfield, C., 157 Whitmore, R. E., 82, 83(196) Whittle, K. J., 345, 346(32), 348(32) Whyte, J. N. C., 186 Wicken, A. J., 277 Wickramazinghe, N. V., 31 Wickus, G. G., 305 Widrnalm, G . , 230 Wiegandt, H., 388, 400, 401, 423, 427(286), 432(173) Wiegant, V. M., 389 Wieniawski, W., 99(70, 71, 72), 100, 1O6(63, 64,66-76), 107(67,68,69, 70, 71), 108(70-73). 109(73-76). 112(63,64,6676), 140(63,64.66-69) Wilberly, S. E., 10 Wilhelms, A., 92, 93(8), 94, 123, 142(8), 144(8) Wilkie, K. C. B., 158, 159(50),160 Wilkinson, J. F., 305 Wilkinson, S. G., 230,286, 293,295(187), 298, 300, 302, 312(97),318(97) Willcox, A., 99, 101(47),102(47) Willers, J. M . N., 217, 219(300a) Williams, G. J., 93, 95, 128,144(22, 23) Williams, R. J., 384 Williams, R. M., 83, 84(199) Williams, T. P. 189 Williamson. F. B., 189, 190(160) Williamson, I. R.,165 Wilson, B. W., 405 Wilson, D. B., 287,323 Wilson, E. B., Jr., 9, 32 Wilson, G., 269 Wilson, S., 390, 391(59) Winchester, B., 235 Windust, J . , 167, 170(106) Winkler, N. M., 292,296(163) Winterbourn, C. C., 393 Wirth, D. F., 362, 363(161), 364(161)

468

AUTHOR INDEX

Wiss, O., 346(47), 347 Witczak, Z. J., 91, 122, 123 Wiiber, G., 252 Wojtowicz, M., 99(70), 100, 106(70, 73, 75, 76, 77, 78). 107(68, 70), 108(70, 73), 109(73, 75, 76, 77), 110(78), 112(68,70, 73, 75-78), 122(78),140(68) Wolf, F.,380 Wolfe, R. S., 198 Wolfrorn, M. L., 30 Wolf-Ullisch, C . , 319 Wong, L. J . , 282 Wong, T. K., 352 Wood, E., 282 Wood, R., 399 Wood, T. M . , 151 Woods, A., 216 Woodside, E. E., 29 Woodward, J. R . , 274, 275(484a) Woolsey, G. B., 86, 87(213) Wrangsell, G . , 230 Wright, A,, 277, 278(2, 3),284,285, 305, 310, 313(60, 81), 314, 315(278), 316(60), 342 Wu, S., 353(92), 354, 366(92) Wu, T. C. M., 290 Wursch, J., 346(47), 347 Wyss, H. R., 86

Y Yadomae, T., 248 Yaegashi, Y., 199 Yagi, Y., 198 Yajima, H., 84 Yalpani, M., 171 Yamada, H., 304 Yamada, T., 208, 241(264) Yamagata, T., 148, 199(8), 205, 206(247) Yarnaguchi, H., 209, 216(270), 237,256 Yarnaguchi, M.,253 Yamaguchi, Y., 179,180(121) Yarnakawa, T., 388, 389(8), 390(8), 394, 399, 401,426,430,437(315) Yarnaki, T.,258 Yarnarnori, S., 285, 288 Yarnarnoto, A., 344,392 Yarnarnoto, K., 237, 287 Yarnarnoto, R., 148, 272(llc), 275 Yamarnoto, S., 247, 266, 269, 275 Yamarnoto, T., 184, 419, 420(264)

Yamamura, Y., 247, 300 Yarnashita, K., 232, 233(323), 237(323, 324), 238(323) Yarnauchi, F., 372(198),373 Yamauchi, R., 267 Yarnazaki, T., 394 Yarnodae, T., 288 Yang, H., 399, 401(164) Yang, R. T., 61 Yaphe, W., 186, 187(145),188(145),190, 251 Yasuda, Y., 232, 372(195),373, 382(195) Yasui, T., 160, 162 Yasurnoto, T., 422 Yeow, Y. M., 380 Yogeeswaran, G., 387.388 Yokobayashi, K., 254 Yokotsuka, T., 182 Yokoyama, K., 288 Yoneyama, T., 288 Yoshida, K . , 205 Yoshida, M., 98 Yoshikawa, M , , 162 Yoshinaga, H., 22, 28 Yoshino, T., 388 Yoshioka, I., 422 Yoshizaka, H., 416 Yosizawa, Z., 205, 210, 211, 212, 213 Young, D. W., 14, 15(53) Young, F. E., 330 Young, G . , 166 Young, J. D., 362 Young, K . , 188 Young, M . N., 293 Young, W. W., Jr., 388 Yu, N., 228 Yu, R. K., 388, 389, 390, 392, 393, 400, 403(170), 405, 407, 430, 437(313) Yuan, R., 313, 315(290) Yuan, Tse-Yuen, R., 285,311(82) Yuasa, R., 316 Yule, K. C., 12.1,135, 137(136) Yung, S. G., 281 Yunker, M. B., 31 Yurchenko, N. N., 336 Yurewicz, E. C., 320 Yurgi, T., 25

L

Zakharova, I. Ya., 292 Zalitis, J . , 290

AUTHORINDEX

Zamoclj, J., 336 Zanetta, J. P., 388(29), 389 Zarkowsky, H., 291 Zechmeister, L., 198 Zehavi, U., 293 Zeleznick, L. D . , 282 Zelsmann, H. R., 63 Zemek, J., 336 Zemell, R. I., 305 Zerbi, G . , 13, 33, 40(44) Zhbankov, R. G., 17, 19, 21, 40,41(152), 43(77), 49(153), 50(70, 153),52, 53(159), 86 Zhukova, I. G., 390, 391(66), 397, 399(138), 403, 409(79), 410(78, 79), 411(79), 412(79), 415(79), 421(79), 422(79), 423(66, 79, 138),424(138), 429(78, 79). 430(192), 434(79),435(79)

469

Ziegler, H., 381 Zikakis, J. P., 374 Zinkel, D. F., 346(45), 347 Zinn, A. 2.. 237 Zollo, F., 422 Zolotarev, B. M., 403 Zoppetti, G., 213 Zorreguieta, A., 276 Zubkov, V. A., 299 Zubkova, 0. B.,35,36(146) Zurabyan, S. E., ll, 17(39), 19(39), 21(39, 71). 25(39), 29(71), 95, 104(34),113, 116(9294), 140(92-94, 95), 141(34), 142(34, 95) Zvezdina, N. D., 424, 427(289, 290), 435(290),436(290)

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SUBJECT INDEX

A

-,

4,6-0-benzylidene-3-deoxy-3-thio-

cyanato-, 143 Acetyl esterase, 162 -, 4,6-O-benzylidene-2,3Acetylglucosaminidase H, endo-P-N-, 370 dideoxy-3-(dimethylaniino)Additive model of atomic interaction, 40, 52 2-thiocyanato-, 143 Adenosine 5’-diphosphate. glycosyl esters, synthesis, 134 280. See also Glycosyl nucleotides Altrose Agar, 186 -, D-, 4-S-acetyl-6-deoxy-4-thio-, syn&draw, 187-190 thesis, 132 Agaropectin, 186-187 -, 6-deoxy-~-,biosynthesis, 296-298 Agarose Altruronic acid, 2-amino-2-deoxy-~-, bioiodine complex, laser-Raman spectroscopy, synthesis, 296-298 84 Amylase related polysaccharides, structnre, enalpha, 252, 254, 257 zymic analysis, 186-190 beta, 255 structure, enzymic analysis, 186-190 Amyloids, 151 Alanine amidase, N-acetylinurainoyl-I.-, 196 Amylopectin Aldohexopyranosyl cyanides, per-0-acetybiosynthesis, transglycosylation reaction, lated, laser-Ranian spectroscopy, 81 256 Aldopyranoses, conformations of, 13 conformation and tautomers, vibrational Aldoses spectra used to analyze, 26 IAiructose-derived, biosynthesis, 287-299 potato, structure, enzymic analysis, 255 u-ribose-derived, in bacterial polysacstructure, enzymic analysis, 253 charides, biosynthesis, 299-300 structure-properties relationships, vibrao-sedolieptulose-derived, in bacterial polptional spectroscopic study, 88 saccharides, biosynthesis, 300-301 Amylose Algaprenol, structure, 346 conformation and tautomers, vibrational Alginicacid spectra used to analyze, 26 Ascophyllutn nodosum, 191, 194-195 *hydrogen bonding, Ranian and infrared Azotobacter oinelandii, 193 spectral study of, 29 FUCUS oesiculosus, 191 iodine complex, laser-Raman spectroscopy Lnrninaria digitafa, 194 84 structure, enzymic analysis, 191-195 laser-Ranian spectroscopy, 82 Allofuranose structure, enzymic analysis, 252-256 -, 3-deoxy-1,2-O-isopropylidene-3-thiostructure-properties relationships, vibracyanato-a-o-, 143 tional spectroscopic study, 88 -, 3-deoxy-1,2:5,6-di-O-isopropylidene-3- vibrational spectra, isotopic substitution thiocyanato-a-o-, 143 studies, 55 -, 5,6-di-O-acetyl-3-deoxy-1,2-O-isoApiogalacturonan. 185 propylidene-3-tIiiocyanato-a-~-,143 Aqueous solutions Allose, D-, biosyntliesis, 296-298 Fourier-transform infrared spectroscopy, Almond glycopeptide N-glycosidase, 216 61 Altropyranoside, methyl a - ~ laser-Raman spectroscopy, 73-75 -, 2,3-di-O-benzyl-4,6-dideoxy-4-thiovibrational spectroscopic studies, 85-86 cyanato-, 143 Arabinan, 183 -, 4.6-0-benzylidene-2.3from plant cell-walls, 359 dideoxy-3-(dimethylarnino)Arabinofuranosidase, a+, 159, 163, 185-186, 2-thiocyanato-, 143 247 471

472

SUBJECT INDEX

Arabinogalactan, 182 soybean, 184 structure, enzyinic analysis, 247 Arabinogalactorhamnogalacturonan,182 Arabinoglucuronoxylan, enzymic analysis, 161 Arabinopyranosyl isothiocyanate, 2,3,4-tri-Oacetyl-a-o-, 141 13C n.m.r., 141 formation of amino acid diastereoisomers using, 117 infrared spectrum, 141 Arabinose, 5-acetamido-5-deoxy-~-,ring isomers, ir spectra, 25 Arabinoxylan corn-cob, enzymic analysis, 160 enzymic analysis, 160-161 oat-spelt, enzymic analysis, 163 soybean, enzymic analysis, 162 wheat-bran, enzymic analysis, 163 wheat-flour, enzymic analysis, 159, 163 Arabinoxyloglucan Nicotiana tabacum, enzymic analysis, 1% tora bean, enzymic analysis, 154 Arthropods, glycolipids. See Glycolipids Asialo-fetuin glycopeptide fraction C, 240 Asialo-orosomucoid, 233 Aspergillus oryzae exo-enzyme, 154, 156

B Bacterial amphiphiles, 277 Bacterial cell-walls, synthesis, 342 Bacterial lipopolysaccharides, 277 0-specific chains, biosynthesis, 312-319 block mechanism, 312-318 monomeric mechanism, 318-319 structure, enzymic analysis, 230-231 Bacterial peptidoglycans carbohydrate chains, assembly, 330-333 structure, enzymic analysis, 195-198 Bacterial polysaccharide chains composed of oligosaccharide repeating units, biosynthesis, 278-339 groups, 277 monosaccharide components, biosynthesis,

286-302 Bacterial polysaccharides biosynthesis, 278-279 glycosyl esters of nucleotides and polyprenyl glycosyl phosphates in, 279-

286

biosynthesis of polymeric chains for, activation of monosaccharides for, 302-

303 biosynthetic classification, 334-335 branched-chain monosaccharides, biosynthesis, 299 capsular, 277 disaccharide fragments, 307 having most common monosaccharides at nonreducing end, 307-308 isomeric, composed of most common monosaccharides, 308-309 enzymic synthesis, from modified precursors, 335-339 exocellular, 277 biosyn thesis block mechanism, 320-323 by unidentified mechanism of chain assembly, 326-327 monomeric mechanism, 323-326 structure, 376 extracellular, 277 Acinobacter, enzymic analysis, 230 Agrobacterium, enzymic analysis, 225,

226 Alcaligenes, enzymic analysis, 225, 226 Klebsiella enzymic analysis, 217-225 phage hydrolysis, oligosaccharides released by, 218-224 phage-induced hydrolysis, 228-230 Rhizobium, enzymic analysis, 225229 furanose monosaccharides, biosynthesis, 298-299 of Gram-positive cell walls, biosynthesis block mechanism, 327-328 monomeric mechanism, 328-329 unidentified mechanism of chain assembly, 329-330 hexose components. See also Hexoses of configurations other than gluco, galacto, and tnanno, biosynthesis, 295298 inter-monomeric linkages in, 305-309 linkage region, 278 monosaccharides modifications of functional groups in, 302-305 structurally related to o-fructose, biosynthesis, 298-299

473

SUBJECT INDEX

structures, 302-303 0-specific. See also Bacterial lipopolysaccharides biosynthesis, 290-293 pentoses, hiosynthesis, 298 polymeric chains assembly, 309-335 mechanisms, 310-312 structure, and mechanism of assembly,

333-335 structure, enzymic analysis, 217-231 Beechwood glucuronoxylans, spectral analysis and identification, 24 Betaprenol, structure, 346 Betulaprenol, structure, 346 Bivalves, glycolipids. See Glycolipids Boric acid, carbohydrate complexation with, infrared and Raman spectroscopic study of, 30 Brachiopods, glycolipids. See Glycolipids Bromelain, pineapple-stem, 232

C Callose, 273 Caramel colorants, spectral analysis and identification, 24 Carbohydrates. See also Food carbohydrates anomeric region, 11, 19 CH,OH group, determination of rotational isomerism, 53 conformaion, vibrational spectra used to analyze, 25-26 conformation and interactions of, vihrational spectroscopic study, 87-88 deuterated, interpretation of spectra of,

53-55 fingerprint region, 11, 17, 19 hydrogen bonding, 15-16 Raman and infrared spectral study of,

28-30 vibrational spectroscopic study, 87 infrared spectra, at low temperatures, 27-

28 infrared spectroscopy, correlated to specific chemical structures, 10 molecular structure, vibrational spectroscopic study, 86-87 orientation, infrared dichroism study, 26-

27

structural analysis of, 11 structure, and atomic coordinates, 13-15 structure factors in, 11-16 structure-properties relationships, 88-89 symmetry operation, I2 tautomers vibrational spectra, analysis of intensities

of, 35-36 vibrational spectra used to analyze, 25-

26 vibrational spectra, 17 frequency region of below 700 cin

1, 17, 21-22 frequency region of 950-700 cm -1, 17, 19-21,43-45 frequency region of I2OO-950 cm - I , 17, 19 frequency region of 1500-1200 cm-l, 17-19,43-46 frequency region of 3600-2800 cni - I , 17,18 Carboxypeptidase Y, 370 K-Carrageenanase. 251 Carrageenans, structure, enzymic analysis, 251-252 Castaprenol, structure, 346 Cathepsin C, 217 Cell-membrane glycoproteins, 232 Cellobiohydrolase. (I+ 4)-P-~-glucan,Trichodenna oiride, 149 Cellobiose aqueous, vs. solid, laser-Raman spectroscopy, 75 vibrational spectra frequency calculations, 49-50 isotopic substitution studies, 54 -, p-, hydrogen bonding, Raman and infrared spectral study of, 29 Cellulase, 151, 273 Cellulose biosynthesis, 360-362 conformation and tautomers, vibrational spectra used to analyze, 25 hydrogen bonding, Raman and infrared spectral study of, 29 laser-Raman spectroscopy, 82,83 orientational measurements in, 27 structure, enzymic analysis, 150-151 Valonia uentricosa, structure, 26 Cellulose I normal coordinate analysis, 46 ~

474

SUBJECT INDEX

vibrational spectra atomic displacements for frequencies of, 46-51 calculated frequencies and computed potential-energy distribution of, 46-49 Cellulose oligosaccharides, conformation and tautomers, vibrational spectra used to analyze, 25 Cephalopods, glycolipids. See Glycolipids Cerebrosides, 437-438 in coelenterates, 411 laser-Raman spectroscopy, 84 niannose-containing, from freshwater bivalves, 420 in sponges, 410 in starfish, 429 Chemical-enzymic synthesis, 335 Chemical reactions, vibrational spectra in study of, 30-31 Chitin biosynthesis, 373-375 Fourier-transform infrared spectroscopy, 63 laser-Raman spectroscopy, 83 orientational measurements in, 27 structure, enzymic analysis, 198 Chitinase, 198 Chitin synthase, 374-375 Chitosan, structure, enzymic analysis, 198 Chondro-4-sulfatase, 200 Chondro-6-sulfatase. 200 Chondroitin 4-sulfate, Fourier-transform infrared spectroscopy, 61 Chondroitin ABC lyase, 199, 205 Chondroitin AC lyase, 148, 205-207 Choiidroitinase AC, 216 Chondroitin B lyase, 205 Chondroitin sulfate ABC lyase, 216 Chondroitin sulfates, structure, enzymic analysis, 198-203 CMP-N-acetyl-neuraminate o-galactosylglycoprotein transferase, 246 Coelenterates, glycolipid content. See Glycolipids Colchicines, N-deacetyl-N-(per-O-acetyl-Dglucopyranosylthiocarbamoy1)(methylthio), synthesis, 102-103 Complex carbohydrates, biosynthesis, in plants, 358-377 regulation mechanisms, 376-378

Crystalline structures vs. compounds in solution, vibrational spectra, 14, 16, 21 orientation, infrared dichroism study, 2627 Curdlan, gelation, Fourier-transform infrared spectroscopy, 62-63 Cyclohexanediols, laser-Raman spectroscopy,

83 Cyclomaltoheptaose, laser-Raman spectroscopy, 82 Cycloinaltohexaose. laser-Raman spectroscopy, 82 Cytidine 5'- (N-acetylneuraminic monophosphate), 283. See also Glycosyl nucleotides Cytidine 5'-diphosphate, glycosyl esters, 280. See also Glycosyl nucleotides Cytidine 5'-monophosphate, glycosyl esters, 280. See also Glycosyl nucleotides

D 3-Deoxyaldulosonic acids, in bacterial polysaccharides, biosynthesis, 301-302 Dermatan sulfate, 199 structure, enzymic analysis, 205-207 Deuterium-substitution method, in assignment of vibrational frequencies, 53-55 Dextran aqueous, vs. solid, laser-Raman spectroscopy, 75 Fourier-transform infrared-difference spectroscopy, 61-62 laser-Raman spectroscopy, 82 Leuconostoc, structure, enzymic analysis, 258-261 Streptococcus, structure, enzymic analysis, 263-264 structure, enzymic analysis, 258-264 vibrational spectra, isotopic substitution studies, 54 Dextranase, 258 Dextranglucosidase, 258-259 Dextrin p-limit, structure, enzymic analysis, 254255 Nageli, structure, enzymic analysis, 255 Di (neoagarobiose) hydrolase, p-O-, 190

475

SUBJECT INDEX Disaccharides C-C and C - 0 bond-lengths in, 14-15 conformational analysis, 14 hydrogen bonding, 15-16 laser-Raman spectroscopy, 75-81 Dolichol biosynthesis, 350 metabolism, 350-352 plant sources, 348 structure, 346 Dolichyl phosphate biosynthesis, 350 metabolism, 350-352

multiplex advantage, 57-58 quantitative analysis of mixtures, 58-60 spectral results, 61-67 time-resolved techniques, 61 Frost resistance, in plants, 383-384 Fructofuranosyl a-D-ghcopyranoside, 6,6‘-

dideoxy-6,6‘dithiocyanato-p-n-, 1,2,3,4,3’,4’-hexa-O-(methylsulfonyl)-, 143 -, 2,3,4,1‘,3‘,4’-hexa-O-acetyl-, 143 -, 2,3,4,lf,3’,4’-hexa-O-benzoy1-, 143 Fructopyranose, p-D-,molecular structure, vibrational spectroscopic study, 87 Fructose spectral analysis and identification, 24 D-

E Echinoderms, glycolipids. See Glycolipids Elsinan, structure, enzymic analysis, 265 Erythrocyte, P. oulgaris lectin receptor-site, 232 Extensin, 382 External symmetry coordinates, 33

F Fast-Fourier-transform algorithm, 7 Fetuin glycopeptide, 240 Ficaprenol, structure, 346 Food carbohydrates, analysis and identification, noncomputer spectroscopic methods, 22-24 Force constants, in normal coordinate analysis, transfer from simple molecules to carbohydrates, 31 Force field, models, in vibrational spectra band assignments, 38-39 Fourier-transform infrared spectroscopy, 79, 56-67 absorbance subtraction, 60-61 advantages of, 58, 66 application to hiological systems, 58 data-processing techniques, 58-61 factor analysis, 60 Fellgett advantage, 57-58 frequency-accuracy advantage, 58 Jacquinot’s advantage, 58 method, 56-58

aqueous laser-Raman spectroscopy, 73-74, 7678 solute-solvent interactions, vibrational spectroscopic studies, 86 structure-properties relationships, vibrational spectroscopic study, 88 Fructose 1,6-bisphosphate, laser-Raman spectroscopy, at varying pH, 81 Fucosidase, a - ~ - 154, , 209, 233 Furanoses, conformation and tautomers, vibrational spectra used to analyze, 25

G Galactan, 182 from plant cell-walls, 359 -, (1 .--) 4 ) - ~ -biosynthesis, , 366 -, D-arabino-D-, structure, enzymic analysis, 247 -, L-arabino-D-, structure, enzymic analysis, 247 Galactanase, p-D-, 247 (1+ 3)-,247 Galactocerebrosides, 437 from marine bivalve, 415 in sea anemone, 411 Galactoglucomannan, 164 Cercis siliquastrum, 180 Nicotiana tabacum, 181 structure, enzymic analysis, 180-186 Galactomannan A. niger, 366

-.

476

SUBJECTINDEX

Caesalpina pulcherima -, 2,3-di-O-benzoyl-4-deoxy-4-thioenzymic analysis, 170-171 cyanato-, 143 hydrolysis, effect of fine structure on, -, 2,3-di-O-benzoy1-4,6-dideoxy-4,6174 di(thiocyanat0)-, 143 Caesalpinu spinosa synthesis, 126 enzymic analysis, 170 -, 4,6-dideoxy-4,6-di(thiocyanato)-, synhydrolysis, effect of fine structure on, thesis, 125 174 -, 2,3,4-tri-O-acety1-6-deoxy-6-thioCaesalpina vesicaria cyanato-, 143 enzymic analysis, 170 Galactopyranosyl isothiwyanate, 2,3,4,6hydrolysis, effect of fine structure on, tetra-O-aCetyl-$-D-, 141 174 Galactosaminidase, endo-N-acetyl-a+-, carob, 176-177 240 enzymic analysis, 167-168.170 D-Galactose hydrolysis, effect of fine structure on, anomers, correlation between CH orienta174 tion and vibrational frequencies Cassiafistula, hydrolysis, effect of fine observed, 21 structure on, 174 C-C and C - 0 bond-lengths in, 15 Cyamopsis tetragonolobus, enzymic analy- D-Galactose dehydrogenase, 232 sis, 170 D-Galactose oxidase, 186, 234 enzymic hydrolysis, effect of fine structure Galactosidase on, 174 a-D-, 165-167 D-galactose distribution, 166 Aspergillus niger, 229-230 Gleditsa triacanthos, 173,175 $-D-. 154,186, 202,208-209, 247 enzymic analysis, 170 -, endo-p-D-, 148,216,240-242,408 hydrolysis, effect of fine structure on, Galactoside, methyl-$-o-, C-C and C-0 174 bond-lengths in, 15 Cleditsia feror, enzymic analysis, 167 Galactosyltransferase guar D-, 203, 371 hydrolysis, effect of fine structure on, UDP-, 244 174 -, 4-p, 202 a-D-galactosidase modified, hydrolysis, Galacturonan, 182 effect of fine structure on, 174 from plant cell-walls, 359 Leucaena leucocephala, 173-175 Galacturonanase, endo-(1 + 4)-a-~-,183enzymic analysis, 168 184 hydrolysis, effect of fine structure on, Gangliosides, 388-390 174 Glucanase from plant cell-walls, 359 -, (1-* 3)(4)-p-D-, 273 Pusa mosami, enzymic analysis, 170-171 -, endo-(I + 3)-a-~-,258 Sophora japonica, enzymic analysis, 165 -, endo-(l- 6)-a-o-, 258 soybean, enzymic analysis, 168 -, endo-(I + ~)-P-D-,267,272 structure -, endo-(1- 4)-$-~-,180 enzymic analysis, 165-178 -, endo-(1 + 6)-p-~-,225-226.272 model, 176 -, eXO-(l+ 4)-a-D-, 262 Galactopyranoside, methyl WD-, eXO-(l+ 3)-p-D-, 149, 266 -, 2,3-di-O-acetyI-4,6-dideoxy-4,6-di(thio- Eisinia bicyclis, 148-149 cyanato)-, 143 Glucans synthesis, 126 D-, based on (1+ 3)-p backbone and (1-, 2,3-di-O-acetyl-6-deoxv-6-thio3)-p chains, structure, enzymic analycyanato-4-O-p-tolyhlfonyl-, 143 sis, 266-273

SUBJECT INDEX a-D-

branched (1+ 4)(1+ 6)-,structure, enzymic analysis, 252-256 enzymic cleavage, 147 structure, enzymic analysis, 252-266

477

-, 1,3,4,6-tetra-O-acetylyl-2-deoxy-2-thioCyanatO-a-D-, 143 -, 1,3,4,6-tetra-O-acetyl-2-deoxy-2-thiocyanato-P-o-, 144

1,2,3,4-tetra-O-acetyl-6-deoxy-6-thiocyanato-a-D-, 143 -, 1,2,3,4-tetra-O-acetyl-6-0-pbased on (1-+ 6)-p chains, structure, enzymic analysis, 275-276 tOlylSUlfOnyl-p-D-, 144 -, 1,3,4-tri-O-acetyl-6-deoxy-6-thiobiosynthesis, 366, 367 cyanato-2-O-p-tolylsulfonyl-a-~-, 143 cyclic (1+ 2)-, structure, enzymic analya-D-GhCOpyranOSe derivatives, vibrational sis, 276 enzymic cleavage, 147-149 spectra, 20 Glucopyranoside, methyl a - D structure, enzymic analysis, 266-276 C-C and C-0 bond-lengths in, 15 -, (1+ 3)(1+ ~)-u-D-, structure, enzyme analysis, 265-266 -, 2-acetamido-3-O-acetyl-2-deoxy-4,6di-0-(methylsulfony1)-, SN2 nu-, (1+ 3)-p-, from plant cell-walls, 359 cleophilic displacement, 125-126 -, 2-0-acetyl-4,6-dideoxy-4-thiocyanato-, -, (1+ 4)-p-, from plant cell-walls, 359 -, (1+ 3)(1+ 4)-p-D-, structure, en144 -, 4-deoxy-4-(thiocyanato)-, synthesis, 126 zymic analysis, 273-275 -, 4,6-dideoxy-4-thiocyanato-2-O-pGlucocerebrosides, 437 tolylsulfonyl-, 144 in sponges, 410 -, 4,6-dideoxy-4-thiocyanato-2-O-pGlucodextranase, 258 Glucofuranose, a-Dtolylsulfonyl-3-O-(trimethylsilyl)-, 144 -, 6-deoxy-l,2:3,5-di-O-isopropylidene-6- -, 2,3-di-O-acetyI-4,6-dideoxy-4,6-di(thiocyanato)-, 144 isothiocyanato-, 141 -, 3-deoxy-l,2-O-isopropylidene-3-thio-, 2,3-di-O-acetyl-4,6-dideoxy-4-thiocyanato-, 144 cyanato-, 143 -, 3-deoxy-1,2:5,6-di-O-isopropylidene-3- -, 2,3-di-O-acetyl-6thiocyanato-, 143 deoxy-4-O-(methylsulfonyl)-6-thiocyanato-, 144 -, 5,6di-O-acetyl-3-deoxy-l,2-O-iso-, 2,3-di-O-benzoyl-4,6--dideoxy-4-thiopropylidene-3-thiocyanato-, 143 cyanato-, 144 Glucofuranoside, methyl p-D-,3-deoxy-3thiocyanato-2-O-p-tolylsulfonyl-5-O-tri-, 2,3,4-tri-O-acetyl-6-deoxy-6tyl-, 143 thiocyanato-,144 -, 2,3,6-tri-O-benzoyl-4-deoxy-4-thio258 Glucohydrolase, exo-(1+ 6)-a-~-, cyanato-, 144 Glucomannan, 164 Glucopyranoside, methyl p-D from plant cell-walls, 359 -, 2,3-di-O-acetyl-6-deoxy-6structure, enzymic analysis, 178-180 thiocyanato-,l44 synthesis, 366 -, 3,4,6-tri-O-acetyl-2-S-(N-acetykhiocarD-Ghconyl isothiocyanate, 2,3,4,5,6penbamoy1)d-thio-, synthesis, 131 ta-0-acetyl-,141 -, 2,3,4-tri-O-acetyl-6deoxy-6thioreaction with diamines, l20 cyanato-, 144 Glucopyranose -, 3,4,6tri-O-acetyl-2-deoxy-2-thio-, 2-acetamido-2-deoxy-~-,laser-Raman cyanato-, 144 spectroscopy, 83 -, 1,3,4,6-tetra-O-acetyl-2-,6dideoxy-2,6 -, 3,4,6-tri-O-acetyl-2-thio-2-S-(thiocarbamoy1)-, synthesis, 131 di(isothiocyanato)-a-D-,141 Glucopyranoside derivatives, mutorotation, -, S-p-D-galactopyranosyl-4-thio-o-, syn2s thesis, 135 p-D-

-,

478

SUBJECT INDEX

Glucopyranosyl bromide Glucosaminide-(1- 4)-P-~-galac-, 6-deoxy-6-thiocyanato-a-~-, synthesis, tosyltransferase, N-acetyl-P-D-, 245123 246 -, 2,3,4-tri-O-acetyl-6-deoxy-6-thioGlucosaminyl-deacetylase, N-acetyl-D-, 215 Cyanato-a-D-, 144 Glucose Glucopyranosyl chloride, 3,4,6-tri-O-aceDtyl-2-deoxy-2-thiocyanato-a-~-, 144 anomers Glucopyranosyl isothiocyanate aqueous solutions vs. crystalline, -, 2-acetamido-4-0-(2-acetamido-3,4,6laser-Raman spectroscopy, 80 tri-O-acety~-2-deoxy-P-~-gluco$, 19-20 pyranosyl)-3,6-di-O-acetyl-P-~-, 142 correlation between CH orientation -, 2-acetamido-3,4,6-tri-O-acetyl-2-deand vibrational frequencies obOXY-P-D-, 142 served, 21 synthesis, 94 spectral differences, 52-53 -, 2,3,4,6-tetra-O-acetyl-P-~-,142, 144 vibrational spectra, calculation of freW n.m.r., 141 quencies, 39-46 formation of amino acid diastereoisomers aqueous using, 117 laser-Raman spectroscopy, 73-74, 76infrared spectrum, 141 78 synthesis, 94 vs. solid, laser-Raman spectroscopy, -, 3,4,6-tri-O-acetyl-2-benzamido-2-de75 OXY-. 142 solute-solvent interactions, vibrational -, 3,4,6-tri-O-acetyl-2-deoxy-2-thiospectroscopic studies, 86 cyanato-a-D-, 141,144 C-C and C - 0 bond-lengths in, 15 -, 2,3,6-tri-O-acetyl-4-0-(2.3,4,6-tetra-O- cryoprotective effect, 89 acetyl-a-D-glucopyranosyl)-P-D-,142 determination of hydration numbers, I3C n.m.r., 141 87 infrared spectrum, 141 monohydrate, C-C and C-0 bond-, 2,3,6-tri-O-acetyl-4-0-(2,3,4,6lengths in, 15 tetra-0-acetyl-P-D-galactosolution, Fourier-transform infrared pyranosy1)-P-ospectroscopy, 61 13C n.m.r., 141 structure-properties relationships, vibrainfrared spectrum, 141 tional spectroscopic study, 88 -, 2,3,6-tri-O-acety1-4-0-(2,3,4,6-tetra-O- vibrational spectra, isotopic substitution acetyl-P-D-glucopyranosyl)-P-D-,142 studies, 54 13C NMR, 141 P-D-, vibrational spectra infrared spectrum, 141 atomic displacements for calculated -, 2,3,4-tri-O-acetyl-6-bromo-6-defrequencies, 41-46 Oxy-a-D-, 141 calculated frequencies, with potential synthesis, 94 energy distributions, 41, 44-45 -, 2,3,4-tri-O-acetyl-6-bromo-6-deobserved and calculated frequencies, OXY-P-D, 144 40-43 Glucosamine sulfatase, N-acetyl-a-D-, 214 hydrogen bonding, Raman and infrared Glucosaminidase spectral study of, 28-29 -, N-acetyl-a+, 214 mutorotation, 25 -, N-acetyl-P-o-, 196,208-209 spectral analysis and identification, 24 from jack bean, 229,230 -, 1,2,3,4-tetra-O-acetyM-de-, endo-N-acetyl-Pa-, 232 oxy-6-thiocyanato-a-~-,synthesis, glycoprotein structure examined with, I23 238-239 D-Glucose-procollagenglucosyltransferase, groups, 237-238 UDP-, 244

SUBJECT INDEX Glucosidase a-D-,

230,234,136

buckwheat, 149

p-D-, 149 almond emulsin, 149 Glucosiduronase, B-D-, 199-200,204, 214 Glucosylceramides, in starfish, 429 Glucosyltransferase, 262 D-, 179 membrane-bound, 284 Glucuronoarabinoxylan, wheat-straw, enzymic analysis, 163 Glycanase, 148 e m action pattern, 149 Glycoconjugates, structure, enzymic analysis, 231-246 Glycoenzymes, in plants, 382 Glycogen hydrogen bonding, Raman and infrared spectral study of, 29 laser-Raman spectroscopy, 83 Glycoglycerolipids, 387 Glycol, laser-Raman spectroscopy, 83 Glycolipids, 342,387 from arthropods, 421-422,436 from brachiopods, 421 from coelenterates, 410-4ll, 436 distribution in marine invertebrates, 436 from echinoderms, 422-434 class Asteroidea, 429 class Echinoidea, 422-428 class Holothurioidea, 434 from freshwater bivalves, 416-421 individual, separation, 395 from marine bivalves, 415-416 from marine worms, 411 from mollusks, 411-421 chss Bivalvia, 415-421 class Cephalopoda, 421 class Gastropoda, 412 class Loricata, 412 neutral, from Asteroides, 429-430 of rice bran, 413 from sponges, 409-410,436 fron tunicates, 434-435 Glycopeptide-N-glycosidase, 239 Glycoproteins, 342-343 biosynthesis, lipid-linked sugars as intermediates, 367-373 in host-pathogen interactions, 380

479

in plants biosynthesis, involvement of lipid intermediates in, 372 linkage between peptide and saccharide moieties, 372 Glycosaminoglycans crystalline, Fourier-transform infrared spectroscopy, 61 laser-Raman spectroscopy, 82 in plants, biosynthesis, 373-375 structure, enzymic analysis, 198-217 vibrational spectra, isotopic substitution studies, 55 Glycosidase, 147-148,231-233 from different sources, 149 Glycosides, 1-thio-p-, laser-Raman spectroscopy, 81 Glycosphingolipids, 387 [2-(methylamino)ethyl)phosphonic group, 397-398 (2-aminoethyl)phosphonicgroup, 397-398 (2-aminoethyl) phosphoric acid group bound to mannose, 398 carbohydrate chain structure chemical analysis, 399-402 determination, 399-409 enzymic analysis, 408-409 mass spectrometry, 402-406 n.m.r. spectroscopy, 406-408 physimhemical analysis, 402-408 composition of, 396-398 distribution, in marine invertebrates, 436 fatty acid composition, determination, 399 fatty acids, 396-397 from freshwater bivalves, 416-417 fucose-containing, 421 mannose-containing, 420,438 isolation, 392-394 mammalian, 387-388 from marine bivalves, 415-416 marine invertebrate, 387 monosaccharides, 397 occurrence, among marine invertebrates, 391-392 perrnethylated mass spectrometry, 404 n.m.r. spectroscopy, 406 of sea snail, 412 separation, 394-395 sphingosine bases, 396,398-399 structure, determination, 398-409

480

SUBJECTINDEX

vertebrate, composition of, 389-390 Glycosylation reaction, 278, 309, 342, 384 in plant glycoprotein biosynthesis, 371 Glycosylceramide, in pearl oyster, 415 Glycosyl nucleotides in biosynthesis of polysaccharide chains of bacterial polymers, 280 primary, 280-283 in bacteria, 281 secondary, 280-281 Glycosyltransferase, 150,306,343 in bacterial polysaccharide chain assembly, 310-311 biosynthetic, specificity, effects on structure, 244 membrane-bound, 283,305 Gram-negative bacteria, lipopolysaccharides, 277 Gram-positive bacteria, cell-wall polymers, 277 Guanosine 5'- (D-mannosyl diphosphate), 282. See olso Glycosyl nucleotides Guanosine 5'-diphosphate, glycosyl esters, 280. See also Glycosyl nucleotides L-Guluronan lyase, 192-194 Guluronic acid L-, biosynthesis, 296-298 -, 2,3-diamino-2,3-dideoxy-~-, biosynthesis, 296-298 Gum arabic, structure, enzymic analysis, 247

H Hen egg-white lysozyme, 195 Heparan sulfate, structure,enzymic analysis, 209-216 Heparan sulfate lyase, 210-214 Heparin, structure, enzymic analysis, 209216 Heparinase. See Heparin lyase Heparin lyase, 210-214 Heparitinase. See Heparan sulfate lyase Heparitin lyase. See Heparan sulfate lyase Heveaprenol, structure, 346 Hexahydro-ero-methylenepolyprenol,structure, 346 Hexahydropolyprenol, structure, 346 Hex-1-enitol -, 1,5-anhydro-~-arabino-,3,4,6-tri-0acetyl-2-deoxy-2-thiocyanato-, 144

-, 3,4,6-di-O-acetyl-l,5-anhydro-2,3-dideoxy-isothiocyanato-~-ribo-3-, 142

-, 3,4,6-tri-O-acetyl-l,5-anhydro-2-deoxy-2-isothiocyanato-~-arabino-, 142 Hex-3-enofuranose, a-D-erythro-, 3-deoxy-1,2:5,6-di-O-isopropylidene-, synthesis, 139

Hex-2-enop y ranoside -, ethyl a-D-erythro-, 4,deoxy-6-O-(methylsulfonyl)-4thiocyanato-, 144

-, ethyl a-D-threo6-azido-2,3,4,6-tetradeoxy-4thiocyanato-, 144 2,3,4-trideoxy-6-0-(methylsulfonyl)-4thiocyanato-, 144 -, methyl a-D-eythro2,3-dideoxy-, synthesis, 135 4,6-O-benzylidene-2,3-dideoxy-, synthesis, 135 Hex-3-enopyranoside -, ethyl o-erythro-, 2,3,4,6-tetradeoxy-2isothiocyanato-6-O-(methylsulfonyl), 142 -, ethyl D-thfeO-, 2,3,4,6-tetradeoxy-2isothiocyanato-6-O-(methylsulfonyl), 142 Hex-Cenuronic acid, 4-deoxy-~-threo-, biosynthesis, 296-298 Hexosaminidase, N-acetyl-Pa-, 204 Hexose, 4-deoxy-~-arabino-,biosynthesis, 296-298 Hexopyranoses C-C and C-0 bond-lengths in, 14-15 hydrogen bonding, Raman and infrared spectral study of, 29 Hexopyranoside -, methyl 3,6-dideoxy-P-D-ribo-, hydrogen bonding, Raman and infrared spectral study of, 29 -, 1-thio-P-Daqueous, laser-Raman spectroscopy, 75 laser-Raman spectroscopy, 81 -, 3,4,6-tri-O-acetyl-2-deoxy-P-D-arabino-, synthesis, 130 Hexose isotopic substitution, in i.r. and Raman spectra band assignments, 37 -, D-gUlUCtObiosynthesis, 287-294 structures, 289

481

SUBJECT INDEX -, L-galacto-, biosynthesis, 294-295

-, D-glUC0biosynthesis, 287-294 structures, 289 -, L-gluco-, biosynthesis, 294-295 -, D-mnnobiosynthesis, 287-294 structures, 289 -, L-munno-, biosynthesis, 294-295 Hexuronic acids, nucleotide-linked, biosynthesis, 289-290 Humic acids, spectral analysis and identification, 24 Hyaluronate lyase, 204 Hyaluronic acid, 198 model molecules, laser-Raman spectroscopy, 80,81 structure, enzymic analysis, 203-205 Hyaluronidase, 199,202, 205 Hydration, 87 Hydration shell, 87 Hydrogen bonding, See akro Carbohydrates, hydrogen bonding in water and aqueous solutions, vibrational spectroscopic studies, 86 Hydrogen-deuterium exchange, 36

I Idopyranoside, methyl a-D-,2,3-di-O-benzyl-4,6-dideoxy-4-thiocyanato-, 145 D-Idose, 4-S-acetyl-6-deoxy-4-thio-, synthesis, 132 a-L-Idosiduronase, 213-214 L-Iduronate sulfatase, 206 L-Iduronic acid, 2 biosynthesis, 296-298 IgE, 232 Infrared dichroism, 26-27 Infrared spectroscopy, 7-8, 16-22 band assignments, 3639 isotopic substitution, 36-38 model-compound approach, 38-39 electro-optical parameters, 35-36 noncomputer results, in analysis of foodstuffs and biological samples, 2224 Inositols, laser-Raman spectroscopy, 83 Interferogram, 57 Interstellar solid material, infrared spectroscopy of, 31 Invertase, membrane-associated isozyme, in plants, 370

Isoamylase, 252-253 Isolichenan, structure, enzymic analysis, 265 Isomaltodextranase. 258,260 Isomaltohydrolase. exo-, 258 Isopullulanase, 257 Isothiocyanates aryl, synthesis, 97 cellulose, synthesis, 97 cycloaddition of, 92 nucleophilic additions, 91 unsaturated, synthesis, 95-96

I Japanese agar, structure, enzymic analysis, 187 Juniprenol, structure, 346

K Keratan sulfate, 198-199 structure, enzymic analysis, 207-209 Ketoses, in bacterial polysaccharides, biosynthesis, 301-302

L Lactoferrin, 233 Lactose analysis and identification, 22-23 isomers, laser-Raman spectroscopy, 80 a-Lactose monohydrate, C-C and C - 0 bondlengths in, 15 Lactosylceramide, 437 in starfish, 429 Laser-Raman spectroscopy, 8-9,6745 advantages of, 73 applications, 85 of carbohydrates, results, 75-85 instrumentation, 70-73 sampling techniques, 70-73 Lectins in host-pathogen interactions, 380 potato, 234 role in plant recognition systems, 379-381 Levans, Fourier-transform infrared-difference spectroscopy, 62 Levoglucosenone, 4 Lichenan, structure, enzymic analysis, 273 Lichenanase, 273-275

482

SUBJECT INDEX

Lipid-linked sugars, in plants lipid moiety, 347-352 occurrence, 347 saccharidederivates, 352-356 structural aspects, 347-352 turnover, 356-358 Lipopolysaccharides bacterial. See Bacterial lipopolysaccharides core region, 278 Lutean, structure, enzymic analysis, 275 Lyase, 148

M ae-Macroglohulin,233 Maltohexahydrolase, exo-, 254 Malto-oligosaccharides, spectroscopicanalysis, 23 Maltopyranoside, methyl p-, C-C and C - 0 bond-lengths in, 15 Maltose aqueous, vs. solid, laser-Raman spectroscopy, 75 p-, hydrogen bonding, Raman and infrared spectral study of, 29 laser-Raman spectroscopy, 82 vibrational spectra, isotopic substitution studies, 54 Maltotetraohydrolase,ero and endo action patterns, 148 Maltotriose, laser-Raman spectroscopy, 82 Mannan from plant cell-walls, 359 D-

synthesis, in plants, 366 yeast cell-wall, structure, enzymic analysis, 248-250 yeast, hiosynthesis, 362-366 -, (1+ ~)-P-D-, enzymicanalysis, 165 Mannanase a-D-, exo-, Arthrobacter, 233 B-D-. 167,169,179-180 A . niger, 170-171 endo-(1--* 4)-, 165 exo-, 165 eXO-(l+ 4)-p-D-, 149 D-Mannan chain, transfer of D-gdactosyl substituents to, 172-173 p-D-Mannan mannobiohydrolase,exo-, 165 a-D-Mannopyranose,1,3,4,6-tetra-O-acetyl-2-deoxy-2-thiocyanato-, 145

Mannopyranoside -, methyl a-D-

2,3,4-tri-O-acetyl-6-deoxy-6-thiocyanato-, 145

3,4,6-tri-O-acetyl-2-deoxy-2-thiocyanato-, 145 -, methyl p-D-, 3,4,6-tri-O-acetyl-2-de-

oxy-2-thiocyanato-, 145 a-D-Mannopyranosylchloride, 3,4,6-tri-0acetyl-2-deoxy-2-thiocyanato-, 145 o-Mannose, anomers, correlatin between CH orientation and Vibrational frequencies observed, 21 Mannosidase a-D-, 209, 234-236 A . niger, 233 jack bean, 232,233 8-0-, 165,168, 209 Mannosylceramide,from freshwater bivalves, 420 a-D-Mannosy1chloride, 3,4,6-tri-O-acety1-2deoxy-2-thiocyanato-, synthesis, 130 D-Mannosyltransferase, 179 GDP-, 244 D-Mannuronan lyase, 192 Melanoidins, spectral analysis and identification, 24 Melanoma, 240 4-0-Methylglucuronoxylans enzymic analysis, 161 white willow, enzymic analysis, 162 Methyl glycosides, hydrogen bonding, 15-16 Michelson interferometer, 56-57 Mixtures, analysisof advantages of Fourier-transform infrared spectroscopy,58 by infrared spectroscopy, 58 Molecular interactions, in aqueous solution, 9

Molecular-mechanicscalculations, 16 Mollusks, glycolipids. See Glycolipids Monoglycosylceramide, from freshwater bivalves, 417 Monosaccharide isothiocyanates,93-123 '3cn.m.r., 139,141 'H n.m.r., 139 reduction by tributyltin hydride, 123 by triethylphosphine, 122 by triethyl phosphite, 122 by triphenyltin hydride, 122

483

SUBJECT INDEX spectroscopic properties, 139-141 UV spectra, 139 Monosaccharides Q and p anomers, 19-20 CH,OH group, possible dispositions of, 18-19 conformational analysis, 14 conformation and frequency calculations for, method, 51-52 conformation and tautomers, vibrational spectra used to analyze, 25 hydrogen bonding, Raman and infrared spectral study of, 29 hydroxyl groups, 18 laser-Raman spectroscopy, 75-81 lipid-linked, in plants, 352-355 Monosaccharide thiocyanates, 123-139 Mucilages, 375-376 Mutorotation, measurement, 25 Mymdextranase, 265 Myeloma proteins, human yG, 232

N Neuraminidase, 208 Normal coordinate analysis, 12, 32-34 computerization, 9 Nucleic acids infrared and Raman spectroscopic studies, 30 laser-Raman spectroscopy, 84-85 Nucleosides, laser-Raman spectroscopy, 84 Nucleotides, laser-Raman spectroscopy, 84 Nucleotide sugars, 280 Nucleotidyltransferases, 280-281

0 0-Hapten, 317 Oligosaccharides laser-Raman spectroscopy, 81-83 lipid-linked in animals, structure, 357 in plants, 355-356 sources, 356 structure, 357 Oligosylceramide, from freshwater bivalves, 417,419-420 One enzyme-one linkage concept, 306,311 Ovalbumin, 232, 239-240

P Papain, 208 Pectic polysaccharides, structure, enzymic analysis, 182-186 Pectin lyase, 183-184 Pectins, analysis and identification, 23 Peptidoglycan Bacillus cereus, 198 Micrococcus lysodeikticus, 195, 197 Staphylococcus aureus, 196 Peptidopolysaccharides, biosynthesis, in plants, 367-373 0-Phosphinicoglycosphingolipids,413 Phosphonoglymsphingolipids, from gastropods, 413-414 0-Phosphonoglycosphingolipids,in gastropods, 438 Phytoglycogen, biosynthesis, branching enzyme, 256 Pinoprenol, structure, 346 Plant cell wall, role of glycoproteins in, 382383 Plant gums, 375 Plant isoprenoids biosynthesis, 351 metabolism, 351 Plant polyprenols. See also Polyprenyl glycosyl phosphates occurrence, 347 structure, 346-350 Podzol, spectral analysis and identification, 24 Point group, 12 Pollen compatibility, 381 Polymerases, specificity toward structure of monosaccharide substrates, in biosynthesis of bacterial polysaccharides, 338-339 Polymers, orientational measurements in, 27 Polyols, complex formation with cations of Group I1 and with borate ions, laser-Raman spectroscopy, 83-84 Polyprenyl diphosphate trisaccharides, enzymes of biosynthesis, specificity toward structure of monosaccharide residues of substrates, 336-337 Polyprenyl glycosyl diphosphates, 285-286 Polyprenyl glycosyl monophosphates, 284285

484

SUBJECTINDEX

Polyprenyl glycosyl phosphates characterization, 3 4 - 3 4 as intermediates in synthesis of complex glycans, 343 solubility properties, 344 Polysaccharides. See also Bacterial polysaccharides; Pectic polysaccharides conformational analysis, 14 enzymes depolymerizing endo action pattern, 147-148 em action pattern, 147,148 Fourier-transform infrared spectroscopy, 63 having (1+ 4)-P-~-glucanbackbone, enzymic analysis of, 150-158 having (l+ 4)-P-~-mannanbackbone, enzymic analysis, 164-182 having P-D-xylan backbone, enzymic analysis, 158-164 in interstellar space, 31 laser-Raman spectroscopy, 75, 81-83 orientation, infrared dichroism study, 2627 from plant cell-walls, 358-359 synthesis, in plants functional aspects, 383 lipid intermediates in, 384-385 used as thickeners, analysis and identification, 23 Porphyran, structure, enzymic analysis, 189190 Proteoglycan aggregate, structure, enzymic analysis, 216-217 Proteoglycan-hyaluronatecomplex, Fouriertransform infrared spectroscopy, 61 Protuberic acid, structure, enzymic analysis, 247-248 Pseudonigeran, structure, enzymic analysis, 265 Zullulan biosynthesis, 367 from plant cell-walls, 359 structure, enzymic analysis, 256-257 Puhlanase, 148, 253-254, 256 Pulmonary glycoprotein, 232 Purpurosamine C, derivative, preparation, 128 Pustulan, structure, enzymic analysis, 275 Pyranose monosaccharides, hydrogen bonding, 15-16

R Raman effect, physical principles of, 67-70 Raman scattering, 67-68 of water, 70 Raman spectra, of carbohydrates, 8 Raman spectrometer, 70 Raman spectroscopy, 8, 16-22 advantages of, 70, 81 band assignments, 36-39 complementarity to infrared spectroscopy, 69 depolarization ratio, 68 electro-optical parameters, 35-36 noncomputer results, in analysis and identification of food carbohydrates and biological samples, 22-24 polarization directions of beams in, 68-69 Rayleigh scattering, 67-68 Recognition systems in animal cells, 378-379 in plants, 378-382 Redundant coordinates, 34 Resonance Raman effect, 84 Rhamnogalacturonan, 182-183 Ribitol, 1,5-anhydro-~-,laser-Raman spectroscopy, 83 Ribofuranose, 3-deoxy-1.2-O-isopropyhdene-3-thiocyanato-u-~-,145 Ribofuranosyl isothiocyanate, 2,3,5-tri-0benzoyl-P-D-, 142 W n.m.r., 141 infrared spectrum, 141 Ribofuranosyl-2-thiothymine,l-fl-o-,synthesis, 98 Ribonuclease, 232 D-Ribose Fourier-transform inhared spectroscopy, 65-66 pure and commercial, infrared spectra, 6566 Rous-sarcoma virus, 232, 2.38

S Saccharides complex formation with cations of Group I1 and with borate ions, laser-Raman spectroscopy, 83-84 synthesis, 341

SUBJECT INDEX Schiff bases, derived from D-glyCOSyl thiosemicarbazide and L-arabinosyl thiosemicarbazide, synthesis, 106-107 Sea urchins sialoglycolipids. See Sialoglycolipids sulfolipids. See Sulfolipids Shafizadeh, Fred, 1-6 career accomplishments, 5 development of thermal analysis methods,

4 education, 1 investigation of cellulose, 1-2 study of synthesis of biologically significant amino sugars, 2 teaching ability, 5-6 at University of Montana Wood Chemistry Laboratory, 3 4 6 work for Weyerhauser, 2-3 work on morphology and biogenesis of cellulose and plant cell walls, 4-5 Sialoglycolipids,388 from Asteroidea, 430-434 carbohydrate chain structure, mass spectrometry, 403 containing sulfated sialic acid, 424,426-

427 distribution, 437 distribution, in marine invertebrates, 426-

437 of echinoderms biological role of, 435-436 structure, 437 and evolutionary position of animals, 436-

437 occurrence, 392 of sea urchins, 423428,437-438 in vertebrates, 437 Sodium hyaluronate, Fourier-transform ink e d spectroscopy, 61 Soil organic matter, spectral analysis and identification, 24 Solanesol, 319 structure, 346 Sorbohranose, 1-deoxy-2,3:4,6-di-O-isopropylidene-l-thiocyanato-tpL-, 1 6 Spadicol, 349 structure, 346 Sponges, glycolipid content. See Glycolipids Starch, laser-Raman spectroxapy, 83 Starfish, glycolipids. See Glywhpids

485

Stokes lines, 68 Storage glycoproteins, in plants, functional aspects, 383 Submaxillary mucin, 240 Succinoglycan depolymerase, 225-226 Sucrose aqueous laser-Raman spectroscopy, 73-74,76-80 solute-solvent interactions, vibrational spectroscopic studies, 86 calcium complexes, laser-Raman spectroscopy> 84 C-C and C- 0 bond-lengths in, 15 cryoprotective effect, 89 determination of hydration numbers, 87 structure-making effect on water, 86 structure-properties relationships, vibrational spectroscopic study, 88 -, 6,6'-dideoxy-1,2,3,4,3',4'hexa-O-(methylsulfonyl)-6,6'-di(thiocyanato)-, synthesis, 138 Sugar colorants, spectral analysis and identification, 23-24 Sugar isothiocyanates, 93 conversion into thioureido intermediates,

97 conversion into substituted thioureides,

105 conversion into thioureido derivatives, 100 as intermediates in synthesis of nucleoside analogs, 97-123 reaction with mines, 97-113 reaction with amino acids, 113-117 reaction with ammonia, 97-113 reaction with carboxylic acids, 113-117 reaction with diamines, 119-121 reaction with diazomethane, 121-122 reaction with enamines, ll7-ll9 reaction with hydrazides, 99-100 reaction with hydrazines, 99 synthesis, method, 93-97 Sugar nucleotides, 341 sugars aqueous, infrared spectra, 18 determination of hydration numbers, 87 freeze-dried, Fourier-transform infrared spectrompy, 63-65 heterocyclic derivatives, 91 infrared spectra, at low temperatures, 28 structure of, anomeric center in, 14

486

SUBJECT INDEX

Sugar thiocyanates, 92-93 synthesis method, 123-139 by s N 2 nucleophilic displacement of sulfonyloxy groups in pentohranoses by thiocyanate ion, 135-139 by sN2 nucleophilic displacement reactions of sulfonyloxy groups in hexopyranoses by thiocyanate ion, 123-135 Sulfolipids, 387 of sea urchins, 428 Sulfonic esters, infrared spectroscopy of, 30 Symmetry operations, 12

T

Uridine (2-acetamido-2-deoxy-~-g~ucosyl diphosphate), 282. See also Glycosyl nucleotides Uridine 5’-diphosphate, glycosyl esters, 280. See also Glycosyl nucleotides

V Valence force-field, 38,39 V-Amylose, vibrational spectra, frequency calculations, 51 Vesicular-stomatitis virus G protein, 234 Vibrational degrees of freedom, 12 Vibrational frequencies, 12 calculations, methods, 31-34 Vibrational spectra intensities, calculation of, 35-36 noncomputer results, 22 Vibrational spectroscopy, background, 10

Talose -, 2-amino-2,6-dideoxy-~-,biosynthesis, 296-298 -, Bdeoxy-L-, biosynthesis, 296-298 W Taluronic acid, 2-amino-%deoxy-~-,biosynthesis, 296-298 Water 1,2,3,4-Thiatriazoles, model, synthesis, 101 Raman scattering of, 70 Thiocarboxamides, synthesis, 109-UO role of, in intensity of sweet-taste sensaThiocyanates, 91 tion, 88 Thiosemicarbazides. synthesis, 106-109 vibrational spectroscopic studies, 85-86 Thiothymine, 1-(tetra-0-acetyl-P-DWilson C F method, 32 glycosyl)-2-, synthesis, 97-98 Wines, i.r. spectroscopy in analysis of, 23 Thymidine 5’-diphosphate, glycosyl esters, Wolfrom, M. L., 1-2 280. See also Clycosyl nucleotides Worms, glycolipids. See Glycolipids Tragacanth, gum, 247 Trehalose, 2,3,4,2’,3‘,4’-hexo-O-acetyl-6,6’X dideoxy-6,6’-dithiocyanato-a-a-, 145 Trifoliin A, 379 Xantham gum Trisialosyllactosylceramide,437 biosynthesis, 376-377 Tunicates, glycolipids. See Clycolipids structure, 376 Xanthan Fourier-transform infrared spectroscopy, U 63 structure, 150-151 UDP-N-acetyl-D-galactosamine:a-Lfucosyl-(l,2)-~-galactose-a-3-N-acety~-~- enzymic analysis, 156-158 X-Ray diffraction, 87 gdactosylaminotransferase, 246 UDP-~-galactose:a-~-fcose-(~,2)-D-&ac- Xylan acetylated, enzymic analysis, 162 tose-a-3-o-galactosykransferase,246 UDP-o-galactose:N-acetyl-(l+ 4)-pDrice-straw, enzymic analysis, 162 D-galactosyltransferase,246 Undecaprenol, 349 Shirakamba wood, enzymic analysis, 162 B-D-, Rhodymenia palmata, enzymic analyUrey-Bradley force-field, 39 sis, 163 Urey-Bradley-Schimanouchi force-field, 39

SUBJECT INDEX hemicellulosic, 158 larch-wood, enzymic analysis, 159 orientational measurements in, 27 from plant cell-walls, 359 Rhodymenia palmata, 158 seaweed, 158 wheat-straw, enzymic analysis, 159 Xylanase p-D-,

163

Schizophyllum commune, 162 -, (1+ 4)-p-D-, 161 Cryptococcus albidus, 160-161 -, endo-(I + 4)-p-o-, 158, 163 lrpex lacteus, 159 Xylofuranose, 5-deoxy-1,2,-O-isopropyhdene-5-thiocyanato-a-~-,145 Xyloglucan Annona muricata, enzymic analysis, 151152

487

bamboo shoot, 156 enzymic analysis, 155 barley, 155-156 cellulase digestion, 152-154 mung bean, enzymic analysis, 154 oat coleoptile, 155 pea, enzymic analysis, 155 Phaseolus coccineus, 155-156 from plant cell-walls, 359 rice endosperm, 155 soybean, 155-156 structure, enzymic analysis, 151-156 sycamore, enzymic analysis, 152 Tamarindus indica, enzymic analysis, 151 Vigna sesquipedalis, enzymic analysis, 154 Xylopyranose, 5-thio-D-, synthesis, 135 P-D-Xylosidase, 159,162-163 D-xylOSyhanSferaSe, 202-203

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