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Advances in Carbohydrate Chemistry and Biochemistry

Volume 44

This Page Intentionally Left Blank

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.

ACADEMIC PRESS, INC. Orlando, Florida 32887

United Kingdom Edition published by

ACADEMIC PRESS INC. (LONDON) LTD. 24-28 Oval Road. London NWI 7DX

LIBRARY OF CONGRESS CATALOG C A R D NUMBER:45-1 135 1 ISBN 0-12-007244-0 PRINTED IN THE UNITED STATES OF AMERICA

86878889

9 8 7 6 5 4 3 2 1

CONTENTS PREFACE.

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

vii

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

vi

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

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

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

vii

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

OBITUARY-FRED

SHAFIZADEH

3

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

OBITUARY-FRED

SHAFIZADEH

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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GARY D. McGINNlS

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.


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.


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


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.


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.


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.


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


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)


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.


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


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.


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


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.


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


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.


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


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.


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


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.


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


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)


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


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


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


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.


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


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.


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


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


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


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 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


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.


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.


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


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.


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-).


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


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.


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


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


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


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.)


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


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.


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.)


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


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.


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.


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.


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


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.


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.


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.


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


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.


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.


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-
9 and 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 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.]


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


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


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


(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.


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


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.


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


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.


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


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.


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


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


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.


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


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.


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


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.


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


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.


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


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


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.


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


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.


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


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)


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


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.


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


+ 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.


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


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.


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


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.


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


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)


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.


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


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.


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


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.


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


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.


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


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.


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.


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


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.


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


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.


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


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.


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


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.


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


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


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


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)


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)


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


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)


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.


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.


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


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.


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


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 (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


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


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


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)


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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